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Notes: Uptake of 3H-thymidine into suspension cultures of mouse marrow cells was stimulated by the addition of serum-free conditioned medium harvested from cultures of mouse L-cells. Characterization of the “conditioning factor activity” (CFA) by gel filtration, ion exchange chromatography, velocity sedimentation, polyacrylamide gel electrophoresis and susceptibility to trypsin digestion indicated that the CFA detected by stimulation of 3H-thymidine uptake is the same as the CFA detected previously by its ability to stimulate colony formation by marrow cells in vitro. The 3H-thymidine uptake assay was used to investigate the kinetics of disappearance of CFA as a function of time in the presence of mouse marrow cells. The CFA recoverable from the cultures decreased rapidly during the first day, and approached background levels by the fifth day. There was no evidence of inhibitory substances in the depleted media. Even if the cultures continued to receive fresh conditioned medium at daily intervals, 3H-thymidine uptake decreased sharply after the fifth day, indicating that the marrow cells had lost their capacity to respond to CFA.

Notes: The mitochondrial-plus-lysosomal fraction of rabbit lung parenchyma was studied by equilibrium density centrifugation in continuous sucrose density gradients (specific gravity 1.035 to 1.250). High concentrations of lysosomal marker enzymes were found both in a broad band at density 1.15-1.18, a density typical for lysosomes, and in a band at density 1.06-1.07. This light density band also had the highest specific activity of phospholipid, which thin layer and gas-liquid chromatography showed to be primarily lecithin with a high content of palmitic acid residues. Electron microscopy of material from the light density band showed a homogeneous array of particles which bear a strong resemblance to the inclusion bodies of the type II alveolar epithelial cell as seen in electron micrographs of rabbit lung tissue sections. These data suggest that the light density band is an isolation of intact type II alveolar epithelial cell inclusion bodies, which previous studies have implicated as the storage site of the phospholipid moiety of pulmonary surfactant.

Notes: Blood leukocytes incubated in vitro with rabbit-marrow cells induced a several-fold increase in basophilic erythroblasts and smaller increases in acidophils and reticulocytes. The main effect was nearly complete in one hour at 37°. Erythropoietin augmented the leukocyte effect; anti-erythropoietin inhibited it with or without erythropoietin. The erythroblast increase came entirely from the marrow cells; the precursor cell class has not been identified, except that division of pre-existing basophils appears to be excluded. Autologous and homologous leukocytes were about equally effective.A method is described of measuring on stained smears changes in relative concentrations of different classes of cells induced experimentally. A method of preparing highly concentrated peripheral blood leukocytes is described.

Notes: Buoyant density gradient separation of Rhesus monkey bone marrow, spleen and blood leukocytes has demonstrated a reproducible and homogeneous light density distribution profile of cells capable of forming hemopoietic colonies in agar culture (in vitro colony forming cells  -  CFC). High resolution density gradient separation performed on a light density fraction of bone marrow produced on average a 100-fold enrichment of in vitro CFC with the most enriched fractions containing the majority of the in vitro CFC population present in the original marrow. Fractions were routinely obtained in which up to 23% of cells formed colonies and 33% were capable of proliferating to some degree upon stimulation. Tritiated thymidine suiciding showed the active proliferative status of the in vitro CFC and application of autoradiography and morphological characterisation to highly enriched density fractions has shown that the in vitro CFC in normal marrow is a transitional lymphocyte. Single cell transfer experiments have shown that in vitro CFC's formed colonies containing both granulocytes and macrophages, formally demonstrating the clonal origin of in vitro colonies and the common origin of granulocytes and macrophages.

Notes: The rate of synthesis of different hemoglobin (Hb) species  -  embryonic, primitive, definitive, and adult  -  in the circulating red cells of the chick embryo have been measured. On the fifth day of chick embryogenesis, there is a marked decrease in the capability of these red cells to synthesize Hb. The rates of synthesis of embryonic and primitive Hb decrease markedly compared to that of definitive and adult Hb. On the fourth day of embryogenesis nucleic acid synthesis drops sharply in these cells. These decreases in metabolic activity preceed the disappearance of the primitive erythrocyte from the chick embryo circulation. It appears that the primitive cell line is specifically shut off with respect to its metabolic activities and possibly destroyed at this time.

Notes: A clonal strain of mammalian cells with increased resistance to acute heat shock at 46° was compared with the heat-sensitive parental line from which it was derived for possible differences that might account for this acquired resistance. Studies on synchronized populations of the two cell strains did not reveal any differential heat sensitivities in the various parts of the cell cycle (G1, S, G2) within either the sensitive or resistant populations. During thermal stress both cell types exhibited marked inhibition of ability to incorporate isotopically labeled precursors into macromolecular RNA, DNA, and protein.However, significant differences were observed between the sensitive and resistant cells in the amount of leakage of materials from the two cell types during heat stress and in their relative rates of recovery after stress. Sensitive cells pre-labeled with 3H-uridine released considerably more acid-soluble (cold, 5% TCA) label-containing materials during heat stress than did pre-labeled resistant cells. This differential release of uridine-containing materials was not paralleled by a generalized differential leakiness to other compounds. In addition, the resistant cells were found to regain the capacity to synthesize vital macromolecules sooner, and at initially faster rates, than the sensitive cells after stress.These results suggest that permeability changes causing decreased leakage of uridine-containing materials during heat stress combined with accelerated rates of recovery of synthesis of essential macromolecules after stress may be important cellular mechanisms in resistance to heat shock.

Notes: The term “contact inhibition of cell division” was borrowed from “contact inhibition of cell movement.” We prefer the term “postconfluence inhibition of cell division” as being more operational and less mechanistically biased; it is operationally defined as a pronounced depression of the mitotic rate in a postconfluent culture which displays a stationary density despite periodic nutrient renewal, the inhibition being locally reversibly by removal of the adjacent cells.The mechanism of postconfluence inhibition is of considerable interest because of the inverse correlation between postconfluence inhibition and the tumorigenicity of a number of cell lines. Several hypotheses, involving direct cell-to-cell contacts or locally restricted diffusion gradiens, could explain postconfluence inhibition.With the goal of discriminating among these hypotheses, time-lapse films were taken of carefully regulated, perfused cultures of 3T3 mouse cells, in which the transition from rapid growth to the stationary phase was recorded. Measurements of cell-to-cell contact, local cell density, and generation times were made on an individual cell level and analyzed with the aid of a computer.We observed that all-around cell-cell contact or a high local cell density present throughout G1 often did not produce immediate inhibition of cell division. We conclude that either (i) simple visible cell-cell contacts or a high local cell density are not the direct cause of postconfluence inhibition of cell division, or (ii) their effects often do not inhibit cell division until after a delay of about one cell generation time. Such a delay may be partly responsible for the 50% overshoot past the stationary density that we observed in 3T3 cultures.

Notes: Antisera have been prepared to clonal cultured rat astrocytic cells (C6 cells) and clonal human astrocytic cells (CHB4 cells). When cultured glial cells are grown in the presence of neuraminidase the serological activity of the cell membrane with homologous antiserum is increased extensively. A similar activation of serological activity can be effected by incubating washed harvested cells with this enzyme. Membrane bound N-acetyl-neuraminic acid is extensively reduced by either incubation. The activity responsible for promoting the enhanced serological activity co-chromatographs and co-electrophoreses with neuraminidase. Proteolytic digestion, unlike neuraminidase treatment, was unable to enhance the serological activity of cultured glial cells.

Notes: Two kinds of extracts of peripheral blood leukocytes induced erythroblast increase and maturation in vitro in rabbit marrow cells. One extract was a secretion or exudate into plasma from intact leukocytes; the other was the supernatant after homogenizing the cells in ethanol. The extracts were less active than the intact leukocytes. The response with the two extracts together was a little greater than with either alone, but less than additive.11-Ketopregnanolone and 3′5′ cyclic AMP were active inducers. Neither increased the effects of leukocytes or their extracts.

Notes: We have investigated the effect of varying La+3 concentrations (0.01 mM to 2.0 mM) on membrane potential and electrolyte composition of Ehrlich ascites tumor cells. La+3 concentrations less than 0.02 mM had no effect. Above 0.02 mM, La+3 induced concentration-dependent loss of electrolytes and water from the cells. At 1.0 mM the effect was maximal and resulted in an 87% reduction in cellular K+, 79% in Cl- and 21% in Na+ within 4.8 minutes. The Na+ loss occurred even in the face of an electrochemical potential gradient favoring Na+ entry. La+3 increased the recorded values of membrane potential; the magnitude of the effect was related to the external La+3 concentration, and was maximal at 1.0 mM. Studies using 140La showed that La+3 binds rapidly to the cell surface and does not enter the cells. The amount of La+3 bound to the cells was related to the external La+3 concentration by a sigmoidal curve and was maximal at about 1.0 mM. The bound La+3 could not be displaced by either added La+3 or Ca+2. Agents known to effect the integrity of the cell membrane, such as phospholipase C, neuraminidase, pronase and Hg+2 were tested for their ability to displace bound La+3. Only pronase displaced bound La+3, indicating that La+3 associates with cell protein. It is hypothesized that La+3 rapidly interacts with membrane protein causing alterations in membrane permeability and capacity to actively transport ions.

Notes: A method is described for measuring lymphopoiesis that enables the production of recirculating and non-recirculating small lymphocytes to be estimated simultaneously. Using this technique, experiments were undertaken to determine whether the production of recirculating cells is influenced by the number present in the recirculating lymphocyte pool. The results suggest that neither a massive lymphocyte transfusion nor depletion of the pool by whole body irradiation or chronic lymph drainage affect the rate at which recirculating small lymphocytes are generated.

