Bibliothek

Notizen: Summary Projections of the somesthetic ascending pathways to the superior colliculus (SC) of the cat have been demonstrated by means of the horseradish peroxidase (HRP) tracing technique. Following HRP injection in one SC, marked neurons were found in the contralateral dorsal horn and in the contralateral dorsal column nuclei, mainly the cuneate nucleus. Moreover, in the contralateral trigeminal complex, the sub-nucleus caudalis of the spinal nucleus of the Vth nerve, and the subnucleus oralis plus the main sensory trigeminal nucleus were labeled. Thus, the anterolateral system and the lemniscal system subserving face and forelimb sensation send afferent fibers to the contralateral SC.

Notizen: Summary In the cat caudate nucleus the same nine types of synapses are found as in putamen and fundus striati. The three parts of the striatum in the strict sense do not differ in the morphological differentiation of synapse types but only in their quantitative distribution. One-third of all synapses in the caudate nucleus are axo-spinous type IV synapses with a curved and divided asymmetric contact. This strongly suggests that the caudate nucleus interneuronal apparatus is dominated by centre-median input, in contrast to the putamen which is controlled by the cortico-striatal input to its internuncial cells and by its strong intrastriatal feedback mechanism. Extensive destruction of the convexity of the cortex and the medial cortex in one hemisphere results in dark degeneration of a large proportion of two of the nine types of caudate synapses: a) the axo-dendritic type VII synapses exciting the large aspiny caudate neurons and b) the axo-spinous type III synapses making contact with the small spiny neurons of the interneuronal cell apparatus.

Notizen: Summary Retrograde tracing experiments using horseradish peroxidase (HRP) have been utilized for demonstrating the origin of efferent projections of the ventral lateral geniculate nucleus (LGNv) in the cat. HRP-positive cells identifiable as origins of thalamic projections were found in LGNv after injections of HRP into the lateral central intralaminar nucleus. The labeled cells appeared concentrated in the medial part of the internal division of LGNv, consisting of medium-sized multipolar cells. Contralaterally, fewer labeled cells were present in the corresponding part of LGNv. In the case of injections of HRP into the midbrain (pretectum and superior colliculus), labeled cells in LGNv were distributed almost exclusively in its external division, composed of mainly small cells. Little overlap of the distribution of HRP-positive cells was seen in LGNv between the thalamic and midbrain injection cases.

Notizen: Summary It has previously been shown that unilateral ablation of the whole auditory cortex in the cat disrupts the precedence effect, and also interferes with the ability of the normal animal to discriminate in the Y-maze between a single sound on one side and a double sound consisting of a signal on both left and right sides. The present work has confirmed these effects and has shown that both can be obtained with lesions confined to AI and AII The ‘one-versus-two’ deficit has invariably been seen in all the animals studied, but a proportion of animals do not show the precedence effect deficit. It has been confirmed that the apparent success of some animals can be due to the training effect of the one-versus-two paradigm, as was proposed in the earlier paper; however it has also been shown that this cannot be the explanation in all cases. It has been demonstrated that cats are able to localize sounds behind them with some success; turning around in the start box to reverse right and left space is therefore a possible strategy for overcoming a unilateral deficit. However, even with the head ‘fixed’ in the forward-facing position, one animal was still able to run well above chance. The size of the lesion does not appear to be a correlate of the performance level.

Notizen: Summary Electron microscopical studies of the thalamic AV-AM nuclei substantiated the presence of two main types of neurons, i.e. principal (or relay) cells and Golgi type II interneurons. Characteristic synaptic islands are found in abundance in the AV-AM. Four different types of synaptic terminals have been identified in these islands: RL-boutons = large axonal terminals with round synaptic vesicles; RS-boutons = small axonal terminals with round synaptic vesicles; F1-boutons = small axonal profiles containing flattened synaptic vesicles, and F2-profiles interpreted as presynaptic dendrite appendages, bearing pleomorphic vesicles, both belonging to the Golgi type II interneurons. — The synaptic relations were studied in normal preparations and after lesions in the mamillary body, limbic cortex and hippocampus. The specific afferents (RL-boutons) — originating from the medial mamillary nucleus — are presynaptic to both relay cell dendrites and “presynaptic” dendrite profiles of Golgi type II interneurons, which in turn are presynaptic to the same relay dendrites (synaptic triads). RS-boutons originate mainly from limbic cortex and hippocampus.

Notizen: Summary A sudden and rapid angular displacement of the limb evokes, in human and monkey subjects, a segmented pattern of electromyographic activity in muscles which are stretched. While the first segment is acknowledged to represent a tendon jerk, it has been proposed that the second segment, occurring with a shorter latency than a reaction time, is mediated by a transcortical loop. The present experiments were conducted in cats to determine the properties of muscle responses to torque perturbations analogous to those used in the monkey, and to determine if the integrity of supraspinal pathways is required for the individual response segments to occur. Torque perturbations which flexed the forearm evoked a segmented response in the electromyogram of the cat triceps muscle. This response typically consisted of three early segments with latencies of 10, 30 and 60 msec which were similar to the M1, M2, and M3 segments described in the monkey. The M3 and occasionally M2 components were depressed when the cat followed rather than resisted the perturbation. A torque pulse of 10 msec duration was sufficient to elicit a near maximal M1 response while torque pulses in excess of 20 msec were required to evoke the M2 response. To determine if any of these components required mediation by the cerebral cortex, experiments were conducted in decerebrate and spinal cats. Similar torque perturbations produced segmented electromyographic responses in the triceps muscles which were indistinguishable in their timing from those observed in intact cats. The torque required to produce the segmented responses was comparable as well. All three segments were dependent upon the activation of receptors in the homonymous muscle and did not require cutaneous input. These observations show that receptor properties and/or spinal mechanisms involved in the stretch reflex are sufficient to produce a segmented response similar to that observed in intact animals.

Notizen: Summary The ascending and intrinsic projections of the superior olivary complex (SO) in the cat were investigated by injection of 3H-leucine and horseradish peroxidase (HRP) in SO and the inferior colliculus (IC), respectively. A topically arranged projection was demonstrated from the nucleus of the trapezoid body (NTB) to the ipsilateral lateral superior olivary nucleus (LSO) with a lesser connection in the opposite direction. The medial superior olivary nucleus (MSO) has a strictly ipsilateral projection, whilst LSO projects symmetrically through the lateral lemniscus (LL) of both sides, to end with topically arranged terminals in the ventrolateral part of the central nucleus of the inferior colliculus (CNIC). Terminal labelling found in the ventral and dorsal nuclei of LL (VNLL and DNLL) probably represents collaterals from bypassing fibres originating in MSO and LSO, respectively. These results were demonstrated by both techniques, whilst in addition the HRP method revealed an ipsilateral and a contralateral projection to IC from VNLL and DNLL, respectively.

Notizen: Summary Single unit recordings were carried out in the reticularis thalamic nucleus (RT) and the ventral lateral geniculate body (LGv) of chronically prepared alert cats under sinusoidal vestibular stimulation in the horizontal plane. Optokinetic stimulation was also used. Of the 57 recorded neurons, 12 present vestibular modulation in the dark, analogous to Duensing's and Schaefer's (1958) type I response in the vestibular nuclei. Responses of 26 cells are similar to response of type II vestibular neurons and 14 units have a type III response; the 5 remaining cells were activated by vestibular stimulation in the vertical sagittal plane. The majority of these cells does not present detectable direct visual responses, but 50% can be driven by Optokinetic stimulation. 74 % of types I, II and III neurons show saccadic responses to vestibular nystagmic saccades in the dark. About 60% present similar saccadic modulations during Optokinetic nystagmus and 55% keep this response for spontaneous saccades in the dark or in front of a striped background. The saccadic responses are constant for a given neuron in all cases of stimulation with latencies ranging from 30 msec prior to the beginning of the saccade to 120 msec after its onset. The histological localization of these units falls on one hand into the caudal part of the RT nucleus (type III neurons) above the dorsal lateral geniculate nucleus and on the other hand within the internal subdivision of the LGv and its rostral limit (all other types). The significance of this new, saccadic and vestibular focus in the feline thalamus is discussed in relation with the two previously known vestibular thalamic relays in terms of interrelations between the vestibular and the visual systems.

Notizen: Summary Different areas of the frontal cortex of the cat were injected with small amounts of horseradish peroxidase. The regions of labeled cells in the mediodorsal nucleus of the thalamus (MD) were related to the injected areas. Distinct relations between subdivisions of MD and of the prefrontal cortex were established: a rather large central sector of MD projects to the gyrus proreus and to the anterior parts of the gyri sigmoideus, rectus, and frontalis. A narrow lateral band of anterior MD neurons projects predominantly to an area on both sides of the sulcus praesylvius, whereas a postero-lateral band sends fibers to a region on the ventral anterior sylvian gyrus. The area between the presylvian sulcus and the sylvian gyrus is apparently free of MD afferents, but not of other thalamic afferents. A fourth sector of MD, situated dorsomedially, projects to the middle parts of the gyri rectus and frontalis. And a fifth sector, located ventrally to the dorsomedial MD sector, projects to the ventral part of the gyrus rectus. The established subfields of MD and of the prefrontal cortex are discussed with respect to previous anatomical research in the cat.

