Library

Notes: The precise mechanism for the neurotoxicity of 2, 5-hexanedione is not known, but cross-linking of neurofilament proteins has been suggested as one possibility. In this study the effects of long-term exposure to 2, 5-hexanedione were studied in the rat nervous system with special reference to regional changes in the quantities of neuronal and glial intermediate filaments. Using enzyme-linked immunosorbent assays the concentrations of 68- and 200-kDa neurofilament polypeptides were shown to be reduced in all brain regions studied. Similar results were obtained in the sciatic nerve. The concentration of glial fibrillary acidic protein was decreased in the cerebellar vermis and the dorsal cerebral cortex, whereas it was increased in the spinal cord, a result suggesting a regional variation in glial sensitivity. The intermediate filaments of the exposed animals were also immunoblotted using polyclonal antisera against the various neurofilament polypeptides and glial fibrillary acidic protein. In all tissues studied, several aggregates with molecular weights higher than those of the monomeric polypeptides were demonstrated. Contrary to clinical observations, these data indicate pronounced effects in both CNS and PNS and call for further studies on CNS effects in humans.

Notes: In this article, the preparation and characterization of polyclonal rabbit antisera against the individual polypeptides of bovine neurofilament (68, 150, and 200 kilodaltons) is described. Selected antisera against the 68- add 150-kilo-dalton neurofilament polypeptides were specific for the corresponding antigen in homogenates of bovine, rat, and human brain as judged by immunoblots. The antisera against the 200-kilodalton neurofilament polypeptide crossjreacted to some extent with the 150-kilodalton neurofilament polypeptide, especially with the human antigen. The most specific antisera were used to develop an enzyme-linked immunosorbent assay (ELISA), and the cross-reactivities between the antisera and the different bovine and rat neurofilament polypeptides were determined. Contrary to the results In the immunoblots, the antiserum against the 200-kilodalton neurofilament polypeptide was subunit-specific, as was the 150-kilodalton antiserum. The 68-kilodalton antiserum displayed a minute cross-reactivity against bovine 150- and 200-kilodalton neurofilaments, but it cross-reacted somewhat more with the rat 150- and 200-kilodalton antigens. Even so, the subunit specificity of the antisera is high enough to enable the development of a quantitative ELISA for determination of the individual bovine or rat neurofilament polypeptides in a mixture. This study is the necessary preparation for such an assay.

Notes: Abstract: In the present study, neuronal and Schwann cell marker proteins were used to biochemically characterize the spatiotemporal progress of degeneration/regeneration in the silicone chamber model for nerve regeneration. Rat sciatic nerves were transected and the proximal and distal stumps were inserted into a bridging silicone chamber with a 10-mm interstump gap. Using dot immunobinding assays, S-100 protein and neuronal intermediate filament polypeptides were measured in different parts of the nerve 0–30 days after transaction. In the most proximal nerve segment, all the measured proteins were transiently increased. In the proximal and distal stumps adjacent to the transection, the studied proteins were decreased indicating degeneration of the nerve. Within the silicone chamber, the regenerating nerve expressed the Schwann cell S-100 protein already at 7 days, whereas the neurofilament polypeptides appeared later. These observations are corroborated by previous morphological studies. The biochemical method described provides a new and fast approach to the study of nerve regeneration.

Notes: Abstract: In this article a fast HPLC technique to separate the individual neurofilament proteins is described. Highly pure fractions of the three neurofilament proteins can be obtained. As much as 50 mg of each neurofilament poly-peptide can be separated from a crude neurofilament protein preparation in one step in less than 2 h. The short separation time is of importance in minimizing degradation, especially of the 150-kilodalton neurofilament poly-peptide.

Notes: Abstract A new, simple one-step procedure [Karlsson et al. Biochem. J. 231, 201–204 (1985)] for the separation of calpains I and II was used prior to the characterization of these enzymes from rabbit brain, using alkali-denatured casein as the substrate. Enzyme activity was dependent on Ca2+ ions and free -SH groups and was maximal around pH 7.4. Incubation of calpains I and II with Ca2+ in the absence of substrate led to a rapid loss of enzyme activity. Enzyme activity was linear at room temperature and millimolar Ca2+ concentrations. However, when incubation ofcalpain I was performed with micromolar Ca2+ concentrations at room temperature proteolytic activity exhibited a lag period of approximately 10 min. This activation period was not as evident with calpain II.

Notes: A theoretical model for predicting the kinetics of ice crystallization inside cells during cryopreservation was developed, and applied to mouse oocytes, by coupling separate models of (1) water transport across the cell membrane, (2) ice nucleation, and (3) crystal growth. The instantaneous cell volume and cytosol composition during continuous cooling in the presence of glycerol were predicted using the water transport model. Classical nucleation theory was used to predict ice nucleation rates, and a nonisothermal diffusion-limited crystal-growth model was used to compute the resulting crystallization kinetics. The model requires knowledge of the nucleation rate parameters Ω and κ, as well as the viscosity η of a water-NaCl-glycerol solution as a function of both the composition and temperature of the solution. These dependences were estimated from data available in the literature. Cell-specific biophysical parameters were obtained from previous studies on mouse oocytes. A sensitivity analysis showed that the model was most sensitive to the values of κ and η.The coupled model was used to study the effect of cooling rate and initial glycerol concentration on intracellular crystal growth. The extent of crystallization, as well as the crystal size distribution, were predicted as functions of time. For rapid cooling at low to intermediate glycerol concentrations, the cells crystallized completely, while at high concentrations of glycerol, partial or total vitrification was observed. As expected, the cooling rate necessary for vitrification dropped with increasing glycerol concentration; when cells initially contained ∼7.5 M glycerol, vitrification was achieved independent of cooling rate. For slow cooling protocols, water transport significantly affected the results. At glycerol concentrations greater than ∼3 M, the final intracellular ice content decreased with increasing glycerol concentration at a fixed cooling rate. In this regime, the cooling rate at which a critical amount of ice was formed increased as more glycerol was used. When less than ∼3 M glycerol was initially present in the cell, an increase in glycerol concentration was predicted to cause an increase in the final intracellular ice content at a given cooling rate. In this regime, the critical cooling rate decreased with increasing glycerol concentration. These predictions clarify previous empirical observations of slow freezing phenomena.