Notes: In extracts of normal and Lesch-Nyhan (LN) heterozygous skin fibroblast monolayer cultures, hypoxanthine-guanine (H-G) and adenine (A) phosphoribosyltransferase (PRT) activities are correlated. These activities vary concomitantly during the life cycle of a culture: Peak activities occur during exponential growth. The ratio of H-GPRT to APRT activity can manifest heterozygosity at the LN locus when H-GPRT activity, per se, is close to and unreliable different from the normal range.If 2 or 8 × 104 cells are planted per 35 mm petri dish, a strain with clearly heterozygous levels of H-GPRT activity in its cell extract may not reveal its genotype by simultaneous measurement of adenine and hypoxanthine incorporation into its acid-insoluble fraction one day after subculture. In contrast, a 1:1 coculture of normal and mutant cells yields the ratio of adenine to hypoxanthine incorporation expected from the behavior of each cell type separately. In heterozygous cultures at the higher population density, the incorporation of hypoxanthine relative to adenine is 28% greater than in those at the lower population density. A 1:1 mixture of normal and mutant cells exhibited a 20% increase in the relative incorporation of hypoxanthine over the same four fold increase in population density. These observations appear to provide a quantitative expression of the phenomenon of metabolic cooperation between contacting H-GPRT-negative and H-GPRT-positive cells.

Notes: Hemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.

Notes: The study of the cytological evolution of the foetal thymus enables us to distinguish an early foetal period (up to 20 weeks) from a late foetal period. The cytological maturation is achieved at 20 weeks of gestation. The thymus in the late foetal phase is comparable to that of the child.The study of the lymphopoietic function shows a higher activity in the foetal thymus up to 20-22 weeks, lymphopoiesis is also slightly increased, in the thymus of the child up to the age of three years. The study of the contribution of the various cell types to the cellularity of the thymus, emphasizes the importance of the medium lymphocytes in the early foetal period. The small lymphocytes though to a lesser extent, are also capable of division in the foetal thymus and in the infantile thymus. Incorporation of uridine is also increased in the early foetal thymus.Cytological composition and lymphopoiesis of the foetal thymus and lymph node, have been compared.

Notes: We have isolated several cell lines which are temperature-sensitive for growth. One of these appears to be temperature-sensitive for cytokinesis. It was isolated from a Syrian hamster cell line by selecting cells which were not killed by 1 μg/ml cytosine arabinoside at 39° but which grew normally at 31°. It shows an increased proportion of binucleate cells when shifted to the non-permissive temperature and time-lapse photomicroscopy shows that a high proportion of attempted mitoses fail at 39°, apparently at the stage of cytokinesis. The cells which have failed to complete mitosis reattach to the plate and have two normal-size nuclei but otherwise behave normally.

Notes: A reduced rate of transport of 2-amino-isobutyric acid (AIB), was found to be associated with M and early G1 periods in synchronized populations of CHO and L cells. A doubling of the transport rate occurred with progress of CHO cells further into G1, and the specific transport activity remained constant during the rest of the cycle, The rate of uridine and thymidine transport also doubled at this point in the cell cycle. There was no change in Km values. The rise in transport rate was blocked by cycloheximide, or when the cells were prevented from leaving metaphase by colcemid.

Notes: Chinese hamster ovary cells with a deficiency in Serine Hydroxymethylase which produces a specific glycine auxotrophy (gly- A) were fused with human cells from a variety of sources and the resulting hybrids analyzed for human gene linkage. Of 102 hybrid clones examined 65 possessed both glyA and lactic dehydrogenase B markers, 35 possessed neither marker. Two clones were found with altered glycine responses which were not linked to LDH-B. The data indicate linkage between genes responsible for serine hydroxymethylase activity and lactic dehydrogenase B. Evidence for absence of linkage between these and a variety of other genes is also presented.

Notes: Although it is well known that thymus function changes with age, it is not known whether these changes are associated with specific thymocyte populations. Since one criterion of specificity is cell size, we studied the size distribution of thymocytes from mice 0.5 days to 30.5 months of age. Body weight, thymus weight, and thymocyte yield were also measured. The mean cell volume of thymocytes from 8.5 to 13 week old mice was 326 μ3, with two detectable subpopulations. Mean thymocyte size was found to change with age. During the first postnatal week, the mean cell volume of the whole thymocyte population increased from 200 to 350 μ3, and the percentage of large cells increased greatly and constituted 90% of the whole population at four days of age. A rather slow decline in mean cell volume with some fluctuation occurred throughout the remaining life span, and at 30.5 months the mean had dropped to about 190 μ3. We suggest on the basis of these data that large thymocytes are involved in the contribution of the thymus to early postnatal development of the immune system and that the age-related functional capacity of the thymus is related to the size of the thymocyte population.

Notes: Suspension cultures of L-929 fibroblasts grown to densities of 6 to 10 × 106 cells/ml through daily centrifugation and resuspension in fresh media, have been maintained for periods up to five months without change in viability or cell size. DNA synthesis and mitosis in these cultures is limited to 5% of the cells per day, a fraction very nearly equal to the fraction of cells rendered nonviable, most likely during the manipulations associated with medium renewal. The kinetics of the flow of cells into the S and M periods following (a) renewal of the medium and (b) dilution of the high density cultures, suggest that the large majority of the cells are in a G0 or early G1 phase, resuming growth readily in response to decreased cell density. This is further indicated by the sequence of the marked shifts occurring in the cell volume distribution spectrum of the high density cultures after dilution. Long term, steady state regulation of growth with retention of intact viability was thus demonstrated in the case of a long established aneuploid cell line. The fact that this occurs in suspension but not in attached cultures, supports the concept that impairment of growth control in such cells affects predominantly regulatory mechanisms located at the cell surface rather than those concerned with intracellular synthesis and metabolism.

Notes: Xenopus embryo cells and HeLa cells were investigated under various conditions to test for coordinate synthesis of high molecular weight (28S and 18S) and low molecular weight (5S) rRNA. Xenopus embryos initiate 28S and 18S rRNA synthesis at gastrulation (Brown and Littna, '64); we found that 5S rRNA synthesis is coordinately initiated with the 28S and 18S rRNAs at the same time in development. Dissociated Xenopus blastula cells were cultured in vitro for several hours to condition the medium; post-gastrula cells were then grown in the conditioned medium to test for the existence of an inhibitor of rRNA synthesis. No inhibitor was detected.Low doses of actinomycin D profoundly inhibit the synthesis of 28S and 18S rRNA in HeLa cells, while 5S rRNA synthesis is less affected by this treatment. Therefore, actinomycin D does not produce a coordinate inhibition of all rRNA species. Similar effects of the antibiotic were found in cultured amphibian cells. Synchronized HeLa cells reinitiating RNA synthesis following mitosis also respond to actinomycin D in a non-coordinate manner.

Notes: Two isogenic acyclic strains of Paramecium multimicronucleatum, syngen 2, expressing mating types III and IV, and a cycler strain, which displays circadian alternation of the two mating types, were treated with proteolytic enzymes, or the inhibitors actinomycin D or puromycin at various stages of the mating reactive period induced by food depletion. Reactivity of acyclic animals was sensitive to proteolytic enzymes, and to both inhibitors before or after the onset of reactivity, suggesting that reactivity is attained and maintained via continuous RNA and protein synthesis, and that protein integrity is essential to the activity of the type-specific substances. A new cell cycle was not necessary for recovery from a block in reactivity.Type III animals were consistently more sensitive to inhibition by puromyin, and required longer recovery periods after all treatments except actinomycin D given after the development of reactivity. Possible explanations of these observations are considered.The daily type changes seen in cycler animals seemed to occur by replacement of one mating type substance with the other. Type-switching was neither prevented nor displaced by exposure to actinomycin D or puromycin during a period before or including a type change, even though reactivity was reduced by more than 95%, suggesting that type-switching is not directly associated with transcription or translation of the type-specific genes.

Notes: A study of aldolases in rat hepatoma clones and subclones has revealed that they synthesize all three forms of aldolase monomers: A (the ubiquitous glycolytic isozyme), B (the form characteristic of the liver) and C, and that in vitro-in vivo passage results in a reversible modulation in aldolase A activity.Three kinds of somatic hybrids, between rat hepatoma cells and either mouse fibroblasts or rat epithelial cells, have been studied. In each case, aldolase B, found only in the hepatoma parent, was absent in the hybrid cells. The absence of aldolase B in the somatic hybrids seems not to be due to trivial factors (species differences, inactivation of all hepatoma aldolase genes, increase in ploidy or loss of chromosomes); it is concluded that extinction of this differentiated function of the hepatoma parent reflects a genetic regulatory phenomenon.

Notes: Livers from five-day chick embryos maintained as organ cultures on Eagle's minimum essential medium (MEM) develop an ultrastructure similar to more mature liver cells, except for glycogen deposits and the smooth endoplasmic reticulum (SER) normally associated with such deposits. The enzymes, glycogen synthetase and glycogen phosphorylase, failed to develop in these cells. The addition of zinc-free insulin (insulin-HCl) to MEM promoted the development of small amounts of SER in the cultured cells, as well as an increase in both glycogen synthetase and phosphorylase activities. The addition of zinc-insulin also stimulated an increase in the activities of both enzymes, and promoted the development of greater amounts of SER and the deposition of glycogen, as well. In addition, both forms of insulin not only prevented the fall of total tissue protein which occurs during organ culture on MEM, but also stimulated net protein synthesis in the explanted liver (Benzo and de la Haba, '71).