Notizen: Summary 1. In the cat visual cortex (VC), electrophoretic glutamate application at a depth corresponding to layer VI may have excitatory or inhibitory effects on relay cells of the lateral geniculate nucleus (LGN). Corticofugal excitation was seen, if the receptive field centers (RFCs) of the VC neurons recorded at the application site were within 2.3 ° of the RFCs of the LGN neurons under test. Inhibitory effects were seen if the RFCs of both cells were further apart up to 3.1 °. Glutamate application at more superficial cortical sites had no effect on LGN-neuron activity. 2. Cross-correlation analysis between spontaneous activities of simultaneously recorded VC and LGN neurons revealed excitatory cortico-geniculate connections in 18 pairs with RFCs separated by less than 1.7 °. In 15 pairs the peak latency of the excitation was 2–5 msec (3.4 msec in the average), 3 pairs showed long cortico-geniculate latencies (13–18 msec). The existence of a fast and slow cortico-geniculate system is suggested. 3. Inhibitory cortico-geniculate interaction was demonstrated with cross-correlation analysis in 8 pairs of which 4 had RFCs separated by more than 1.7 °. The onset latency of the inhibition was 2–7 msec except for 2 pairs with about 20 msec latency. 4. Most of the LGN neurons which were affected by cortical glutamate application or which showed an excitatory or inhibitory connection with a VC neuron were sustained cells, while the majority of VC neurons which were recorded in the effective glutamate application sites or which showed a significant interaction with LGN neurons in the cross-correlogram were binocularly driven and complex, with mostly large RFCs (mean diameter 3.5 °). They responded briskly to moving small spots as well as to moving slits. 5. It is concluded that the corticofugal excitatory effect is transmitted through monosynaptic links from VC neurons located in layer VI (complex cell) to LGN relay neurons (mostly sustained-cell) and this system is organized in a precise topographical manner. 6. In an Appendix neuron pairs which showed a positive correlation in the geniculo-cortical direction were described. The findings may support the view that complex as well as simple cells are driven monosynaptically from geniculo-cortical afferents of the sustained or transient type.

Notizen: Summary In the anesthetized cat, the posterior canal nerve (PCN) was stimulated by electric pulses and synaptic responses were recorded intracellularly in the three antagonistic pairs of extraocular motoneurons. Pure reciprocal effects were obtained in the motoneurons innervating the antagonistic pair of ipsilateral oblique muscles and the antagonistic pair of contralateral vertical rectus muscles. These responses consisted of low threshold disynaptic excitatory postsynaptic potentials (EPSPs) in either the contralateral superior oblique (c-SO) (trochlear) or contralateral inferior rectus (c-IR) motoneurons and of disynaptic inhibitory postsynaptic potentials (IPSPs) in either the ipsilateral inferior oblique (i-IO) or ipsilateral superior rectus (i-SR) motoneurons. In addition, disynaptic IPSPs were also found in (i-SO) motoneurons. Mixtures of low threshold (dior trisynaptic) EPSPs and IPSPs were found in all other extraocular motoneurons except for the contralateral lateral rectus (c-LR) motoneurons. These results may afford a basis for the characteristic eye movements induced by vertical canal nerve stimulation.

Notizen: Summary 1. The activity of cat semicircular canal and otolith afferents was studied during yaw and roll rotations, respectively, to examine their dynamic behavior. 2. A sinusoidal analysis of the canal afferent activities showed that their dynamic characteristics are similar to those of second order vestibular neurons, except for a two to three-fold lower absolute gain. This agrees with earlier studies using angular acceleration steps. 3. Both divisions of the eighth nerve were sampled so as to examine afferents from both the utriculus and sacculus. Within the range of inputs used (± 25 degrees lateral tilt), the presumed saccular afferents (inferior division) showed either a γ- or β-response. However, the gain of their response was generally much less than for the afferents of the superior division (mostly utricular). This behavior is to be expected on the basis of receptor orientations and the components of gravity acting upon the macular receptors. 4. In response to ramp changes in angular position, some otolith units showed a phasic-tonic response pattern, i.e., an overshoot followed by an adaptation to a new steady state level of activity. The majority of units showed predominantly tonic responses proportional to displacement. 5. During sinusoidal rotations the predominantly tonic units showed small phase leads of 0 to 15 degrees at 0.025 Hz which remained constant or decreased to 0 to −15 degrees at 1.0 Hz. The gains were flat or increased by up to 2 fold. The phasic-tonic units showed greater phase leads, 10 to 50 degrees, and gains which increased from 2 to 8 fold. 6. This behavior of otolith afferents suggests that they can provide information about both the magnitude and the rate of change of linear acceleration stimuli.

Notizen: Summary 1. The electrical activity of single trochlear motoneurons (TMns) and axons of second order vestibular neurons presumably terminating on these motoneurons were studied during natural stimulation of semicircular canals and otolith organs in cats anesthetized with Ketamine. 2. Null point analysis showed that TMns received an excitatory canal input from the contralateral posterior canal, and labyrinthine lesion experiments suggested that the functionally synergistic, ipsilateral anterior canal provides an inhibitory input. A small number of motoneurons showed orthogonal canal convergence. 3. In addition to the canal projections most TMns received an otolithic input. Firing rate was proportional to lateral head tilt and was of the β type. Most units also responded to pitch with an increase and decrease in firing rate on nose-up and nose-down positioning, respectively. Lesion experiments indicated that the otolith responses are the results of reciprocal innervation of TMns by contralateral (excitatory) and ipsilateral (inhibitory) otolith projections. 4. During sinusoidal rotation in yaw (canal only stimulation) the mean phase lag re acceleration of the response of TMns increased from 60 ° at 0.025 Hz to 126 ° at 1.0 Hz. In roll (canal plus otolith stimulation) the phase lag of TMn responses measured 180 ° and 130 ° at 0.025 and 1.0 Hz, respectively. Phase-lags measured in Vi and Vc axons were less by ca. 15 °. 5. The otolith contribution to TMn responses in roll was calculated by vectorial subtraction of the yaw from the roll responses: A phase lag of 10 (0.025 Hz) to 90 ° (0.5 Hz) re. displacement was noted and gain was constant over the same range. Similar lag dynamics were revealed in TMns when studied during ramp displacement of the head. 6. The possible functional role of central canal-otolith convergence and the differences between the responses of primary vestibular afferents and secondary vestibular neurons and TMns will be discussed.

Notizen: Summary It is known that kittens reared in ways that restrict movement while visual stimulation is received exhibit deficits in visually guided behavior. Presumably, the behavioral dysfunction is due to a lack of sensorimotor coordination during visual exposure. The current investigation was undertaken to study this effect both physiologically and behaviorally. Two groups of kittens were normally reared until they were nearly 4 weeks old. They were then placed in a darkroom and exposed daily for 1–2 hours while one eye was occluded. On alternate days, alternate eyes were covered. One group was restrained in a body cast while a given eye was exposed, but the kittens were free to move about while the other eye received visual experience. A second control group was alternately occluded, but freely mobile during all exposures. An additional cat was also unrestrained during daily rearing periods and neither eye was ever occluded. Behavioral tests showed clear deficits when the “passive” eye of the restrained-unrestrained group was used. Deficiencies were found in visually guided paw placing, pursuit eye movements, and jumping behavior to a platform. Responses of single cells were studied in area 17 of the visual cortex. Ocular dominance distributions showed marked reductions in binocularity for alternate occluder cats. The eye that had received exposure while animals were active in the restrained-unrestrained group also tended to predominate but the difference was not statistically significant.

Notizen: Summary In the previous study it was shown that cats were capable of making rapid and accurate adjustments in the force they applied to a lever in accord with information provided by a compensatory display. In the present study, isometric responses were examined in greater detail to determine 1. if a general control policy (or model) governing responses of different magnitudes could be inferred from the relations among output parameters and 2. if the earliest output measures were scaled to the preceding sensory events. The force adjustments elicited by the sudden motion of the display showed a linear relation between the peak force and the peak of its first derivative, dF/dt. Similarly, the peak d2F/dt2 was a linear function of dF/dt. By contrast, the times required to achieve the peak force and the peak dF/dt were largely independent of their magnitudes. These adjustments were produced by a burst of EMG activity in agonist muscles which coincided with the rising phase of dF/dt. The observations suggest that such motor outputs are determined by a pulse-step control policy. The amplitude of the pulse would control the rate of rise of dF/dt (and therefore also the peak force since the rising phase of dF/dt was of constant duration), and the step would control the level of the terminal steady state force. Both the peak force and the preceding peak dF/dt were highly correlated with the amplitude of the perturbation. Changes in display gain, which altered the required relation between input and output magnitudes, resulted in a gradual readjustment of the output parameters. It was concluded that the motor outputs were scaled from their inception to requirements dictated by the initial sensory information. The selection by the cat of the appropriate scaling function was contingent upon its previous experience with the device.

Notizen: Summary A versatile tracking task has been developed to assess the competence of feline subjects in using sensory information to initiate and guide limb movement. In the present study we investigated the response latency and the factors which affect it in order to define temporal constraints on the underlying processing of information. The paradigm makes use of an electromechanical display of the difference between the output of transducers in a manipulandum (controlled by the cat) and a target level (controlled by the experimenter). Because of inertia and friction, a step change in target level required 230 msec to fully shift the display. The animals were trained to match the position of the manipulandum (or the force they applied to it isometrically) to the target level for a food reward. Target perturbations elicited rapid position or force adjustments of appropriate direction and magnitude at short latency. Under isometric conditions, the latency of the response was an inverse function of the extent of display motion and of the peak rate of force change attained during the adjustment. Asymptotic values of the response time, measured from the perturbation to the first change in force applied to the lever, were typically between 50 and 70 msec. Since the motor responses were invariably initiated while the display was still moving, the animals must have used information contained in the derivatives of its motion. In the absence of visual cues, deflection of the vibrissae by the sudden motion of the display provided two of the four animals with sufficient information to initiate and guide their movements without changes in response latency. When vision was allowed, section of the vibrissae produced a persistent increase in response times of 15–20 msec. This difference in latency is compatible with the delays due to retinal transmission. The short time interval elapsing between stimulus and response strongly suggests that the topography and the metrics of the underlying transformations are adjusted by gating and/or biasing processes which precede the stimulus. It is concluded that sensory information can generate purposeful movements through pathways which include only a small number of central relays in series, independently of its modality.

Notizen: Summary The current source density (CSD) method in its one-dimensional approximation is used to analyze the field potentials in visual areas 18 and 17 of the cat, which were elicited by stimulating electrodes in the optic chiasm (OX), the optic radiation (OR) or in the respective cortical area itself. The CSD analysis reveals the basic pattern of excitatory postsynaptic activity. 1. In both visual areas the basic specific excitatory activity flows along three different intracortical pathways, all starting in layer IV: The first pathway relays activity from layer IV to supragranular pyramidal cells via strong, local connections to layer III and from there through long-distance connections to layer II. The second pathway conveys activity from layer IV to layer V, where it mainly contacts apical dendrites of layer VI pyramidal cells. This infragranular polysynaptic activity is not clearly resolvable into separate components, suggesting that it is conveyed by various groups of axons, among them long-distance horizontal connections. The third pathway has one synaptic relay within layer IV and then conveys activity to layer III. In addition, monosynaptic activity is revealed in layers VI and I. 2. In A 18 one coherent, fast-conducting group of afferents induces this basic activity pattern. In A 17 no such fast conducting input is resolvable; the supragranular activity is induced by a small group of afferents with intermediate conduction velocity, which terminate in the upper part of layer IV. The infragranular activity is induced by afferents with slower and widely scattered conduction velocities, which terminate in the lower part of layer IV. The layer VI input is very prominent in A 17 and also has a wide latency scatter. 3. The supragranular activity is more prominent in A 18 than in A 17 and the respective layers appear thicker, in accordance with anatomy. In A 17 the infragranular activity prevails and layers IV and VI appear very broad, again in accordance with anatomy. 4. Comparison of the CSDs with the original evoked potentials shows that the surface evoked potentials over A 18 reflect the three dipolar sink/source distributions of the coherent monosynaptic activity in layer IV and of the two prominent polysynaptic activities in layers III and II. The widely scattered activity in the lower part of layer IV in A 17 and all infragranular activities in both areas generate smaller, partly closed-field potentials; those are not discernible from the strong far-field potentials which originate from the supragranular activity and — especially in A 17 —from farther distant events.

Notizen: Summary The distribution of the interhemispheric projection from area 17 and 18 was studied using the anterograde degeneration technique. Besides the classical visual areas (17, 18, 19), area 21 and several visual areas in the middle suprasylvian sulcus also received visual callosal input. In the four terminal areas of the middle suprasylvian sulcus the projection was found to be focused on representations of the vertical meridian including the area centralis, as in the classical visual areas. An increase of the width of visual field represented in the zone of callosal terminations can be seen from area 17 through area 18 to area 19 and possibly this trend continues in the suprasylvian visual areas.

Notizen: Abstract The effects of the ergolene derivatives bromocriptine, dihydroergotoxine, methysergide, d-LSD, CF 25-397, and 29-712 on the metabolism of the biogenic amines in the brain of the rat were investigated. All six ergolene derivatives were found to increase the concentration of 4-hydroxy-3-methoxyphenylethylene glycol sulphate in the brain stem, i.e., to increase the turnover of noradrenaline (NA). Since in brain homogenates the agents inhibited the binding of 3H-dihydroergocryptine to α-adrenoceptors, but only weakly inhibited the binding of 3H-alprenolol to β-adrenoceptors, it is suggested that the increased turnover of NA may be a consequence of a blockade of α-adrenoceptors by ergolenes. All of the ergolenes increased the concentration of serotonin (5-HT) in the cortex, but only bromocriptine and 29-712 increased the concentration of 5-hydroxyindoleacetic acid (5-HIAA), the other compounds decreasing the concentration of this metabolite, i.e., inhibiting 5-HT turnover. Reserpine-induced PGO waves in the cat were inhibited by all six compounds, bromocriptine and 29-712 being the least active. Both of these findings suggest that the ergolenes possess serotonergic activity. The increase in the concentration of 5-HIAA after bromocriptine and 29-712 may be secondary to some action on other systems. The actions of the ergolenes on the metabolism of dopamine (DA) in the striatum are more complex. Bromocriptine, 29-712, and, to a much lesser extent, dihydroergotoxine reduced the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC), i.e., they inhibited DA turnover. These findings are compatible with the proposed dopaminergic activity of the drugs. CF 25-397 caused a slight increase in the DOPAC concentration at high doses, and d-LSD and methysergide caused pronounced increases. At doses below 1 mg/kg i.p., d-LSD decreased the DOPAC concentration. This biphasic effect of d-LSD may be due to interaction with different types of DA receptors or may reflect some secondary action of the compound. The profiles of activity of the various ergolenes are discussed. Bromocriptine and 29-712, wich have similar profiles of activity, can be clearly differentiated from the other ergolenes. CF 25-397 seems to be a potent and, at low doses, specific serotonergic drug.

Notizen: Abstract The effect of the new 1,5-benzodiazepine clobazam on visual evoked potentials (VEP) and spontaneous EEG in the conscious rabbit and on spinal polysynaptic reflexes in the decerebrated cat was studied in comparison to the 1,4-benzodiazepine diazepam. Clobazam was half as potent as diazepam in depressing the amplitude of visual evoked potentials in the nonanaesthetized rabbit, whereas the depressing effect on spinal polysynaptic reflexes in the decerebrated cat was only 1/7–1/30 of the diazepam effect. The action of clobazam and diazepam on VEP also showed differences in time course, i.e., the peak effect of clobazam lasted from 1 to 6 h after application, whereas the effect of diazepam appeared after 10min and declined already after 1 h. Both compounds had similar effects on computer-analyzed spontaneous EEG in the rabbit (power spectrum analysis), with an increase of power in the beta-band (13–39 Hz) and a decrease in the alpha (8–13 Hz) and theta (4–8 Hz) bands.

Notizen: Summary Following stereotactically performed lesions in nucleus reticularis tegmenti pontis (N.r.t.) degenerating fibers are traced to the contralateral N.r.t., to the pontine nuclei, through brachium pontis to restricted areas of the cerebellar nuclei and to most parts of the cerebellar cortex where they terminate in the granular layer. Most degenerating fragments are found in the contralateral half of the cerebellum with the greatest density in the vermal lobules VI and VIIA and in the flocculus. Following injections of HRP in the various cerebellar lobules labeled cells are mainly present within limited groups in the N.r.t.. Injections in vermal lobules VI-VIII B give rise to labeled cells within circumscribed areas in the dorsal and ventral parts throughout the rostrocaudal extent of the N.r.t.. In cases with injections in lobule IX or the ventral paraflocculus labeled cells are found ventrally in the rostral half of the N.r.t., while following injections in the vermal lobules I-V labeled cells are mainly found in the ventral and caudal part of the N.r.t.. Following injections in the intermediate and lateral parts of the anterior lobe, Crus I and II, the paramedian lobule and the dorsal paraflocculus labeled cells occur within groups in medial and lateral parts throughout the rostrocaudal extent of the N.r.t.. Following injections in the flocculus labeled cells are found in a very distinct group in the dorsal and rostral part of the N.r.t., While an injection in the nodulus (lobule X) gave rise to a smaller group of labeled neurons in the dorsolateral corner in the caudal part of the N.r.t.. Labeled cells within processus tegmentosus lateralis (p.t.l.) are only found following injections in lobules VI-VIIIA, Crus I and II and the dorsal paraflocculus. From what is known about afferents to the N.r.t., it is concluded that no cerebellar lobule gets information from one only of these sources via the N.r.t.. Visual information can probably be mediated from the superior colliculus via the N.r.t. to the flocculus and to a minor extent to the vermal lobules VI-VIII B, and from the pretectum via the N.r.t. to both vermal and lateral parts of the cerebellum.

Notizen: Summary The light microscopical morphology and proteinase activities are studied during the first phases of implantation in the cat, i.e. at 12, 13 and 14 days post coitum (d.p.c.). Timed matings are used to obtain exact data on the time course of implantation events. Size and shape of the blastocysts and the topographical relationships between trophoblast, zona pellucida and endometrium are studied on cryostat sections. These observations indicate that the zona pellucida is being removed at 12 d p.c. by dissolution which starts at the abembryonic pole and lateral of the embryonic disc. Since the zona has, in spite of the considerable expansion of the blastocyst, a thickness of 8–10 μm at this stage, it must have undergone a process of swelling or material must have been added invisibly. Invasion of the trophoblast into the endometrium begins between 13 and 14 d p. c. and is fully under way at 14 d p. c. Widening of the glandular lumina in the neighborhood of the blastocysts at 12–13 d p.c. indicates an early preimplantation interaction between the blastocyst and the endometrium. Amino Acid Arylamidase (Aminopeptidase) activity is found, in histochemical tests, to be high in the trophoblast but low in the endometrium in all three investigated stages. Proteinase Activity is studied with a highly sensitive gelatin substrate film test. Moderate to medium activity is found in the trophoblast at 12–13 d p.c. Very high proteinase activity is present in the invasion zone at 14 d p.c. Experiments with a large number of specific proteinase inhibitors in vitro and preliminary investigation of pH dependence show that it is mainly due to a cathepsin-B-like endopeptidase. This enzyme can be traced to both the trophoblast and the uterine epithelium. The disintegrating zona pellucida shows, at 12 d p.c., only little gelatinolytic proteinase activity. A trypsin-like endopeptidase as described for the rabbit blastocyst could not be identified with certainty in the cat but there is some indication that it might be present at 12 d p.c. Considerable trypsin-like proteinase activity is found in scattered endometrial stroma cells at all stages. The possible physiological role of the described proteinases in implantation is discussed.

Notizen: Summary The blood, yolk sac, liver, spleen, and bone marrow from 65 timed embryos of 22 cats were examined using light microscopic means in six different age categories. Nucleated primitive erythroblasts derived from the yolk sac are mature by the 19th day and represent 98% of the circulating blood cells, some of them found circulating even at birth. Definitive yolk sac erythropoiesis comprises a span of up to 30 days. On the 36th day, hematopoietic contribution drops to 15%. Neutrophils and the first thrombocytes are present on the 17th day, eosinophils and lymphocytes by the 25th day. Hepatic hematopoiesis most likely begins with definitive erythropoiesis on abouth the 20th day; granulopoiesis occurs in the liver on the 25th day. Blood forming tissue in the liver amounts to 28% which drops to 4% at birth. Splenic hematopoiesis begins on about the 36th day but contributes little to the blood. Bone marrow activity begins at mid-term and supplies about 50% of the blood cells on the 45th day. Hematocrit values increase from 22% on the 36th day to 47% at birth, thus exceeding the normal value of adult cats. The red blood cell number increases from 0.8 million/mm3 on the 25th day to 3.8 million on the 45th day and 6.3 million at birth. The total leukocyte count (880 on the 45th day and 6.480 at birth) must be calculated from the differential count of nucleated cells. Primitive erythroblasts represent the most common nucleated cells on the 25th day; on the 36th and 45th day, definitive erythroblasts predominate, but are outnumbered by leukocytes at birth. On the 36th and 45th day, lymphocytes are the predominating cell type in the white blood picture. The contribution of the hematopoietic organs to the feline prenatal blood formation is shown graphically.