Notes: The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.

Notes: A protein kinase that catalyzes the phosphorylation of histone was partially purified from rat thymus, and the rate of histone phosphorylation was stimulated three- to fourfold by 1 × 10-6 M adenosine 3′,5′-monophosphate (cyclic AMP). Thymic protein kinase was more active than the enzyme from spleen. Histone fractions f1, f2a, f2b, and f3 were all capable of serving as phosphate acceptors for the thymic protein kinase, and the rate of phosphorylation of each fraction was stimulated by cyclic AMP. The ability of various 3′,5′-mononucleotides to stimulate protein kinase activity was compared. Inosine 3′,5′-monophosphate (cyclic IMP) was the most effective substitute for cyclic AMP.The cellular distribution of cyclic AMP-dependent protein kinase and adenylate cyclase activities in the thymus was determined. Cyclic AMP-dependent protein kinase activity is present in both small thymocytes and residual thymic tissue. The specific activity of protein kinase from residual tissue, both for basal and cyclic AMP-stimulated enzyme, was greater than that of enzyme from small thymocytes. In contrast to this, adenylate cyclase activity is predominately localized in the thymocytes.

Notes: Treatment of adult mice with rabbit anti-mouse thymocyte serum decreased the number and frequency of alloantigen-sensitive units responsible for graft-versus-host reactions and prolonged the survival of skin allografts. Whereas alloantigen-sensitive units were suppressed directly in vitro, they were not apparently suppressed directly in vivo since the fall-off of their numbers and/or function did not occur during the first day after serum injection. Treatment of prospective recipients of thymus cell grafts impaired the production of alloantigen-sensitive units by transplanted primitive progenitors. Differentiation with proliferation of alloantigen-sensitive units was less affected.Similarly, treatment of prospective recipients of thymus cell grafts with antilymphocytic serum impaired the production of specific inducer cells responsive to sheep erythrocytes by transplanted more primitive cells, presumably antigenreactive cells. Production of new precursors of anti-sheep erythrocyte hemolytic plaque-forming cells by transplanted bone marrow was not affected. Thus, antilymphocytic serum impairs the generation of immunocompetent cells of thymic origin by altering a hemopoietic inductive environment necessary for an early stage in differentiation.

Notes: Removal of the parathyroid glands caused a marked and permanent lowering of the mitotic activity in rat bone marrow, which was followed by a pronounced decline (ca. 40%) in the size of the marrow's nucleated cell population. These changes occurred only when the hypocalcemia induced by the lack of parathyroid hormone (PTH) was maintained by feeding the animals a calcium deficient diet. Since the overall marrow hypoplasia was almost entirely due to strikingly large reductions (ca. 70%) in the size of the erythroid and lymphoid subpopulations, it is concluded that PTH and calcium are major physiological regulators of the proliferation of erythroid and lymphoid cells.

Notes: Calcium-influx associated with the action potential was studied in squid giant axons using an EDTA-free preparation of aequorin as a probe. Associated with an individual action potential there was a transient increase in aequorin luminescence and the time course of this increase was examined on a kinetic basis. The luminescent response associated with a train of action potentials was compared with that expected on the basis of superposition of individual responses. The analyses of luminescence curves produced by long trains of action potentials were complicated by failure of superposition. A long pulse of inward current was found to produce a very large enhancement of luminescence.

Notes: Activities of two membrane associated enzymes, viz. 5′-nucleo-tidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.1) are known to be altered by glucocorticoid treatment of cells in culture. In certain HeLa cell lines, their activity is elevated, while in others, it is decreased. In order to confirm the data obtained previously with cell homogenates, cells were fractionated essentially as described by Bosmann, Hagopian and Eylar ('68) after 96 hours exposure to 0.5 μg/ml prednisolone. This treatment resulted in changes both in the specific activity and in the distribution of the two enzymes among the isolated membrane fractions. The alterations in specific activity in the individual membrane fractions reflected changes previously observed in preparations from whole cells. A shift in distribution of total enzymatic activity to fractions of higher density occurred in both cell lines in response to the steroid. These results may be indicative of cellular membrane alterations as a consequence of steroid treatment.

Notes: The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1-10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.

Notes: The effect of dibutyryl adenosine cyclic 3′:5′ monophosphate and testosterone on growth, response to serum and transport properties of Chinese hamster ovary cells was studied. There was a marked depression of growth in the presence of both compounds. A change of medium was sufficient to permit a partially synchronized burst of growth of the treated cells either in the presence or absence of dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone. However, in the presence of these compounds a second round of cell division was prevented. Initiation of the cell cycle by cells exposed to dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone displayed a greater serum requirement than untreated cells. It is concluded that serum and cyclic AMP could have antagonistic interactions in growth regulation.The treated cells had a reduced ability to accumulate amino-isobutyrate and glutamine, but no difference was observed with uridine uptake. The data suggest that a functional alteration of the membrane is induced by the exposure to dibutyrl adenosine cyclic 3′:5′ monophosphate plus testosterone.

Notes: Medium calcium ions were essential to the development of the lymphocyte transformation response induced by PHA. Magnesium ions could not substitute for calcium ions. Calcium exerted its influence during the induction phase of the process before DNA synthesis began since its removal after the response had fully developed did not alter subsequent levels of nucleic acid synthesis. The early development of RNA synthesis was prevented by low calcium levels indicating that calcium either directly influenced the initiation of this process or some prior event(s).

Notes: Chinese hamster cell lines have a strong tendency to maintain as modal chromosome number the euploid one characteristic of the species. By following the evolution of karyotypes in cloned subpopulation it has been possible to show that these pseudodiploid constitutions are not simply established due to a selective advantage but they are actually generated in populations where pseudodiploid classes had been absent. The possible role of the mitotic apparatus in keeping and restoring the euploid number is discussed.

Notes: In glucose exchange transport into red blood cells the rate of glucose uptake showed two pH dependent maxima, with the larger at approximately pH 7.5 and the smaller one at pH 3.In the studied pH range the relation between the rate of glucose uptake and the substrate concentration followed Michaelis-Menten kinetics. While the maximal velocity (V) reflected the pH changes of the media, the Michaelis constant (Km) remained constant.The dissociation constants of the groups of the free carrier and the carrier-glucose complex were the same. The pK of the acidic group was 5.2 and of the basic group 9.5.Glucose was not bound to groups of the carrier which dissociated protons in the pH range of three to nine.By rearranging the equation for the pH dependence of the relative influx a more definitive graphic determination of the pK values was produced.

Notes: Chinese hamster mitotic cells (95% in mitosis with 85% in metaphase and 10% in anaphase) were shaken loose from monolayer asynchronous cultures and stored at 1°C for up to 28 hours. During this period, the mitotic index did not decrease and the cells remained cytologically normal. However, over a four-hour period, metaphase cells located within 1.8 minutes of anaphase, 10% of the metaphase population, were able to move into anaphase; this point of 1.8 minutes corresponds in time to that reported for the spindle activation marker. When the cells were warmed to 37°C, they were delayed in entering G1 and S (35 and 70 minutes, respectively, after a 4-hour treatment). This delay of 70 minutes was maintained for three cell cycles, during which a high degree of synchrony was maintained. Cold treatment for 12 hours produced delays into G1 and S of 50 and 110 minutes, respectively. A fraction of the metaphase cells (12 or 50% after treatments of 4 or 12 hours, respectively) evidenced chromosomal aggregation, were unable to complete cytokinesis, and appeared in the next division as tetraploid cells. These tetraploid cells were unable to survive and produce macroscopic colonies. It is concluded that this decrease in viability is caused by irreversible effects on the spindle and/or centriolar components in metaphase cells located prior to the spindle activation marker.

Notes: The ADP-stimulated (State 3) respiration of myocardial mitochondria with glutamate-malate, glutamate-pyruvate, palmitylcarnitine and β-hydroxybutyrate as substrates declined in rats after the age of 20 months. There was no significant decline in pyruvate-malate, α-oxoglutarate, palmityl-CoA, succinate and ascorbate cytochrome c oxidation. Skeletal muscle mitochondria from senescent animals showed a similar decline in glutamate-malate oxidation but not in palmityl-CoA, palmitylcarnitine, succinate and ascorbate-cytochrome c oxidation. The controlled oxidation with ADP-limiting (State 4) and the ADP/O ratio were not affected. The results indicate an alteration in the subtle regulatory capacity for mitochondrial oxidation in senescent rats. It is suggested that the alteration may be in certain anion transport and associated functions across the mitochondrial membrane or dehydrogenase activity.

Notes: Self-diffusion coefficient measurements of water in untreated ovarian eggs of Rana pipiens using nuclear magnetic resonance indicate that cytoplasmic water has reduced translational mobility compared with pure water. Using a simple two-state model, we find that ∼67% is “relatively immobile.” Consideration of the nuclear magnetic resonance spin-lattice and spin-spin relaxation times indicates that the decreased mobility can largely be ascribed to hydration. Our value for the self-diffusion coefficient (6.8 × 10-6 cm2/sec) is lower than those reported by other investigators using isotopic water exchange techniques on frog eggs chemically treated to remove the membrane. However, the results reported here are in agreement with unpublished data on untreated frog eggs implying that chemical treatment has modified the cytoplasm in some manner.