Notizen: Summary The Golgi architecture of the Fascia dentata and hippocampus is described in the cat. The main cell types are like those found in other species. The initial collaterals of granule cell axons were demonstrated and are commented on. The interneurons proved to be of several types and subtypes not observed in lower species. The intrinsic fibers and few of the afferents — both in the dentate fascia and in the hippocampus — are discussed.

Notizen: Summary Electronmicroscope studies have been performed on the greater splanchnic nerve and the nerve to the medial head of gastrocnemius muscle of control and acrylamide poisoned cats. Degeneration of unmyelinated as well as of myelinated fibres was observed in both nerves. In cats severely poisoned with acrylamide, some very large unmyelinated axons undergoing early degeneration were seen in the splanchnic nerve. In the nerve to medial head of gastrocnemius, there was a decrease in the proportion of large diameter unmyelinated axons.

Notizen: Summary An 18 month old cat had a 6 month history of abnormal high stepping gait, ataxia, muscle twitching and loss of sensory perception. The clinical course was interrupted by several episodes of temporary remission. The essential pathological features were those of a polyradiculoneuritis with segmental demyelination, focal mononuclear cell infiltration and hypertrophic onion-bulb formations. The condition is compared with chronic relapsing polyradiculoneuritis in the dog and in man.

Notizen: Summary A method based on an intracerebral coordinate system and contrast ventriculography was developed for Stereotaxic surgery in the cat. The intercommissural (CA-CP) line was used as the principal reference because its length showed little variability in animals within a wide weight range. The Stereotaxic coordinates of the substantia nigra (SN) in relation to the CA-CP line and the midline were evaluated in serial sections. Stereotaxic operations with the use of ventricular radiography were performed in different parts of the SN in 18 cats. Histological verification showed that the selected target regions within the SN were very accurately reached in all animals operated with this method. The precision of the Stereotaxic method based on the intracerebral coordinate system compared to the Horsley-Clarke method is discussed.

Notizen: Summary Light microscopic autoradiography and electron microscopy were used to trace the nigrothalamic projections and to study the sites of termination of this pathway in the cat. Injections of tritiated amino acids or electrolytic lesions were placed in the substantia nigra pars reticularis (SNr). An accumulation of radioactivity was found in the ventral medial nucleus and in the ventromedial part of the ventral anterior nucleus. At the ultrastructural level degenerating medium size synaptic boutons and medium size myelinated fibers were observed in these nuclei. The boutons contained clear pleomorphic vesicles and formed symmetrical type synaptic contacts with regular type dendrites and vesicle-containing dendrites. The present findings indicate that the ventral medial nucleus is the principal site of termination of nigrothalamic projections in the cat.

Notizen: Abstract Complete vascular isolation of the hindlimbs was performed in vagotomized cats under Sodium Pentobarbital anesthesia. The hindlimbs were perfused at constant flow with blood kept at a constant and physiological $$P_{0_2 }$$ , $$P_{{\text{C}}0_2 }$$ , and pH values by means of a specially designed pump-oxygenator system. The animals were hyperventilated with different CO2 mixtures (0%, 5%, 7% and 10% in O2) thereby changing blood gases and pH levels in the upper body but not in the hindlimb vascular bed. At body $$P_{{\text{aC}}0_2 }$$ (mm Hg) of 13.7 ±1.0 (means±SE), 30.6±1.05, 40,4±0,9 and 58.4 ±2.9 the hindlimb perfusion pressure (mm Hg) was, respectively 124±7.6, 138±7.4, 156±11.9 and 187 ±15.1. These changes in perfusion pressure were still present after complete peripheral chemoreceptor denervation but were abolished after section of the spinal cord at the T5 level. Since hindlimb perfusion pressure fell when body $$P_{{\text{aC}}0_2 }$$ was lowered below physiological levels it is concluded that part of the neurogenic vascular tone of the hindlimbs is maintained by a CO2 mediated stimulation of supraspinal structures.

Notizen: Abstract The cervical vagus nerve (VN) was electrically stimulated in Nembutal-anaesthetized cats. The responses recorded from the cerebellar surface were found in lob. V and VI in a bilateral sagittal strip perpendicular to the longitudinal axis of the folia. At a longer latency, potentials were also found in the paramedian lobule. Field potential analysis confirmed the existence of a sagittal strip in the deep parts of the lobules. The distribution of these potentials and their field potential profiles indicate that they are transmitted through the climbing fiber (CF) system. Experiments with local anaesthetics and deafferentation support our view that the potentials described in the vermis by Dell and Olson (1951) could be originated extracerebellarly. A strong parallelism was found between the amplitude of the cerebellar responses and the amplitude of the group B1 component of the vagal afferent potentials, having a conduction velocity between 4 and 20 m/s. The pathway by which the vagal afferents reach the inferior olive and the functional significance of these afferents are discussed.

Notizen: Summary The presence and distribution of myocardial α-adrenoceptors in different parts of the heart of various mammalian species was investigated. For this reason experiments were performed in isolated cardiac preparations of rats, guinea pigs and cats. In order to obtain more information about the nature of the cardiac α-adrenoceptors additional experiments were undertaken at different temperatures. These studies were aimed to show whether or not a conversion of β-to α-adrenoceptors or vice versa takes place. Moreover, we analyzed the influence of hypothyroidism on the sensitivity of α- and β-adrenoceptors of preparations from rats fed with propylthiouracil. Finally, we tried to find out whether stimulation of these α-adrenoceptors leads to the formation of the cyclic nucleotides cAMP and cGMP. The following results were obtained: 1. In right ventricular strips of rats, guinea pigs and cats phenylephrine stimulated α-adrenoceptors. After blockade of β-adrenoceptors the respective pD2-values of phenylephrine were 5.32, 5.99 and 5.33. The doseresponse curves obtained in the presence of α-adrenolytic drugs were shifted to the right without depression of the maximum. In the rat, in addition, the pA2-values for the α-adrenolytic drugs yohimbine (5.79) and phentolamine (7.85) were determined. They were at least 0.5 log units higher than those found in the rat for other α-adrenoceptors (Van Rossum, 1965) thus supporting the view that the population of cardiac α-adrenoceptors is different from that of other organs. In left ventricular strips of guinea pigs the pD2-value for the α-mimetic effect of phenylephrine was of the same order of magnitude as that obtained in the right ventricle. 2. In the papillary muscle of the right ventricle of guinea pigs and cats phenylephrine stimulated α-adrenoceptors. 3. In the left atrium of the rat, phenylephrine stimulated myocardial α-adrenoceptors (pD2: 5.58). Also in this preparation the pA2-values for yohimbine (6.36) and phentolamine (8.21) were different from those found for other α-adrenoceptors in the rat (Van Rossum, 1965). Likewise in strips from the left atrium of the cat myocardial α-adrenoceptors are present. 4. In spontaneously beating right atria of the rat a clear-cut positive chronotropic effect mediated by stimulation of α-adrenoceptors could not be demonstrated although in 6 out of 15 preparations a small positive chronotropic effect became evident. No positive chronotropic effect at all was obtained by stimulation of α-adrenoceptors in the cat right atrium. 5. In ventricular strips as well as in left atria from hypothyroid rats the pD2-value for the α-effect of phenylephrine was increased, in the atrium more than in the ventricle, while the pA2-values for yohimbine and phentolamine were not significantly different from the controls. Under these conditions a distinct positive chronotropic effect mediated by stimulation of α-receptors was found in the spontaneously beating rat atrium. 6. After stimulation of α-adrenoceptors in atria and ventricular strips of normal and propylthiouraciltreated rats, as well as in strips of guinea-pig ventricles or cat atria no elevation of cAMP and cGMP was observed. 7. The concept of a single adrenoceptor convertible from α- to β-type or vice versa was not supported by our experiments. These were carried out at different temperatures on left atria and on ventricular strips of rat hearts using the irreversible α-adrenoceptor blocking agent phenoxybenzamine. 8. The present experiments provide evidence for the existence of α-adrenoceptors in the myocardium of various mammalian species. Their stimulation produces positive inotropic effects without increases in heart rate.

Notizen: Summary Gene frequency surveys conducted in Alexandria and Cairo reveal genetic profiles which are extensions of those that characterize the cat populations of European cities. For nine selected comparisons with Alexandria, regression analysis indicates that a linear function best describes the relationship between Nei's and Cavalli-Sforza's genetic distance indices and geographic distance.

Notizen: We have tested 21 different basis sets of synthetic DNA circular dichroism spectra and have slected one for use in spectral analyses of natural DNAs. This “standard” set consists of spectra of eight polymers: poly[d(A-A-T)·d(A-T-T)], poly[d(A-G-G)·d(C-C-T)], poly[d(A-T)·d(A-T)], poly[d(G-C)·d(G-C)], poly[d(A-G)·d(C-T)], poly[d(A-C)·d(G-T)], poly[d(A-T-C)·d(G-A-T)], and poly[d(A-G-C)·d(G-C-T)]. This basis set, applied according to the first-neighbor polymer procedure of Gray and Tinoco, allows a more uniformly accurate spectral analysis of six natural complex DNAs and eight (A+T)-rich satellite DNAs for base composition and first-neighbor frequencies than was previously possible. We find that spectra of poly[d(A)·d(T)] and/or poly[d(A-C-T-)·d(A-G-T)] are not generally required for good analysis results but we show in this and the following paper that these spectra are needed for the most accurate analyses of some satellite DNAs.

Notizen: The interaction of counterions with a suitably long, charged oligomer appears susceptible to treatment in the context of polyelectrolyte theory by the introduction of an end-effect parameter that reflects the reduced association of counterions with the terminal regions of the oligo-ion. Use of a physically reasonable value for the end-effect parameter provides excellent agreement between theory and the experimental data of Elson, Scheffler, and Baldwin [J. Mol. Biol. 54, 401-415 (1970)] on the dependences of melting temperature on salt concentration and chain length for a series of hairpin helices formed by d(TA) oligomers. The differences in behavior expected for hairpin, dimer, and oligomer-polymer helices are discussed. The salt dependence of the end-joining equilibrium investigated for λ DNA by Wang and Davidson [Cold Spring Harbor Symp. Quant. Biol. 33, 409-415 (1968)] is treated as an oligomer-polymer interconversion. The dependence of equilibrium constant for this reaction on counterion concentration is in good agreement with that predicted by theory for an end-region totalling 24 nucleotides, the known length of the λ ends.

Notizen: Solvent denaturation is developed along thermodynamic lines rather than from multiple-binding theory. Almost all the relations derivable from site-binding theory have their counterparts in the thermodynamic formulation showing that the details of binding models may be sufficient but are not necessary for the general description of solvent denaturation. Equations are derived for the effect of denaturant concentration on stability at constant temperature and on tm. It is recommended that the thermodynamic treatment be used instead of binding models unless stoichiometric interactions are demonstrable experimentally.

Notizen: The optical properties of the 1,N6 - etheno derivative of ApA (εApεA) have been studied. Absorbance and CD measurements suggest that (1) neutral salts tend to unstack this molecule and (2) the stacking interaction is weaker than in ApA. εApεA is found to be quenched strongly with respect to the monomer. (εAMP); this quenching is solvent dependent (1M NaCl 〉 5M NaClo4〉40%glycerol) and increases with the ratio of temperature to viscosity (T/η) in each case. Fluorescence lifetime measurements also reveal a temperature- and solvent-dependent decay which is nonlinear on a semilog plot. In the presence of 95% glycerol, this decay return to linearity. These data have been considered from two points of view: (1) two-state pictures which are based on thermodynamic least-squares fit to quatum yield and CD curves, together with two exponential fits to the decay curves and (2) a dynamical model in which relatives fluorophore motion leads to deexcitation via intramolecular collision. A simple model of type (2) gives qualitative agreement with the observed behavior.

Notizen: The native antibiotic polymyxin B, carrying five positive charges, displaces the monoacetylated on from its adsorbed state at the interface between negatively charged lipid layers ans water. A simulation model for asdorption, governed by electrostatic forces, is presented. The model adequently representes the competitive adsorption kinetics and equilibria.

Notizen: In order to study the role of D-amino acid residues in keeping the stable β-sheet conformation and in the antimicrobial activity of gramicidin S (GS), the four analogs of GS containing D-Ala, L-Ala, Gly, and Aib (α-aminoisobutyric acid) in place of D-Phe were synthesized. D-Ala-and Gly-containing analogs showed antimicrobial activity, while those containing L-Ala and Aib showed no activity. Conformation of these analogs and their derivatives were studied by comparison of ORD and CD spectra and by slective methylation method. It is concluded that the biologically active analogs have β-sheet conformation while inactive analogs have a much different conformation from that of GS. This indicates that D-Ala-Pro and Gly-Pro sequences favor taking a β-bend form but L-Ala-Pro and Aib-Pro sequences do not because the presence of L-side methyl group on the α-carbon atom of LAla and Aib residues destabilizes the β-bend form. This would explain why the inactive analogs which take a different conformation from that of the active ones result in the loss of activity.

Notizen: Earlier studies by the authors showed that the collagen fibrils in rat-tail tendon have a bi-modal distribution of fibril diameters from a time shortly after birth through to the onset of maturity at about 3-4 months. Present work has extended those observations for rats up to the age of 2 years. Histograms of the fibril diameter distributions for mature tail tendon and direct electron microscope observations show that the fibrils break down as the tendon ages. Further work on the constant diameter subfibrils of diameter 140 Å described previously, has confirmed that these are part of the elastic fibers present in tendon at all ages. It has been shown that there is relatively little variation in the collagen fibril diameter distribution as a function of the position of the specimen in the tail, and as the measured percentage of the area taken by the collagen fibrils present at any particular point. Estimation of the fibrillar collagen content of rat-tail tendon as a function of age indicates that it increases steadily from birth and reaches a maximum at the onset of maturity, beyond which the fibrillar collagen content appears to remain constant.

Notizen: We have made quantitative estimates of some of the energetic factors to be considered in packaging of double-stranded DNA in virus particles. Numerical calculations were made using parameters appropriate for T4 bacteriophage. The unfavorable factors, and the Gibbs free energies per mole virus at 20°C associated with them, are bending, 1.5 × 103 kcal/mol; conformational restriction upon condensation, 5.1 × 102 kcal/mol; polyelectrolyte repulsion, 2.1 × 105kcal/mol; and melting or kinking, 6.9 × 103 kcal/mol. These must be counterbalanced in the assembled phage by noncovalent bonding interactions between protein subunits in the phage-head shell; by interactions between the DNA and polyvalent cations, especially putrescine and spermidine; nad perhaps by repulsive excluded volume and electrostatic interaction between the DNA and acidic polypeptides. Indeed, a rough estimate of the standard free energey of interaction between T4 DNA and the putrescine and spermidine contained in the head is --2.1 × 105 kcal/mol. In the absence of the other two sources of stabilization, each head protein subunit must contribute about 210 kcal/mol of binding energy.

Notizen: A theoretical basis for thin-film dialysis involving binding between a ligand and nondialyzing species is presented. A general differential equation that applies to the case of equivalent, noninteracting sites is derived relating [A]F, [A]T, [P]T, and K. Numerical solutions to this equation are used to develop a series of escape curves corresponding to specific values of the parameters [P]T, [A]i, K, and k0. A general method for determining an equilibrium binding constant from thin-film dialysis data is given. A comparison of thin-film dialysis results predicted by this theory with literature data shows essential agreement.

Notizen: The normal and vacuum uv spectra of cytosine, 5-azacytosine, and 6-azacytosine thin layers were determined. The spectra were resolved into a series of Gaussian bands and the experimental band positions and oscillator strengths were compared with the calculated values. The calculations were carried out with the PPP-SCF-CI method. The agreement of the calculations with those of others and with the experiment is discussed.

Notizen: To obtain information on the solvent-solute interactions in the dinucleoside monophosphates pertaining to the dimeric subunits of RNA and DNA, we have computed the accessibility of a water molecule to the oxygen atoms of the subunits following the method of Lee and Richards [J. Mol. Biol. 55, 379-400 (1971)]. The solute molecules (dimeric unit) is represented by a set of interlocking spheres of appropriate van der Waals radii assigned to each atom, a solvent (water) molecule is rolled along the envelope of the van der Waals surface, and the total surface accessible to the solvent molecule - and hence the solvent accessibility of various atoms of the solute molecule for different conformations - are computed. From the calculated atomic accessibilities, solvation maps in the (ω′,ω) space have been constructed, keeping ψ at 60°, 180°, and -60°. The C(3′)-endo sugar system in the case of DNA subunit have been considered. The solvation maps describing the solvatability of single and groups of atoms give significant information on the backbone conformational domains that are preferred for solvent interaction, thus adding knowledge to the relative stability of the various possible conformations. The B-DNA-type conformer exposes three polar atoms - namely, PO1, O(3′), and O(1′) - to external solvent, whereas the A-DNA- and C-DNA-type conformers expose only one polar atom - O(3′) and O(1′), respectively - to the solvent. The O(2′) atom of the furanose ring system in the RNA subunit could give added stability via solvent association or interunit hydrogen bonding with or without a bridging water. The superposition solvation maps describing the accessibility of a group of polar atoms help to interpret a good number of phosphodiester conformations observed in a energetically less favored conformational domains in the tRNAPhe crystal. Another intresting fact that results from this study is the prediction that the trans oriented of ω is the most favorable conformations of random-coil polynucleotides in solution.

Notizen: The Conformational properties of synthetic S-peptide analogs, in which the residues in the N-terminal sequence 1-6 were progressively deleted or replaced with amino acids of lower helical propensity, were studied by CD. Increasing the concentration of guanidine hydrochloride and decreasing the temperature were found to produce progressive destruction of ordered conformations, in the parallel with the increasing solubility of the peptide unit, while increasing the concentration of trifluoroethanol and decreasing the temperature produced the opposite effect. The maximum helicity determined in the these sets of experiments is found equal to or greater than that determined in the formation of the ribonuclease S′ complexes. With some peptides the maximum value of predicted helical conformation is reached, and the tendency of tertiary structure to reduce the maximum possible helicity is evident. We discuss the validity of the procedure by which conformational information, drawn from measurements in helicogenic solvents, is related to the state in native protein.

Notizen: A thermal perturbation curve (TPC) is defined to be the derivative of the fractional degree of saturation, f, with respect to temperature, considered as a function of the natural logarithm of free ligand concentration, y. The theoretical framework for the use of such curves in the thermodynamic analysis of ligand binding to macromolecules is presented. The thermal perturbation curve either provides or complements the information obtained from the derivative binding isotherm ∂f/∂y. For a single set of identical and independent sites the TPC is identical to the derivative binding isotherm. Analysis of such a curve directly yield ΔH0 and ΔG0 for the binding reaction. In actual experimental work, however, the TPC can only be approximated because of “self-buffering” effects relations between the parameter of the approximate curve and the thermodynamic quantities have been developed. This technique is applied to the proflavin-α-chymotryspin system to demonstrate its usefulness. The general features of thermal perturbation curves for cases of multiple sets of independent sites and cooperatively interacting sites have also been developed. The analysis of thermal perturbation curves in combination with other methods should provide a more powerful approach to the characterization of ligand-macromolecule interactions.

Notizen: The conformation of three sequential copolypeptides, poly(L-tyrosyl-L-lysine), poly(L-tyrosyl-L-lysyl-L-lysine), and poly[L-tyrosyl-(L-lysyl)2-L-lysine] have been studied by a variety of techniques, including CD, ir spectroscopy, analytical ultracentrifugation, and x-ray diffraction. Depending upon the pH and sovent composition, poly(L-tyrosyl-Llysyl-L-lysine) and poly [L-tyrosyl-(Llysyl)2-L-lysine] can adopt either the α-helical or random-coil conformation, while poly(L-tyrosyl-L-lysine) forms either inter- or intramolecular β-structures.