Notes: The levels of carbohydrates were measured in cells before and after transformation with DNA and RNA-containing oncogenic virus. The most consistent finding was a markedly lower content of L-fucose in all transformed cells relative to their normal counterpart. The cell pellet from a line of Chinese hamster cells transformed with polyoma or SV-40 viruses showed a marked decrease in the level of carbohydrates. However, when the carbohydrate levels were calculated for the whole system (cells plus wash) the transformed cells except for L-fucose had almost the same amount of carbohydrate as the controls. The levels of various carbohydrates in cells transformed by Rous sarcoma virus were the same or were elevated above the controls. Here consideration of the washes did not alter the results. Possible explanations for the results and causes of error in these comparative studies are discussed.

Notes: The response of unfertilized sea urchin eggs stimulated with continuous D.C. was studied using cinematographic techniques. As reported by previous workers, the initiation of the response was anodal in polarity and followed a typical strength-duration curve. However, the anodal response may be swelling and/or contraction, depending on the amplitude of the applied stimulus, i.e., the magnitude of the contraction is directly related to the applied stimulus and is a graded response. It is suggested that the phenomena of anodal swelling and anodal contraction represent a continuum of responses based on ion exchange.

Notes: The sedimentation behaviour of single human red blood cells fixed with glutaraldehyde at pH 7.4 and 6.4 was studied and compared to resiflts previously reported for normal fresh cells. The cells fixed at pH 7.4 were observed to have normal shapes while those fixed at pH 6.4 were more spherical and less disc-like. Fixation of glutaraldehyde removed “membrane flicker,” resulting in increased stability as indicated by a decrease in the number of orientation changes per minute from 2.93 ± 0.16 (SEM) to 1.74 ± 0.10 (SEM). An orientation change was defined as a change of 45° in any direction. Fixation also increased the edge-to-flat time preference ratio from 2.5 to 4.7, and increased the sedimentation velocities in all three orientations, despite a measured 2.5% decrease in mean cell density.Fixation of cells at pH 6.4 showed that the decrease in stability and preference for the on-edge orientation was associated with an increase in the sphericity of the cell.

Notes: 3T3 and SV-40 transformed 3T3 mouse fibroblasts were cultured in media with serum and antibiotics plus ammonia (NH3 z NH4+) added as NH4C1. Both cell lines cultured without added ammonia showed normal morphology and multiplication even though ammonia in the medium at the end of the culture period ranged from 35 to 48 μg/ml. Ammonia concentrations being significantly higher in media removed from cells at the end of the culture period than in media incubated identically without cells, verified that cells released substantial quantities of ammonia in addition to components of the medium which underwent spontaneous breakdown. Both cell lines showed changes in morphology and highly significant reductions in cell multiplication which increased progressively as the concentration of added ammonia on the initial day of culture was increased to 35μg/ml. Control 3T3 cultures released significantly greater quantities of ammonia per cell than control cultures of transformed cells but their multiplication was more adversely affected by added ammonia. There were downward shifts in pH of the culturing medium for both cell lines as culture age increased at all concentrations of added ammonia, However, significant reductions in cell multiplication resulted from additions of ammonia that did not produce significant changes in extracellular pH. The data show that studies upon the effects of pH of the medium on cultured cells require control of ammonia concentrations.

Notes: Normal and virally transformed mouse cells respond differently in vitro to high concentrations of potassium. A higher potassium concentration is required to inhibit multiplication of SV40 transformed 3T3 cells to the same extent as that of normal 3T3 cells. This potassium effect correlates to specific activity of Na-K dependent ATPase in membranous fraction, normal 3T3 cells having the higher enzyme activity being more sensitive to potassium than SV40-3T3 cells which have the lower specific activity of the enzyme.Contact inhibition of growth and changes of concanavalin A binding sites which are characteristics of viral transformation influence specific activities of Na-K dependent ATPase and of adenyl cyclase. Incubation with trypsinized concanavalin A causes SV40-3T3 to show contact inhibition of growth and at the same time, higher specific activities of both enzymes than the observed in untreated cells. Cellular content of cyclic 3′,5′-AMP of contact inhibited 3T3 is higher than that in transformed SV40-3T3, but no difference is detectable in ATP content.

Notes: Addition of AMP to cultures of Tetrahymena pyriformis caused an increase in glycogen content of the cells and a small inhibition of growth. Adenine, adenosine, ADP, and ATP also increased glycogen content. Inosine and GMP were less effective; cytidine and uridine were ineffective. The increase in glycogen content was also observed in cultures supplemented with ribose, fructose, or glycerol, and when glyconeogenesis was increased by partial anaerobiosis. Adenine itself did not serve as a glycogen precursor, nor could the lipids of the cell have been the source of the carbon for the increased glycogen. The specific activity of glycogen from cultures supplemented with labelled amino acids was lower in AMP-treated cells than in controls. AMP-treatment had little effect on 14CO2 production from labelled glucose, acetate, or pyruvate, but baused a marked inhibition of the oxidation of labelled glyoxylate. It was suggested that AMP increases the rate of glyconeogenesis from precursors other than amino acids and interferes with malate synthase activity or malate transfer from peroxisomes to mitochondria.

Notes: Effects of ionizing radiation and of sulfhydryl reagents on the 45Ca binding of red cell membranes were studied. Corresponding effects of these agents on potassium leak from intact red cells were also determined. Essentially all the 45Ca associated with the ghosts appeared to be bound. Calcium binding could be described by assuming two independent groups of binding sites with dissociation constants of about 6 × 10-4 m and 2 × 10-4 m. The total binding capacity was about 2.5 × 10-4 moles/g ghost protein. Membrane calcium was decreased by radiation and by the two sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and N-ethyl maleimide (NEM). The tightly bound calcium fraction appeared to be most affected by these agents. Changes in potassium leak evoked by varying doses of agents appeared to parallel effects on membrane calcium. These investigations suggest that the increased cation permeability observed after exposure or red cells to radiation or sulfhydryl reagents may be related to alterations in the calcium-binding properties of the cell membrane.

Notes: Erythropoiesis was suppressed in CDF1 female mice using one of three different techniques: hypertransfusion, daily injections of low doses of actinomycin D, or post-hypobaric polycythemia. Spleens and bone marrows of these mice contained increased numbers of stem cells as determined by transplantation into lethally irradiated syngeneic mice. These stem cells were capable of producing both erythrocytic and granulocytic progeny. An increase in the number of multipotential stem cells or increases in the numbers of both unipotential granulocytic and erythrocytic stem cells is compatible with these observations and implies that hematopoietic stem cells may have a basic rate of division that is in part independent of stimuli to differentiate. The implications of these findings for the possible modification of antineoplastic drug-induced bone marrow toxicity are discussed.

Notes: Pig kidney cells exhibit resistance to puromycin when cultured at high cell densities, growing progressively in otherwise lethal concentrations of drug. Comparison of population growth of cultures inoculated at different densities indicates that survival in puromycin is a function of cell density. Uptake of puromycin from culture media is less at higher cell densities resulting in decreased inhibition of protein synthesis by puromycin. Enhanced survival in puromycin at high population densities is attributed to reduced accumulation of puromycin from the culture media.

Notes: The distribution of sedimentation velocities was determined, by a photoelectric method, for human erythrocytes at low concentrations in Ringer solution. The light absorption at 414 nm was measured, as a function of time, 10 mm below the top of the column. From the frequency distribution of cell velocities that of Rs √ρ-σ was found; Rs being the Stokes' radius, ρ the cell density and σ the density of the solution. Cell density was measured by the phthalate method and the mean Stokes' radius was found to be 2.58 μm. The size distributions showed some skewness but were in good general agreement with those measured by Celloscope counter, and with reported measurements from photomicrographs of cells in hanging drop suspensions. The skewness was much less than that encountered with electric sensing zone instruments (e.g. Celloscope) and the sedimentation method, being based on entirely different premises, provides an important check on such data. The skewness is due to a bias in the orientation of human erythrocytes during sedimentation. This bias may be a characteristic of biconcave cells; it could be absent in many species and reliable measurements of size distribution would then be obtained.

Notes: The effect of di- and trivalent cations on the membrane potential of the Ehrlich ascites tumor cell has been investigated using micro-electrode techniques. In solutions free of multivalent cations the average membrane potential for 46 cells was 8.3 ± 0.5 mv (SE). However, the potentials were not stable and decayed with a half-time of about six seconds. Addition of Ca++ decreased the rate of decay and concomitantly increased the membrane potential. The magnitude of these effects was a function of the Ca++ concentration. At the optimum concentration (2 mM), the half-time of decay was increased to 12 seconds and the membrane potential was raised to 17.8 ± 1.7 mv (SE). The related alkaline-earth cations, Sr++, Ba++ and Mg++ had similar effects on both the stability and magnitude of the membrane potential. The effect of La+++, which was qualitatively similar to that of the divalent cations, was also concentration dependent. However, 100-fold lower concentrations were adequate to achieve comparable effects. Moreover, membrane potentials were stable for up to ten minutes in La+++-containing solutions. Variations in intracellular Cl- content induced by temperature changes were paralleled by changes in membrane potentials. However, the potentials were not those expected for a simple Cl- electrode.

Notes: Rates of secretion of milk constituents (fat, protein, lactose, Na+ and K+) in the lactating goat were measured under normal circumstances and after injections of ouabain. In all experiments a close association was noted in the secretion rates for protein, lactose and K+. Under the influence of ouabain, the concentration of Na+ in the milk tended to rise and that of K+ to fall. The rate of milk fat secretion varied independently from the rates for the other constituents. It is reasonably assumed that the principal mechanism of milk protein secretion is by emptying of Golgi vesicles through the plasma membrane. The close correlation in rates for protein, lactose and K+ supports the contention that all three are assembled in Golgi vesicles and secreted by the same mechanism.