Notizen: Molecular motion in solid poly(L-alanine), Poly(L-leucine), poly(L-valine), and polyglycine has been investigated through measurement of the portion spin-lattice relaxation time at 30 and 60 MHz between 110 and 350°K. Rapid random reoriention of sied-chain methyl groups provides the dominent source of relaxation in the first three; activation energies are 10.5 ± 1 1, 8.5 ± 1 kJ/mol, respectively, significantly lower than in the monomeric crystals. Relaxation times in poyglycine are two orders of magnitude longer than in the monomeric crystals. Relaxation times in polyglycine, significantly lower than in the monomeric crystals. Relaxation times in polyglycine are two orders of magnitude longer and are attributed mainly to segmental motions of the polymer chains. Evidence of nonexponential recovery of nuclear magnetization was encountered in the first three homopolyamino acids but not in polylycine, and was attributed to the correlated time to characterize these motions gave quite good agreement with the data; some improvement was obtained for two polymers using a Cole-Davidson distribution of correlation times. For biopolymers using a Cole-Davidson distribution of correlation times. For biopolymers generally it is concluded that rapid methyl group reorientation is a common dynamical feature and an important source of nuclear magnetic relaxation.

Notizen: The karl Fischer reagent method has been used to study the drying of whale skeletal muscle myoglobin. Drying curves for metmyglobin were obtained by mechanical pumping (moderate vacuum) and diffusion pumping (high vacum) at 20, 10, 0, -10, and -20°C. Strongly held water is present in about the same amounts as were previously observed with lysozyme. Nearly all of this strongly held water is still present during the drying of acid denatured metmyoglobin. This suggest that a core structure containing strongly held water is still present at pH 4.0. Comparison of the drying of metmyoglobin, deoxymyoglobin, and oxymyoglobin indicates that the iron-coordinated water of methyoglobin is the second most strongly held water of the molocule.

Notizen: Mixtures of bacteriophage T4D particles with up to six tail fibres attached were seprated and analyzed in a quasielastic light scattering-band electrophoresis apparatus. The electrophoretic mobilities and diffusion coefficients of the seprated bands were determined during the same experiment. Species differing in mobility by 0.05 × 10-4 cm2/V sec were resolved. Henry's electropheresis theory for spheres indicates that each tail fiber contributes about 140 effective positive charges to the phage structure. Estimate of the charge using the permanent dipole moment and the electrophorectic mobility (Bontje et. al. (1977) Biopolymers 16, 551-572) gives an effective charge of + 230 to -250 per tail fiber. The charge distribution on the fiberless particles was estimated to be -3380 on the head and -290 on the tail if the dipole moment was assumed positive and -1400 on the head and -1000 on the tail for a negative dipole. Tail-fiber attachement does not proceed to completion in our invitro system, as substantial distributions as a function of tail-fiber input indicates the reaction is a random, noncooperative process.

Notizen: The contribution of the 1-6 N-terminal sequence to the conformational properties of the S-peptide (the 1-20 sequence of ribonuclease A) was assessed by determining in the ribonuclease S′ system the helical content and the binding capability of synthetic [Orn10]-S-peptide analogs, in which lysine1, glutamic2 and threonine3 were progressively deleted, alanine4 and alanine5were alternatively replaced by serine, and alanine6 was substituted by serine or proline. Both the deletion of the three N-terminal residues and the alanine6/proline replacement produces the loss of the helical structure up to lysine7. No or minor effects are found in all other cases. From the comparison of the binding data, the energy for the conformational stabilization of the N-terminal region was calculated to amount to 1.4 kcal/mol. The results are discussed in comparison with the known x-ray data of the enzyme, with some predictive rules of secondary structure which were applied to this region and with the known phylogenetic variance of the residues in this region.

Notizen: In the Polymerization of phenylalanine N-carboxyanhydride (NCA) in No2Oh initiated by MeNHBzl, L-,D-, and DL-NCA As were polymerized at the same rate, and no stereoselectivity was observed. When the same experiment was carried out in HCONEt2, however, L- and D-NCA were both polymerized at a rate which was about twice as large as that of DL-NCA. In this case, the polymerization is stereoselective, ascribable to a preferable reaction between the optical enantiomorphs of the terminal residue of the growing chain and the NCA of the same chirality. On the other hand, the polymerization initiated by SarNMe2 and MeNH(CH2)2CONMe2 were stereoselective in NO2Ph and HCONEt2, but they were not stereoselective in m-(MeO)2Ph. These findings indicate that the polymerizations initiated by a strong base in highly dipolar solvents are stereoselective. Apparently, the reaction between a chiral, cyclic terminal of growing chain and a chiral, cyclic activated NCA in the activated-NCA mechanism is highly stereoselective. In addition, from a kinetic investigation on on the copolymerization between L- and D-NCAs, the penultimate chiral centers were also suggested to contribute to the stereoselection. Stereoselection by the α-helical conformation of the growing chain and by a chiral, linear terminal amine have been considered so far, and the contribution from the present type of stereoselection must have been overlooked.

Notizen: Transient electric birefringence measurements of the bacteriophages T2L0 and T6 were performed under such conditions that the tail fibers are extended. The data obtained are compared to previously reported data for T4B. For all T-even phages the degree of extension of the tail fibers is a function of pH, ionic strength, and temperature. For T4B, much higher ionic strengths are needed than for T2L0 and T6 to accomplish complete tail-fiber extension. The rotational diffusion coefficients of the phages with fully extended fibers are equal to 120 ± 3 sec-1, 132 ± 5 sec-1, 157 ± 4 sec-1 for T2L0, T4B, and T6, respectively. The respective optical anistropies are - (2.66 ± 0.05) × 10-4, and - (3.07 ± 0.15) × 10-4. The differences in the rotational diffusion coefficient and optical anisotropy arise because the conformation of the fully extended tail fibers is different for the three phages. The tail fibers of T2L0 project further into the solution (away from the head) than do those of T4B and T6. The apparent permanent dipole moments of T2L0 and T6 decrease with increasing ionic strength. This decrease is caused by the screening of the surface charges on the phage body by the counter-ions in the solution. The biological relevance of this decrease is illustrated by the fact that the adsorption rate of T6 phages to E. coli B bacteria shows a similar dependence of ionic strength. Evidence is pressented that the tail fibers may move more or less independently of the phage body when an electric field is applied to the suspension.

Notizen: Die Rheinanliegerstaaten haben im gemeinsamen Bemühen um die Qualität des Rheinwassers schon 1963 die „Internationale Kommission zum Schutze des Rheins gegen Verunreinigung“ (IKSR) ins Leben gerufen. Die IKSR überwacht die Wasserqualität und gibt einen jährlichen Bericht mit Analysendaten heraus. Außerdem erarbeitet sie Vorschläge für Maßnahmen zum Schutze des Stroms. In Ergänzung der jeweiligen nationalen Gewässerschutzmaßnahmen haben die Rheinanliegerstaaten 1976 das Übereinkommen zum Schutze des Rheins gegen chemische Verunreinigung (Chemieabkommen) abgeschlossen. Das Chemieakbkommen, durch das die Ableitung bestimmter gefährlicher Stoffe aus den Stoffgruppen und -familien: Queck-silber- und Cadmiumverbindungen, organische Halogen-, Phosphor- und Zinnverbin-dungen, cancerogene Stoffe so weit wie möglich verhindert und aus weiteren Stoffgruppen und -familien, darunter den meisten Metallen, streng überwacht und eingeschränkt werden soll, hat einen langfristigen präventiven Charakter. Das Chloridabkommen soll dagegen sofort zu Maßnahmen führen. Zu diesen Abkommen der Rheinanliegerstaaten treten eine Reihe weiterer internationaler Abkommen und Vereinbarungen mit Auswirkungen auf den Rhein, verschiedene Richtlinien der Europäischen Gemeinschaft, ein geplantes Abkommen der Länder im Europarat, ein Abkommen zum Schutze der Nordsee und des Nordatlantiks gegen die Verschmutzung vom Lande aus.

Notizen: We have measured the ultraviolet CD spectra for covalently closed and linear forms of phage PM2 DNA in solution. We find that increased concentrations of salt or ethanol (up to 50% ethanol by weight) depress the long-wavelength positive CD bands in the spectra of both forms of DNA, although the spectrum of the native covalently closed DNA always has a slightly larger magnitude of these bands than does the spectrum of the linear DNA. In addition, both DNAs are equally capable of undergoing a transition to the A conformation in 70-80% ethanol at low Na+ concentrations. Thus, the constraint imposed by the covalent closure of a DNA molecule does not seem to hinder its conformational response to these changing solution conditions. Lang [(1973) J. Mol. Biol. 78, 247-254] has found by electron microscopy that T7 DNA has an inherent ability to condense into compact particles, suggested to be supercoils of multiple order. Both covalently closed and linear forms of PM2 DNA also become condensed when the DNA, in 0.2M ammonium acetate and 1 mM EDTA, is exposed to ethanol and subsequent drying on specimen grids [Lang, D., Taylor, T. N., Dobyan, D. C. & Gray, D. M. (1976) J. Mol. Biol. 106, 97-107]. Under similar conditions, in solutions of 0.2M ammonium acetate and 1 mM EDTA to which ethanol is added, we have measured the CD spectra of both covalently closed and linear forms of DNA. Below ethanol concentrations at which the DNA obviously precipitates, the CD spectra of both forms have reduced long-wavelength positive CD bands.

Notizen: The polymeric cobalt(II) porphyrin complexes were prepared from cobalt(II) protoporphyrin IX dimethyl ester(Co(II)P) and copolymers of 4-vinylpyridine and styrene(PSP), and their binding ability of molecular oxygen was studied in toluene solution. The five- and six-coordinate structure of CoP-PSP complexes were confirmed by esr spectra. The esr parameters for the CoP-PSP complexes were not affected by the molecular weight and the vinylpyridine-unit content of PSP-ligand. The 1:1 dioxygen-Co complex was reversibly formed when the solution of CoP-PSP was exposed to oxygen atmosphere at low temperature. While the visible spectra and esr parameters for the dioxygen complexes of CoP-PSP were the same as those of the CoP-pyridine complex, the equilibrium constant for the oxygen binding increased with the vinylpyridine-unit content of the PSP-ligand. The larger entropy change was observed for the oxygenation in the CoP-PSP system especially, of which the vinylpyridine-unit content was large.