Notes: The surfaces of cells from the early embryo of the chick were examined with the electron microscope using histochemical techniques in order to determine the distribution of cell surface material. The use of lanthanum nitrate as a stain for protein-polysaccharide showed that in whole embryos the surface layer was present in areas of close membrane apposition, but relatively sparse where the membranes bounded large intercellular spaces. On cells freshly dissociated with EDTA, this technique demonstrated a very uniform layer over the surface, possibly indicating some redistribution. Close examination of the lanthanum stained material in whole embryos revealed the presence of periodicities about 180 Å in diameter.In correlation with a progressive reduction of electrophoretic mobility, demonstrated previously, from embryonic stage 1 to stage 5, some decrease in the affinity of the surface for lanthanum and colloidal iron was observed.

Notes: Upon addition of bleomycin (BLM) to suspension cultures of Chinese hamster cells (line CHO), cells closer to prophase than 56 minutes continue dividing at the normal rate, whereas cells at earlier positions in the cell cycle either fail to reach mitosis altogether (at 200 μg/ml) or enter mitosis and divide at a reduced rate at lower drug concentrations. At 100 μg/ml of BLM (the rate of cell division slowed to a doubling time of 167 hours), initiation and termination of DNA synthesis occur at normal rates, resulting in an accumulation of cells with a G2 DNA content in the first 130 minutes of G2. Bleomycin effects are not readily reversible. The rates of incorporation of leucine, uridine, or thymidine into cells treated for six hours with 100 μg/ml of BLM were 90, 85, and 80%, respectively, of the values obtained in control cultures, suggesting that the effects of BLM on cell-cycle traverse cannot be correlated with gross inhibition of macromolecular synthesis.

Notes: The relative proliferative capacity of haematopoietic cell populations derived from 22-week-old adult bone marrow and 14-18 day foetal liver has been studied in lethally irradiated syngeneic recipients by means of chromosome markers. Although starting at a disadvantage in terms of the number of colony-forming units (stem cells) injected, the foetal liver-derived populations steadily increased their relative numbers in the myeloid and lymphoid tissues over a period of several weeks until a plateau was reached. It is suggested that stem cells in foetal liver have, on average, a higher intrinsic capacity for self-renewal than do those in bone marrow, and that this capacity falls to the adult level within about ten weeks of transfer.

Notes: Anthramycin and actinomycin D, two different types of DNA-binding antibiotics, were compared for their effects on RNA synthesis in suspension cultures of mouse leukemia L1210 cells. RNA was labeled with radioactive uridine and selective effects on the synthesis of different classes of RNA were studied by sucrose gradient sedimentation of RNA purified from whole cells or from nucleolar and nucleoplasmic fractions.Two major differences were noted. (1) Whereas actinomycin produced two phases in the inhibition of uridine incorporation, the rapid phase being complete within a few minutes, anthramycin produced only a slow progressive inhibition. (2) Whereas actinomycin selectively inhibits nucleolar 45s RNA synthesis, anthramycin inhibits this RNA equally to the inhibition of the same size RNA in the nucleoplasm.Both antibiotics caused a shift towards lower molecular weight (slower sedimentation) in the distribution of nucleoplasmic RNA molecules synthesized in the presence of drug. When the two antibiotics were compared at concentrations producing equal extents of inhibition of nucleoplasmic RNA synthesis, anthryamycin produced the greater shift. The shift in sedimentation anthramycin produced the greater shift. The shift in sedimentation was not due to a slowing of RNA chain growth rate, since the change in sedimentation persisted when uridine incorporation time was increased so as to compensate for the reduction in RNA synthesis rate.The selective inhibition of nucleolar RNA synthesis by actinomycin could be due to possible differences in the properties of the different RNA polymerases, or to differences in the initiation rates for transcription. The absence of selectivity in the case of anthramycin might be related to the near irreversibility of its binding to DNA. The shift towards lower molecular weight of the RNA synthesized is compatible with (but does not constitute strong evidence for) premature termination of RNA chains.

Notes: Treatment of polymorphonuclear leukocytes with sialidase release about one-half of the total sialic acid of the cells. Such treatment affects neither phagocytic ingestion nor glycolysis. These results are in contrast to earlier data in the literature that indicate suppression of both functions mentioned upon desialation of these cells. The presence of an inhibitor of glycolysis that contaminated the crude enzyme previously used is suggested.

Notes: A multiplication-stimulating activity for chicken embryo fibroblasts has been purified approximately 6000-fold from calf serum by a three step procedure. The partially purified multiplication-stimulating activity stimulated DNA synthesis, mitosis, and growth of cultured chicken embryo fibroblasts. In addition, the active step 3 material was shown to have non-suppressible insulin-like activity and to be an inhibitor of trypsin activity.The activity appeared to be associated with a small protein and was heat stable, but sensitive to dithiothreitol and to periodate.

Notes: Prostaglandin type E1 (PGE1) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10-8 picomoles per cell as produced by an exposure to 0.5 μg of PGE1 per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10-8 picomoles per cell) by exposure to a greater PGE1 concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE1's early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.

Notes: The density distribution and cation composition of red blood cells from newborn puppies have been studied. The density distribution of red cells from a newborn puppy in a bovine serum albumin density gradient resembles a normal distribution with a peak density at a region less than that found for adult dog red cells. In two weeks the whole distribution shifts toward a more dense region, and a second cell peak appears so that the distribution becomes bimodal. This second cell peak is smaller than the original peak, and it appears at a region of lower density. In nine weeks the distribution becomes a normal one again, but the peak density corresponds to the peak density of the second cell peak which first appeared at two weeks. Evidence has been obtained to show that fetal red cells are located in the more dense cell peak and neonatal cells are in the less dense second peak. These results were obtained by labeling fetal cells with Cr51 and neonatal cells with Fe59. The analysis of the cation content of these cells shows that fetal cells contain more K and Na and have a higher K/Na ratio than adult red cells. Furthermore, neonatal cells contain considerably less cation and hemoglobin than do fetal cells. From a study of the cation and hemoglobin content of red cells appearing in various density fractions it is concluded that fetal cells lose K and Na during the first two weeks after birth. Thus, the change in the density disribution of the erythrocytes is thought to be due to two factors: (1) An increase in the density of fetal cells due to the loss of K and Na and, hence, water during the first two weeks after birth, and (2) the entry of less dense neonatal cells into the circulation.

Notes: The cellular response to an intraperitoneal injection of antigen (tetanus toxoid) was studied in reconstituted animals in order to determine the mechanism of control of eosinophil granulocytopoiesis. Antigen treatment of the marrow cell donors did not consistently increase the number of spleen and bone marrow colonies in recipient animals or change the percentage of eosinophil or other hemopoietic colony types. Antigen pre-treatment of the irradiated recipients increased the percentage of eosinophil-containing colonies in the spleen and femoral bone marrow without significantly changing the total number of either spleen or marrow colonies. Antigen treatment of both the bone marrow cell donor and recipient produced a further increase in the percentage of eosinophil-containing colonies in the marrow cavity, but not in the spleen. Antigen treatment of the irradiated recipient increased the number of eosinophilic cells (but not the total number of cells) in both the peritoneal cavity and the bone marrow. Antigen treatment of both the marrow donor and recipient produced a further increase in the number of eosinophilic cells in the peritoneal cavity, but not in a single femur. Since antigen treatment of the marrow recipient, or recipient and donor, but not of the marrow donor alone, results in increased eosinophilic cell and colony numbers, the effect of antigen appears to be mediated through some host factor(s), perhaps the eosinophilic hemopoietic inducing microenvironment (HIM), rather than directly on the hemopoietic stem cells.

Notes: Cell division, net Na+-K+ and amino-acid transport of cultured Ehrlich ascites is reversibly inhibited by Ouabain at a final concentration of 1 × 10-3M. A line of Ehrlich ascites cells resistant to the growth inhibiting effects of Ouabain has been developed. These cells behave similarly to Ouabain-sensitive cells in the following respects doubling time, S phase time, chromosome number, cell surface charge density, rate of incorporation of C14 Uridine and 3H-Thymidine, sensitivity to Digoxin and Digitoxin, steady state Na+, K+ levels and rate of loss of K+ and gain of Na+ in cold.Ouabain resistant cells differ from sensitive cells only with respect to the effect of ouabain on active Na+, K+ transport. Although Ouabain inhibits active Na+, K+ transport in sensitive cells it has no significant effect in resistant cells.

Notes: Studies of human erythrocyte adhesion to glass have demonstrated consistently greater adhesion with serum-containing media than with a comparable concentration of plasma. This serum-plasma difference is explained by the adhesion-inhibiting property of plasma fibrinogen. The fibrinogen effect is probably mediated through its firm binding to glass, since no adsorption onto the red cell surface could be demonstrated.The ability of more red cells to adhere to a foreign surface after plasma coagulation (the formation of serum from plasma) may be significant in the red cell surface interactions necessary for the formation of a fibrin-red cell thrombus.

Notes: A Coulter-orifice pulse-height analyzer system was used to measure volume spectra of mammalian cells in suspension at different times after the addition of an equal volume of water. In appropriate hypotonic medium, cultured mammalian cells rapidly increase in volume and then shrink, more slowly, approaching their initial volumes within 20 to 30 minutes at 37.5°C. The shrinking phase was found to be reversibly inhibited by ouabain and inhibited in both K+-free and Na+-free solutions; neither choline+ nor Li+ could substitute for extracellular Na+ in supporting the shrinking phenomenon but Rb+ and Cs+ were fairly good substitutes for K+.Under conditions similar to those with which the shrinking phenomenon was observed with cultured cells, it was not found with either human or mouse red blood cells.Two methods were used to determine intracellular Na+ and K+ content in osmotically shocked cells and in unshocked controls. An isotope equilibration method was employed with L5178-Y mouse lymphoblasts and a chemical determination by flame photometry was used with Ehrlich ascites tumor cells. The K+ content was significantly reduced and the Na+ content was unchanged or somewhat increased in cells which had returned to their original volumes in hypotonic medium. The K+ content was even more reduced but the Na+ content was greatly increased in cells which were osmotically shocked in the presence of ouabain.

Notes: Electrophoretic patterns of newly synthesized proteins have been compared for hamster embryo fibroblasts in asynchronous cultures, mitotically synchronized cultures, and stationary phase cultures. Only proteins with molecular weight between 30,000 and 150,000, comprising 60-70% of the total cell proteins and excluding histones and collagen were included in the comparison. Although no significant differences could be detected between such patterns for cells at different stages of the cell cycle, significant differences were detected between patterns for cells in stationary phase and for proliferating asynchronous or synchronous cells in any stage of the cell cycle. These differences amounted to at least 5% of the newly synthesized cellular proteins. Much larger differences were detected between patterns from a nuclear fraction of proliferating and resting cells. These results indicate that normal cells in stationary phase are arrested in a state distinct from any phase of the normal cell cycle and may provide a biochemical marker for resting cells.

Notes: Two embryonic hemoglobins are found in chick erythrocytes during early development by column chromatography on DEAE-cellulose and by electrophoresis on starch gel and gelatinized cellulose acetate. Beginning during the sixth day of incubation these are replaced by three different hemoglobins. Following hatching, chicks lose the most alkaline component, so that in the adult two hemoglobins are again found. Chromatographic fractionation on phosphonic acid cellulose also revealed an increase in the number of hemoglobins present in chicks during their development. We report the presence of at least 11 hemoglobins whose relative concentrations rise and fall during chick ontogeny. Failure to recognize the multiplicity of chicken hemoglobins is believed to be the major source of the confusion regarding this subject in the present literature.

Notes: Sublines derived from V79 and B150 Chinese hamster cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and thymidine kinase (TK) respectively. When these strains are cultivated together without virus treatment in HAT medium, hybrid colonies with normal enzymic patterns arise in high frequency. The ratio of proliferating hybrid colonies to the minority cell inoculum in mixed cultures is approximately 1/1000. This observation has been used to examine the effect of x-irradiation pretreatment of one cell type on the frequency of hybrid formation in mixed cultures. B150 cells (TK-) were irradiated in monolayer cultures at dose levels of 200-4000r (180 KV, 15 ma). After trypsinization, the irradiated cells were mixed with unirradiated V79 cells (subline 129, HGPRT-) and cultivated in HAT medium. The per cent decline in hybrid frequency as a function of x-irradiation was compared to the relative decline in viability of x-irradiated B150 cells when assayed in cultures alone. These curves diverged markedly, with the frequency of proliferating hybrids decreasing much less rapidly with radiation dose than survival of B150 cells. By contrast, when B150 cells were subjected to acute heat shock for graded intervals and subsequently mixed with untreated V79-129 cells, both viability of B150 cells and hybrid formation declined at the same rate. Our results suggest that lethally irradiated cells can still fuse with normal cells, and may contribute functional chromosomes to the hybrid karyotype. At limiting doses of x-irradiation this technique may be useful as a means for induced segregation of marker chromosomes.

Notes: Growth of cultured human fibroblasts in low oxygen resulted in reciprocal changes in the levels of cytochrome oxidase and several glycolytic enzymes. After five days' growth in low oxygen, cytochrome oxidase specific activity fell to 40% of the level of control cultures, while lactic dehydrogenase (LDH), aldolase, and triose phosphate dehydrogenase (TDH) levels were increased by 2- to 3-fold. These changes were accompanied by a change in the LDH isoenzyme pattern resulting from an increase in the proportion of LDH A subunits; the aldolase electropherogram was unchanged. When fibroblasts were grown for five days in medium containing chloramphenicol, cytochrome oxidase specific activity fell to 10% of control values, but LDH, aldolase and TDH specific activities and LDH and aldolase electropherograms did not differ significantly from controls. These findings are interpreted to indicate that the increased accumulation of LDH, aldolase and TDH induced by low oxygen is not mediated by the rate of accumulation of cytochrome oxidase.

Notes: Mechanisms which determine sodium and potassium content and volume of rat thymic and human chronic lymphocytic leukemia (CLL) lymphocytes have been studied. The deleterious effect of cell isolation on monovalent cation content was proven by comparing thymus sodium and potassium concentration to that of thymocytes prepared from autologous hemithymus. In vivo distribution ratios of sodium-24 and potassium-42 between thymus water and plasma water were very similar to the distribution ratios of non-radioactive isotopes (sodium-23 and potassium-39). The similar lymphocyte: thymocyte ratio of (a) cell volume (1.48), (b) cell sodium plus potassium (1.47) and (c) cell water (1.50) demonstrated the close correlation of lymphocyte volume with monovalent cation content and water content. Steady-state CLL lymphocyte sodium (32 ± 1.9 mM) and potassium (131 ± 5.1 mM) and thymocyte sodium (31 ± 1.2 mM) and potassium (136 ± 3.9 mM) were similar; however, these steady-state levels were maintained by quantitatively different membrane functions. Radiopotassium and radiosodium uptake by thymocytes was more rapid than by CLL lymphocytes. Ouabain-sensitive potassium influx was 2.4 times greater in thymic (8.70 ± 2.28 mmoles/cm2/min × 10-8) than in CLL (3.24 ± 0.45 mmoles/cm2/min × 10-8) lymphocytes. Potassium exodus was also slower in CLL lymphocytes as compared to thymocytes. Ouabain-sensitive sodium accumulation and ouabain-insensitive sodium accumulation were also slower in CLL lymphocytes than in rat thymocytes. Half-maximal ouabain inhibition of sodium entry was 7.5 × 10-3 M in thymic and CLL lymphocytes. The inhibitory effect of ouabain on sodium and potassium transport was easily reversible. Oligomycin inhibited ouabain-sensitive potassium accumulation in both lymphocyte types. Four lines of evidence indicate the presence in the lymphocyte of a system of leaks and pumps, the latter subserved by a ouabain and oligomycin-sensitive (sodium-potassium) ATPase: (a) steady-state monovalent cation gradient (K ∼ 20:1, Na ∼ 5:1), (b) the inability to maintain normal sodium and potassium gradients at cold temperature and in the presence of ouabain, (c) the effect of ouabain and oligomycin on active potassium influx and (d) the restitution of steady-state sodium and potassium concentration after cell isolation, ouabain treatment and cold exposure. CLL lymphocytes as compared to rat thymocytes have a decreased rate of ouabain-insensitive sodium uptake and potassium exodus requiring a reduced rate of active sodium extrusion and potassium accumulation to maintain steady-state cation content. Ouabain-sensitive ATPase is difficult to locate in lymphocytes in vitro possibly because it comprises a very small proportion of membrane ATPase since magnesium activated ecto-ATPase in intact lymphocytes is 1500 to 2500 times that of the intact erythrocyte. The inhibition by ouabain of blast transformation, mitosis, amino acid accumulation and nucleic acid synthesis in vitro, may reflect the importance of ouabain-sensitive ATPase and monovalent cation transport in the function of lymphoid cells.

Notes: Circadian rhythms are demonstrated in the tongue of adult rats for both the mitotic index of the basal epithelium and the uptake rate of injected 3H-thymidine by the tongue tip. The animals were entrained to a light-dark cycle for four weeks prior to the experiments with the light phase extending from 0600 to 1800 hours (CST). The daily fluctuation is approximately 300% for the mitotic index and 185% for the uptake rate of 3H-thymi-dine. The highest mitotic index occurs at 1100, and the highest uptake of 3H-thymidine occurs four hours earlier at 0700. The least activity for both parameters occurs during the first part of the dark span of the light-dark cycle. Estimates of several other rhythmic parameters are determined by a computerized method.

Notes: In adult sheep, the hepatic sinusoids have an endothelial lining with numerous gaps and fenestrae and are surrounded by a complete basal lamina. In newborn lambs, the sinusoids have a relatively thick endothelium with fewer gaps, and the space of Disse contains a partially formed endothelial basal lamina. In both adult sheep and lambs, perisinusoidal cells appear to form a network around the sinusoid. In portal areas, the endothelium of the smaller capillaries is fenestrated, but the larger capillaries have unfenestrated walls and are enmeshed in processes of pericytes. Projections of endothelial cells partially or completely occlude the lumen of some portal capillaries, and of a large majority of sinusoids scattered throughout the lobule.

Notes: Lens regeneration was inhibited in adult Triturus v. with 1500 r of x-irradiation. Implants of irradiated or unirradiated segments of dorsal iris were then implanted into either irradiated or unirradiated eyes. When irradiated eyes received irradiated implants, only minor degrees of lens regeneration were seen from host and implant. Unirradiated eyes receiving unirradiated implants often formed two normal lenses, one from the host and one from the implant. Irradiated eyes receiving unirradiated implants usually formed one normal regenerate arising from the implant. Unirradiated eyes receiving irradiated implants usually formed only one normal regenerate, originating from the host. The results indicate that inhibition of lens regeneration by x-irradiation is due to effects solely on the iris, and that unirradiated neural retina, under the experimental conditions used, does not stimulate recovery of the regenerative ability of an irradiated iris. These results are discussed in relation to the question of neural-tissue-mediated recovery of regeneration systems from x-irradiation.