Notizen: The low-temperature dynamic mechanical relaxation of ox Ligamentum nuchae elastin, swollen with water and ethylene glycol at various swelling ratios, has been investigated by means of the Rheovibron DDV II viscoelastometer, at frequencies from 3.5 to 110 Hz. The peak location on the temperature scale, its intensity and activation energy have been studied as a function of solvent content and it has been found that the two solvents influence the dynamic mechanical behavior in a quite different way. A similar interaction of the solvent molecules with the backbone peptide groups is assumed for both solvents, and the differences in the mechanical spectra, which are more marked the higher the solvation degree, are explained in terms of the different size of the protein-solvent relaxing units.

Notizen: The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.

Notizen: The single-strand helix-coil transition in various oligo- and polyadenylates is characterized by means of an improved cable temperature-jump technique. In all the polymers studied {poly(rA), poly(dA), poly[A(m2′)] and poly[A(e2′)]} helix-coil relaxation is observed in the time range from 30 to 1000 nsec. Relaxation-time constants observed at wavelengths λ〈280 nm (τα) are different from those found at λ 〉280 nm (τβ), indicating the presence of more than two conformational states. The time constants τα increase in the series poly(dA), poly[A(m2′)], constants τβ/τα is approximately 2.5, except in poly(dA) where τβ/τα ≈ 9. Relaxation measurements with r(A)n- oligomers show a decrease in conformational mobility with increasing chain length. The relaxation curves also demonstrate that “internal” residues have lower reaction rates than residues at the ends of the oligomer chain. Measurement in D2O reveal a solvent isotope effect for τα of +87% for poly(rA), and of +53% for poly(dA), whereas no isotope effect is found in τβ. The absence of “slow” relaxation processes in the model compound 9,9′ -trimethylenebisadenine shows that the relatively low rate of the single-strand helix-coil transitions is due to the coupling of base stacking with the folding of the sugar-phosphate chain. The absence of a seprate relaxation process (corresponding to τβ) in 9,9′-trimethylenebisadenine, as well as in the dinucleotides ApC and CpA, suggests that this relaxation process is dependent upon the presence of both the sugar-phosphate chain and of adjacent adenine bases. The experimental data provide evidence that there is more than one ordered conformation in various single-stranded oligo- and polyadenylates and that the transition between these conformations is influenced by the sugar conformation.

Notizen: An algorithm has been developed that permits one to find all possible conformations of the sugar-phosphate backbone for any given disposition of DNA base pairs. For each of the conformations thus obtained, the energy of the helix was calculated by the method of atom-atom potentials. Several isolated regions in the space of the bases′ parameters (Arnott's parameters) have been found for energetically favorable helical structures. Two parameters, the distance of a base pair from the helix axis, D, and the windling angle, τ, allow one to subdivide possible conformations into the families of closely related forms. Two regions (ravines) on the (D, τ) map correspond to the know A and B families. In the B family a continuous transition has been obtained in which the double helix undergoes increasing winding, while the base pairs are moving toward the major (nonglycosidic) groove.Interrelationships between the variables, characterizing the spatial structure of the double helix, D, τ, TL and χ, when going along the bottom of the B ravine, were also obtained.Besides the Known A and B families, several new ones were found to be energetically possible. Among these the strongly underwound helices with the negative D values, as well as the forms with the C4-C5 angle in a trans position, should be mentioned.Biological roles of the different double-stranded conformations, in particular, in protein-nuclei acid interaction are discussed.

Notizen: The theoretical basis for the statistical mechanical deconvolution of a thermally induced macromolecular melting profile is presented. It is demonstrated that all the thermodynamic quantities characterizing a multistate macromolecular transition can be obtained from the average excess enthalpy function, 〈ΔH〉, of the system, without any assumption of the particular model or mechanism of the reaction.Experimentally, 〈ΔH〉 is obtained from scanning calorimetric data by direct integration of the excess apparent molar heat capacity function, ΦCp. Once 〈ΔH〉 is known as a continuous function of the temperature, the partition function, Q, of the system can be calculated by means of the equation \documentclass{article}\pagestyle{empty}\begin{document}$$Q_{\left( T \right)} = \exp \smallint _{T_0}^T {\left( {\frac{{\left\langle {\Delta H} \right\rangle }} {{RT^2}}} \right)} dT $$\end{document} From the partition function all the thermodynamic quantities of the system can be obtained. It is shown that the number of discrete macroscopic energy states, the enthalpy and entropy changes between them, and the relative population of each state as a function of temperature can be calculated in a recursive form.

Notizen: The high-resolution 1H-nmr study of the ferrichrome cyclohexapeptides, in d6-DMSO solutions, has been extended to the amide NH spectral region. A total of ten diamagnetic analogues of ferrichrome that differ in the coordinated metal ion (Al3+, Ga3+ or Co3+), the primary structure, the nature of the bidentate hydroxamate moiety, or the isotope compositions (14N, 15N) have been investigated. The 3JαNH values reflect regiorous conformational isomorphism throughout the complete suite of analogues, quite independent of the residue occupancy of each site. Totally resolved amide multiplets have been obtained in most cases and the four-line (doublet of doublets) appearances of glycyl NH resonances has been observed for the first times; these data enabled stereospecific assignment and accurate analysis of the NH-CαH proton spin systems. The high resolution was made possible by the use of a suitable spectral deconvolution shceme at 360 MHz. The fine structure, extraordinarily well displayed in the 15N-peptide spectra, provides a series of parameter values whose consistency has been checked by computer simulation. Since the crystallographic structure for two of the ferric peptides is known to 0.002-Å resolution, a 3J vs θ correspondence could be confidently established. A Karplus curve was derived from the combined x-ray and nmr data: \documentclass{article}\pagestyle{empty}\begin{document}$$ ^3 J_{\alpha {\rm NH}} = 5.4\cos ^2 \theta - 1.3\cos \theta + 2.2{\rm Hz} $$\end{document} It is suggested that seriously nonplanar amides can exhibit 3JαNH values higher than predicted by the ferrichrome curve.

Notizen: The general formulas describing the low-angular dependence of polarized (Vv) and depolarized (Hv) light-scattering intensity by a chain macromolecule of arbitrary form with anisotropic polarizability are derived. It is shown that the value dHv/dtHvo (where t = [〈R2〉S/L]2, S being the scattering vector) is an indication of the chain-flexibility mechanism. This permits one to distinguish between the models of a wormlike chain, regular zigzag, or statistical zigzag. The results of numerical calculations useful for the interpretation of experimental data are presented.

Notizen: The kinetics of the fluorescence decay of the energy donor in a homologous series of oligopeptides each containing at its ends a donor and an acceptor of electronic excitation energy was investigated in solvent mixtures of different viscosities. The repeating unit in the peptides was N5-(2-hydroxyethyl)-L-glutamine and the chromophores used as donor and acceptor were naphthalene and dansyl, respectively. The number of units in the peptides studied varied from four to nine. The solvents used were mixtures of glycerol and trifluoroethanol in various proportions. The decay rate of the donor fluorescence increases when the solvent viscosity decreases. This behavior is due to the disturbance of the equilibrium end-to-end distribution of distance of the excited molecules by the energy transfer process, which is more favorable foe short than for long distances. The subsequent rearrangement towards the equilibrium distribution by diffusion of the molecular ends relative to one another enhances the efficiency of the energy transfer. Assuming a modified Fick equation to describe this diffusion motion, the fluorescence decay data were analyzed in terms of a diffusion coefficient describing the Brownian motion of the molecular ends. The diffusion coefficients thus evaluated increase systematically upon decreasing the solvent viscosity. For example, for the oligopeptides studied it changes from unmeasurably small values in glycerol solution to values varying between 10-8 to 10-7 cm2/sec at room temperature in a glycerol trifluoroethanol solvent mixture of viscosity of 8 centipoise. The values obtained for the diffusion coefficient are smaller by about an order of magnitude than the values expected for the diffusion coefficients of the free chromophores in solvents of comparable viscosity. It is thus concluded that the backbone of the polymeric chains possesses appreciable internal friction which exerts resistance to the Brownian motion of the polymer chains. The diffusion coefficient of the end-to-end motion is systematically smaller for the shorter than for the longer chains. For example, at room temperature in a solvent mixture of 8 centipoise it is 3×10-8, 5×10-8, 7.6×10-8, and 8.5×10-8 cm2/sec for oligomers containing four, five, eight, and nine N5-(2-hydroxylethyl)-L-glutamine repeating units, respectively. The internal friction thus impedes the motion of the molecular ends more effectively in the shorter chains than in the longer ones.Analysis of the energy-transfer experiments in solvents spanning a wide range of viscosities shows that the orientational factor appearing in Förster's equation for energy transfer does not interfere perceptibly with the evaluation of the end-to-end distances from the fluorescence decay data when naphthalene and dansyl serve as donor and acceptor, respectively. This is due to the fact that both the donor emission and acceptor absorption in the range of spectral overlap are characterized by more than one transition dipole moment, as is evident from the corresponding polarization data.

Notizen: Raman spectroscopic studies have been carried out on polymers of L-valine ranging in degree of polymerization (DP) from 2 to 930. The spectrum of the hexapeptide (DP = 6) is closely similar over the entire range 40-1750 cm-1 to those of polymers with much higher DP, and the structure is clearly shown to be that of the antiparallel pleated sheet (β-structure) by the amide I and III frequencies. The formation of a little α-helical structure occurs in polymers with DP above 500, although the amount does not appear to be a linear function of DP. The α-helical structure is unstable and readily destroyed in samples cast from trifluoroacetic acid solution. It is stabilized by the incorporation of L-alanine, a strong helix-former; polymers of the latter may in turn be forced into a α-structure in copolymers sufficiently rich in L-valine.

Notizen: The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λmax) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λmax, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λmax of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.

Notizen: Group-specific polysaccharides isolated by means of a cetavlon procedure are immunogenic in man and induce protective immunity against meningococcal meningitis. Minute quantities of the polymers in solution can act as vaccines. We now report the first characterization of a fractionated (C-1) group C polysaccharide in 0.4KM KCl and 0.05M sodium acetate by means of light-scattering spectroscopy. Independent measurements of refractive index increments, absolute scattered intensities, angular scattering intensities and line widths as a function of scattering angles and delay times at different concentrations using incident wavelengths of 632.8 nm from a He-Ne laser and of 488 nm from an argon-ion laser yield information on aggregation properties, molecular weight (Mr), radius of gyration 〈r0g〉1/2z, translational diffusion coefficient 〈D〉0z, and second virial coefficients A2 and B2 of C-1 polysaccharide.At relatively high ionic strength (0.04M KCl + 0.05M sodium acetate), we obtain for the C-1 polysaccharide in solution Mr = 5.15 × 105, 〈r2g〉1/2z = 345 Å, A2 = 1.25 × 10-4 ml/g, 〈D〉0z = 1.16 × 10-7 cm2/sec with a corresponding Stokes radius of 240 Å and B2 = 4.4 ml/g. A2 and B2 are the second virial coefficients from intensity- and diffusion-coefficient measurements. The C-1 polysaccharide aggregates in solution and behaves hydrodynamically like random coils. Viscosity and sedimentation studies further confirm our conclusions that the fractioned C-1 polysaccharide aggregates in solution and EDTA can partially break up those aggregates. However, the system remains polydisperse even after adding an excess amount of EDTA. The weight-average molecular weight of the C-1 polysaccharide in solution depends upon ionic strength and exhibits a minimum at ∼0.2M KCl. Finally, viscosity, light-scattering, and sedimentation results all show that the aggregated macromolecular system behaves like random-coiled polymers with no measurable shape factors.

Notizen: We extend fluorescence correlation spectroscopy to systems that undergo translation or laminar flow in a sample cell. We include theoretical and experimental results; we consider uniform and nonuniform velocity profiles. Concentration correlation analysis extracts microscopic rate parameters from measurements of the spontaneous concentration fluctutations, which occur even at equilibrium. Fluorescence is one of the most sensitive means of monitoring these fluctuations. Analysis of flowing or translating systems (1) offers a method of measuring number concentrations of selected species, for example, of aggregates or polymers, (2) provides a nonperturbing velocity probe, (3) sometimes allows one to circumvent photolytic degradation, (4) has proved extremely helpful in testing and aliging apparatus for fluorescence correaltion measurement and in verifying theoretical analyses, and (5) may be required for interpretation of results obtained on systems in motion, even though that motion is undesired or initially unsuspected. We include both theoretical and experimental results for combined Poiseuille flow and diffusion in the geometry which is of most practical interest. Theoretical expressions for the much simpler cases of nondiffusive Poiseuille flow as well as uniform flow or translation with or without diffusion constitute limiting cases which are displayed explicitly.

Notizen: In the preceding article1 we demonstrated that the partition function of a system is experimentally accessible from scanning calorimetric data. In this article the general results of the deconvolution theory are applied to the general case of cooperative transitions in macromolecules. It is demonstrated that, in the limit of very large systems, all the relevant molecular averages and molecular distribution functions can be directly obtained from the experiment. In doing this, the method of the grand partition function is used. It is shown that in the case of homopolymers, only one parameter, the stability constant, needs to be explicitly specified for a complete description of the system. Since the partition function is directly evaluated from the experiment, no special assumptions or artificial constraints directed to obtain a mathematically solvable model are required. This result offers the unique opportunity of having direct experimental access to statistical averages of systems in which the partition function cannot analytically be solved. Consequently, the theory can be extended to cooperative transitions occurring in two and three dimensions by introducing cluster distribution functions.

Notizen: The pyridoxal Schiff bases of the polypeptides poly(L-lysine), poly(L-ornithine), and poly(L-α,γ-diaminobutyric acid) were prepared and investigated in water/methanol by CD spectra and equilibrium dialysis experiments. Only the poly(L-α,γ-diaminobutyric acid) derivative is characterized by a relevant optical activity similar to that found in pyridoxal enzymes. The stereospecific interactions between the pyridoxylideneimine group and the polypeptide chain prevent the hydrolysis reaction of the aldimine bond.

Notizen: The protein turkey-heart cytochrome c is used as a model protein to study charge effects in sedimentation equilibrium experiments in three-component solutions. Data are given for the dependence of the apparent M (1-υρ) on ρ in solutions of KCl, RbCl, CsCl, and triethylamine hydrochloride. The results show the Donnan effect to have a significant influence on the apparent molecular weight, found by extrapolation of the data to a solution density of one. The apparent molecular weights are for protein at infinite dilution. A theoretical treatment is presented where the magnitude of this effect can be predicted accurately from the formal net charge of the protein as computed from the amino acid composition. The results are shown to be important in computing the preferential hydration of the protein in concentrated salt solutions. For such systems the Donnan effect should be subtracted from the total interaction coefficient for multicomponent system in order to obtain the preferential hydration.

Notizen: The cupric complexes of poly(Nε-acetoacetyl-L-lysine), [Lys(Acac)]n′ poly(Nδ-acetoacetyl-L-ornithine), [Orn(Acac)]n′ and poly(Nγ-acetoacetyl-L-diaminobutyric acid), [A2bu-(Acac)]n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.

Notizen: The Mettler/Paar precision density meter DMA-02D has been used to determine the concentration of saturated solutions of amino acids at 20.0, 25.0, and 29.8 °C. The technique has proven itself an elegant and precise method. The solubilities of all of the amino acids with the exceptions of proline, lysine, and cystine have been measured. The Gibbs free energies of transfer from saturated water solution to 1M Na2SO4 and to 1M Gu·HCL along with the van't Hoff heats and entropies have been calculated. The van't Hoff heats have been compared with the calorimetrically determined heats for some of the amino acids. The Lumry-Rajender relation between the entropy and heats has been observed. The process of transfer of the amino acids from water to the solvents is primarily enthalpic rather than entropic.

Notizen: The effect of magnesium ions on the parameters of the DNA helix-coil transition has been studied for the concentration range 10-6-10-1M at the ionic strengths of 10-3M Na+. Special attention has been given to the region of low ion concentrations and to the effect of polyvalent metallic impurities present in DNA. It has been shown that binding with Mg++ increases the DNA stability, the effect being observed mainly in the concentration range 10-6-10-4M. At[Mg++]〉10-2M the thermal stability of DNA starts to decrease. The melting range extends to concentrations ∼10-5M and then decreases to 7-8°C at the ion content of 10-3M. Asymmetry of the melting curves is observed at low ionic strengths ([Na+] = 10-3M) and [Mg++] ≤ 10-5M. The results, analyzed in terms of the statistical thermodynamic theory of double-stranded homopolymers melting in the presence of ligands, suggest that the effects observed might be due to the ion redistribution from denatured to native DNA. An experimental DNA-Mg++ phase diagram has been obtained which is in good agreement with the theory. It has been shown that thermal denaturation of the system may be an efficient method for determining the ion-binding constants for both native and denatured DNA.

Notizen: Studies on the cross-linking of a tripeptide (t-butyloxycarbonyl-L-alanyl-D,L-2-amino-6-heptenoyl-L-alanine methyl ester) have shown that it is possible to form specific cross-links in good yields through Schiff base formation of the ε amino group of lysine. The heptenoic acid residue has been ozonized to an aldehyde and condensed with the ε amino of lysine in the compounds alpha-t-butyloxycarbonyl-L-alanyl-L-lysine methyl ester and alpha-t-butyloxycarbonyl-L-lysine methyl ester to form the cross-link, lysinonorleucine. This compound has been stabilized by reduction with sodium borohydride and quantitated on the amino acid analyzer. This technique converts from 60 to 98% of the available aldehyde to lysinonorleucine.

Notizen: The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit 〉 bovine ≫ ovalbumin; with anti-ovalbumin, it is ovalbumin ≫ rabbit. Since the frequencies of the amide I band appear near 1655 cm-1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.

Notizen: The Raman spectra of guanylyl (3′-5′) guanosine (GpG) in solution in H2O and D2O at pH 3-7 have been recorded at various temperatures between 0 and 80°C. The results are consistent with the existence in the lower temperature range of stable aggregates formed by the stacking of GpG tetramers. The aggregates melt cooperatively near 60°C, which results in important changes in the spectra. Among these, a large increase in intensity of some of the bands assigned to the guanine residues shows that unstacking of the bases occurs at the melting. Also apparent in the spectra are changes in the intensity and frequency of band attributable to molecular groups involved in intermolecular hydrogen bonding between adjacent molecules in the complex. The melting temperature of GpG decreases by approximately 15°C upon lowering the concentration from 5 × 10-2 to 5 × 10-4M, as shown by Raman, calorimetric, CD, and uv measurements. The experimentally determined ΔH and ΔS for the melting transition are 9 Kcal/mol and 28 e.u./mol, respectively. The aggregation of GpG in 1.5 × 10-3M solutions was found to be very slow. The half-time of the process, which roughly follows first-order kinetics, is approximately 3 min at 10°C and 21 min at 35°C. The negative energy of activation associated with this reaction (-143 Kcal) indicated that the process involves intermediates whose concentrations decrease the temperatures raised, thus slowing down the overall process. The rate of disaggregation of GpG upon dilution to very low concentration is also extremely slow, indicating that the GpG aggregates, once formed, are very stable.

Notizen: The Raman spectra of poly(L-lysine) with various structures, ionized poly(L-glutamic acid), and deuterated N-methylacetamides have been observed using visible and the 257.3-nm laser lines as the light source. Most of the Raman bands with significantly enhanced intensities in the uv-excited spectra of the polymers have been assigned to the vibrations associated with the C=O and C-N stretching modes, the amide I, II, III, I′, II′, and III′, with reference to the results obtained for simple amide molecules including the deuterated N-methylacetamides. Several amide frequencies have been newly identified and the structures of the polymers have been discussed through the comparison of the Raman and ir amide frequencies.