Notes: The possibility that chondrocytes of the proximal fragment of Meckel's cartilage may participate in resorption of the extracellular substance of the cartilage and outlive its removal, has been investigated in vitro and in vivo. Mandibles from 18 day in utero mouse foetuses were cultured for 14 days on an an antibiotic-free chemically defined medium. When tested histochemically for acid phosphatase heavy deposits of reaction product were evident in cells in areas where resorption appeared to be occurring. When maintained on medium containing 1.0 μg/ml hydrocortisone, groups of chondrocytes hypertrophied and were reactive for acid phosphatase, and this was accompanied by loss of intervening extracellular substance. These changes were intensified by increasing the oxygen tension of the environment and by further supplementing the medium with 1.0 μg/ml triiodothyronine. Chondrocytes in the vicinity of sites of resorption could incorporate 3H-proline and 3H-thymidine. In vivo, chondrocytes in the vicinity of resorbing areas in two to three day post-partum animals were highly reactive for acid phosphatase, and could incorporate 3H-thymidine, 3H-proline, and 3H-uridine. These observations have been interpreted to suggest that resorption of the proximal fragment of Meckel's cartilage is not necessarily accompanied by death of the chondrocytes, and that the chondrocytes may participate in removal of the extracellular substance. Furthermore, the response of the chondrocytes of Meckel's cartilage to hydrocortisone in vitro appears to differ from the response that has been reported to occur in a number of other cells in vitro.

Notes: The enzymes elastase, chymotrypsin, pepsin, pronase, hyaluronidase, and collagenase were utilized on thin Epon sections to obtain further information on the nature of elastic fibers. Results showed that the central amorphous material was selectively removed by elastase and after hydrogen peroxide pretreatment it also was removed by chymotrypsin, pepsin and pronase. None of the enzymes removed the peripheral microfibrils, but pepsin did accentuate the density and periodic beading of these structures. The failure of proteases to remove the microfibrils under these conditions is discussed.

Notes: The spheno-occipital synchondroses of 20 cynomolgus monkeys have been examined histologically and radiographically. The findings by these two methods have been compared and correlated with the dental age of the monkeys. The radiographic examination was carried out on the isolated clivus region while serial sections were used for histological examination. Comparison of the synchondrosis by the two methods resulted in six disagreements when the synchondroses were classified as open, partly closed or closed. It is suggested that this difficulty is due to superimposition of shadows on the radiographs and small degrees of histological union not being recognised radiographically. It is further suggested that care should be taken in determining the age at closure when radiographs alone are used as the criterion. The dental age at initial closure appeared to vary widely in some cases occurring at an early mixed dentition stage and in others as late as the adolescent dentition. The closure of the synchondrosis was not related to the eruption time of any particular tooth. The pattern of closure did not conform to the classical type, i.e., from above downwards nor indeed was any consistent pattern revealed in either the radiographs or histological sections.

Notes: The tongue of the echidna, Tachyglossus aculeatus is divided structurally and functionally into two distinct parts; a free rostral portion covered by stratified squamous epithelium bearing filiform papillae and a fixed caudal portion formed by a trapezoidal pad which bears highly specialized keratinized spines. These spines are similar in their morphology to the horny teeth of the living cyclostome Myxine and the hard keratin of rat filiform papillae.The gross and light microscopic structure of the lingual spines is described and their use as a masticatory organ (by occlusion with a similar set of spines on the palate) is discussed in relation to the animal's diet and method of feeding.

Notes: Changes in the ependymal surfaces of the floor of the third ventricle and lateral recess have been studied in male rats by electron microscopy after the intraventricular administration of epinephrine or dopamine. In control rats the floor of the third ventricle has essentially a smooth contour interrupted only by occasional bleb-like protrusions or microvilli. In the floor of the lateral recess this surface gradually becomes more irregular due to the appearance of increased surface modifications. A further increase in the number of complexly-folded microvilli, pinocytotic vacuoles and coated vesicles occurs on the epen-dymal surface of the roof of the lateral recess and adjacent ependyma. These morphological findings are consistent with the concept that the ependymal cells of the lateral recess may serve as important sites for the absorption of materials from the CSF.Increased numbers of bleb-like protrusions were observed on the ependymal surface of the floor of the third ventricle five minutes after the intraventricular administration of either epinephrine or dopamine. The ependymal surface of the roof of the lateral recess also showed bleb-like protrusions and a complexly-folded microvillus border. Fifteen minutes after either the epinephrine or dopa-mine administration all of the ependymal surfaces studied appeared as in control rats. The increase in bleb-like protrusions noted in the floor of the third ventricle after the injection of epinephrine or dopamine are suggestive of a secretory process rather than a manifestation of an absorptive function in this region.

Notes: In the adult goldfish, Carassius auratus (a stomachless freshwater teleost), there is a striking proximal-to-distal gradient in the microscopic appearance of the intestine. This gradient is correlated, moreover, with differences in the site of absorption of lipid and protein. The proximal regions (intestinal bulb and anterior intestine) have a greater surface area, manifested by elongated mucosal ridges in which the epithelial cells have regularly arranged closely spaced microvilli. The distal region (posterior intestine) has less overall surface area, but the epithelium exhibits the typical ultrastructural features of pinocytosis, namely extensive invagination of the luminal surface membrane and massive accumulation of vesicles and vacuoles in the apical cytoplasm. In addition, a conspicuous PAS-positive supranuclear “body” is visible with the light microscope. Administered triglyceride is absorbed exclusively by the epithelium of the anterior regions, where presumably, it must be hydrolyzed prior to uptake. Protein (horseradish peroxidase), on the other hand, is absorbed primarily in the most distal region, where the epithelium appears to be equipped for the uptake of large intact molecules. These regional differences are discussed in relation to comparable differences in the mammalian intestine, especially during development.

Notes: This study of primitive bronchial epithelium in human fetuses revealed cells of distinctive ultrastructural appearance. These cells contained cytoplasmic granules similar to neurosecretory-type granules and are termed fetal Kultschitzky-type cells (K-type cells) of lung. The fetal K-type cells appear similar to certain cells in mature lungs and in other tissues that have an endocrine function. The advanced cytoplasmic differentiation and large numbers of K-type cells in early stages of fetal development suggests that these cells might also have an important, probably endocrine function.

Notes: The innervation of the acinar cells of the rat parotid gland was studied using potassium permanganate fixation. Adrenergic terminals, characterized by the presence of small granular vesicles, and cholinergic terminals, containing small agranular vesicles, made close contact with the acinar cells. Both adrenergic and cholinergic terminals were observed in contact with the same cell, and subsurface cisterns of endoplasmic reticulum were found in relation to the contact areas of both types of endings. In addition, both adrenergic and cholinergic axons were found within the same Schwann cell sheath in the connective tissue spaces.

Notes: During a carbohydrate-rich diet the liver contains a large amount of evenly distributed glycogen. During a fat-rich diet glycogen is located mainly in zone 1 of the liver acinus. These results suggest that different acinar enzyme patterns can be induced by different diets. The acinar glycogen patterns remain unchanged during prolonged starvation.The curve of glycogen percentage vs. starvation time has the same general shape as the curve representing the changes in resistance during the general-adaptation-syndrome.

Notes: Rabbit anti-rat-brain serum immunoglobulins injected into pregnant rats on the ninth, tenth, eleventh or twelfth day of gestation resulted in a fetal resorption rate 15 to 30 times higher than that found in normal untreated pregnant rats. Human serum immunoglobulins obtained from normal postpartum mothers produced a similar percentage of fetal resorption when injected by the same routes into pregnant rats of the same gestational age. In neither of the above experiments were malformations detected among the delivered 20-day fetuses. Injection of human serum immunoglobulins obtained from mothers of children with spina bifida manifesta into pregnant rats along similar routes and periods gave comparable fetal resorption rates. Injection of the above immuno-globulin into the lumen of the uterus adjacent to implantation sites gave a low fetal resorption rate and produced varying degrees of skeletal and soft tissue malformations among the viable survivors.The only difference which could be discerned between normal postpartum immunoglobulins and those obtained from mothers of spina bifida manifesta children was characterized in the latter by a two-fold increase in the IgG levels and the immunoelectrophoretic reactivity of its immunoglobulins with human spinal cord antigens. The developmental defects observed were: 1. Cranial-thinning and bleb formation of skull bones; widening of the foramen magnum; descent of the obex closer to the foramen magnum. 2. Skeletal-delayed or inhibited calcification of the bodies and spinous processes of the thoracic and lumbar vertebrae; widening of the vertebral canal and central canal of the spinal cord.

Notes: A light microscopic study was carried out on the Golgi complex of the absorptive epithelial cells of the small intestine of adult male rats. Silver impregnation methods (Da Fano's, Aoyama's, and Elftman's) were used. Deposits of silver in the Golgi complex were heaviest in the lower half of the villi. Thereafter, a progressive diminution in the reaction occurred. In the uppermost quarter of the villus no reaction was visible. Villi of the duodenum, midgut, and terminal ileum all exhibited this phenomenon regardless of variations in fixation method, concentration and pH of the silver solutions, exposure time to the silver solutions, and various dietary regimens (high fat, no fat, and starvation). The change observed in the silver reaction of the Golgi complex probably reflects a structural or functional change of this organelle as the absorptive epithelial cells migrate along the villi toward the extrusion zones.

Notes: The sartorius muscle of ten normal subjects was studied by elec-tromyography. It is most active during flexion of the hip and only slightly active during lateral rotation and abduction of the hip. The muscle is usually more active during knee flexion than during extension, but this is not universal. Apparently in some persons it plays a role in knee extension in addition to its knee flexion action, depending on knee position and variations in insertion. Sartorius appears to be a regulator in hip flexion and lateral rotation during the swing phase of gait.

Notes: Mature female golden hamsters, which had demonstrated regular reproductive cycles, when exposed to 23,000 feet simulated altitude for eight or more days became acyclic. Their ovaries, which were polyfollicular, suggested that LH release or synthesis by the hypophysis had been blocked. Human chori-onic gonadotrophin (which has LH activity) caused similarly exposed animals to ovulate. When animals which were exposed to high altitude for nine days were returned to control altitude, they ovulated five days later. Starvation for nine days, which has no significant effect on adrenal weight but does cause increased LH dependent ovarian interstitial development at control altitude, failed to do so at 23,000 feet. It is hypothesized that high altitude selectively blocks LH secretions by the hypophysis.High altitude also interrupted pregnancy when treatment lasted four or more days and was begun before day 12. Since follicular development of pregnancy continued normally but corpora lutea regressed and both FSH and prolactin are needed to maintain the hamster corpus luteum, this study suggests the possibility that prolactin secretion is blocked in the pregnant animal at high altitude.

Notes: The mesenchymal specificity of epithelium from the submandibular salivary gland of embryonic mice was re-investigated employing standard epithelio-mesenchymal separation and recombination techniques. For this purpose, tissue recombinants were constructed with epithelium and mesenchyme of the submandibular salivary gland and the seminal vesicle, preputial gland, and urogenital sinus. Salivary glands were dissected from 13-day and the accessory sexual glands from 13- to 19-day old embryos. Tissue recombinants were grown for two to three weeks in the anterior chamber of the eye of adult male hosts.Mesenchyme from accessory sexual glands supported the growth, branching, and differentiation of epithelium from the salivary gland. After three weeks of intraocular growth, most of these heterotypic recombinants were indistinguishable from intraocular grafts of this gland. However, recombinants constructed with mesenchyme from the urogenital sinus or preputial gland exhibited an age dependent developmental response. Mesenchyme from 15- to 16-day old embryos supported normal morphogenesis of the salivary gland, whereas atypical epithelial differentiation occurred when 13- or 14-day old mesenchyme was employed. On the other hand, the reciprocal recombinants composed of mesenchyme from the salivary gland and epithelium derived from accessory sexual glands resulted in abortive epithelial differentiation regardless of the age of the tissues. These results suggest that the mesenchymal specificity of epithelium from the salivary gland may be related to the morphogenetic sensitivity of the heterotypic mesenchyme to androgens.

Notes: Histologic changes in the region of the squamocolumnar junction of the uterine cervix in ovariectomized C3H/HeJ mice were studied at 6-12 hour intervals after the injection of 2.3μg estradiol benzoate. Mitotic activity, especially in the basal layer of stratified squamous epithelium, increased as time elapsed after estrogen administration and produced a concomitant increase in stratification of squamous epithelium; some mitotic activity was present in the uterine columnar epithelium throughout the period of observation. The histologic character of the squamocolumnar junction changed from gradual transition to an abrupt demarcation, as in intact mice during the estrous cycle. Subcolumnar cells in the transition zone of untreated animals formed a unilayered reticulum which after estrogen injection developed into a reticulum of stratified squamous epithelium. Extensions of stratified epithelium which develop during chronic estrogen treatment apparently develop from the stratified reticulum. There was no evidence to indicate any contribution of metaplasia to estrogen-induced epidermization.

Notes: Wounds ˜ 1 mm wide were made in the middle third of the spine in both scapulae of 25 middle-aged Wistar-strain rats weighing ˜ 500 gm. Wounding of the body of the scapula was avoided. Some of the animals were injected with tetracycline, and sections from their scapulae were viewed in ultraviolet light. After one or more weeks of healing, bony callus was deposited on the dorsal surface of the blade of the scapulae in the vicinity of the wounds. Discrete deposits of bony callus were also deposited on the corresponding costal surface of the blade of the scapulae. These deposits, which were in no way connected with those on the dorsal surface, did not exhibit continuity with the wound in the spine. This observation has been interpreted as suggesting the passage of an ‘osteogenic message,’ whose nature can only be guessed, from the vicinity of the wound on the dorsal surface of the scapula to the corresponding costal surface. Lacunae of osteocytes in the scapulae, lying deep to periosteal and endosteal callus, fluoresced. This suggests that these osteocytes were activated by the trauma, and deposited mineral salts on the walls of their lacunae.

Notes: Different acinar glycogen patterns are observed in rat liver after different diets. Experiments were carried out to determine if these patterns depend on the concentration of blood glucose in the various zones of the acinus or on the direction of the blood flow. It is possible to investigate this problem by reversal of blood flow in a perfusion apparatus. Livers were perfused with Krebs-Henseleit solution, the same volume of bovine erythrocytes, and 379 mg% glucose, 2.5% albumin and different quantities of heparin. Perfusion in the normal direction is possible through the vena porta and in the reversed direction by perfusing through the vena cava. The reported results show that glycogen localization depends on the acinar enzyme pattern for glycogen synthesis rather than the direction of blood flow.

Notes: The effect of stress-induced release of ACTH or suppression of ACTH release on concentric whorls of rough endoplasmic reticulum has been studied in the adrenal gland of the Mongolian gerbil. The border zone between the zona fasciculata and reticularis appears to be a zone of transition, and stress may transform these cells into typical zona fasciculata cells. Absence (hypophysectomy) or reduction (testosterone, dexamethasone) of ACTH however causes neither atrophy nor hypertrophy of whorls. The stress of saline injections for five days caused disappearance of whorls and a rise in blood cortisol to 1.5 times that in controls. Whorls were replaced by a focal accumulation of smooth endoplasmic reticulum. Testosterone or dexamethasone as well as transaural hypophysectomy prevented the disappearance of whorls in spite of the stress of injection. Testosterone caused a 50% reduction in blood cortisol levels whereas both dexamethasone and hypophysectomy reduced blood cortisol to 25% of that in controls.

Notes: Degeneration of unknown etiology was noted in the spinal nerve roots of aging rats. These animals (Charles River CD® rats) were included in a study of long-term effects of ionizing radiation. The initial lesion was a de-myelination of an individual axon or a few, isolated, scattered axons in the ventral roots of rats between 18 and 20 months of age. As the lesion progressed, the dorsal roots showed involvement, and by 24 months the usual finding was marked degeneration in ventral roots with degenerative changes of a lesser degree in the dorsal roots. When degeneration was complete, connective tissue filled the regions normally occupied by neural tissue. The lesion was unrelated to the radiation procedure, occurring with equal frequency in both irradiated and non-irradiated animals. Furthermore, lesions occurred with equal frequency in both males and females. The one factor to which the lesion appeared to be related was age since it was not observed in animals less than 18 months old but was seen in 66.7% of those between 18 and 20 months and in 96.0% of those older than 20 months. These data indicate that this lesion needs to be considered when using aging laboratory rats.

Notes: Examination of the morphology in the submandibular salivary gland, adrenal gland and renal cortex of androgen-insensitive, tfm/ Y mutant mice has revealed predominantly female characteristics. A sexual dimorphism in alkaline-phosphatase (AP) activity was seen in the submandibular salivary gland of this strain of mice; the AP activity in the mutants was similar to females. There was no dimorphism in renal AP activity.

Notes: Based on differences in light absorption the stages of the rat seminiferous epithelial wave were identified by a transillumination technique. The most marked difference was encountered in the boundary between stages VIII and IX where the mature spermatozoa are released into the tubular lumen. Other characteristic transitions were observed between stages VI and VII as well as stages XII and XIII. All stages could be divided into four major, easily recognizable zones. Within the zones the stages could be identified with an accuracy of ± 1 stage. The characteristic changes take place in the tubular center and are due to the development of the acrosome in the spermatid. The acrosome appears to contain material with a strong light absorption capacity.Based on these observations a method was developed to isolate identified fragments of the rat seminiferous tubule in native condition. These fragments were used to quantify the activity of three previously described enzymes: specific tubular acid phosphatase III and IV as well as aminopeptidase III.The tubular acid phosphatase IV showed a high activity in stages corresponding to the final maturation process of the spermatid, whereas acid phosphatase III was evenly active in the different stages. Also aminopeptidase III showed a peak of activity during stages V-VII coincident with that of acid phosphatase IV, The possible role of these enzymes in the morphological maturation of spermatid and the destruction of the residual body is discussed.

Notes: Histochemical and autoradiographic studies using 35S-sulfate indicate that the majority of the cartilage cells in the developing mandibular condyle of the young mouse are active, vital cells. Concomitant with the increase of hypoxic conditions within the deeper layers of the cartilage, an increase in sulfated glycosaminoglucuronoglycans synthesis takes place. Hypertrophic chon-drocytes in the premineralized and mineralized zones reveal marked 35S-sulfate uptake in comparison with the less differentiated cells in the chondroblastic and perichondrial zones. These observations of radiosulfate activity support the concept that calcification processes in the condylar cartilage are not necessarily accompanied by degeneration and death of the hypertrophic chondrocytes. The radiosulfate activity of the surviving chondrocytes in the vicinity of the ossification front indicates possible modulation into osteoprogenitor cells.