Synaptically activated increases in Ca2+ concentration in hippocampal CA1 pyramidal cells are primarily due to voltage-gated Ca2+ channels.

Changes in intracellular Ca2+ concentration ([Ca2+]i) in the soma and dendrites of hippocampal CA1 pyramidal neurons were measured using intracellularly injected fura-2. A large component of the [Ca2+]i elevation caused by high frequency stimulation of the Schaffer collaterals was correlated with the Na+ spikes triggered by the excitatory postsynaptic potentials (EPSPs). These spikes were generated in the soma and proximal dendrites and stimulated Ca2+ entry through voltage-gated Ca2+ channels. Suppressing spikes by hyperpolarizing the soma or by injecting QX-314 revealed a smaller nonspike component of Ca2+ entry. A substantial fraction of this component was mediated by the action of the EPSPs on voltage-gated Ca2+ channels, because it persisted in 2-amino-5-phosphonovaleric acid and because it was usually reduced when Ca2+ channel activity was suppressed by hyperpolarization. Ca2+ entry through the N-methyl-D-aspartate receptor channel could not be detected with certainty, perhaps because it was highly localized.     

Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody.

The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene. By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no. 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised. The binding of no. 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated. Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope. No. 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them. Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c. Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host.     

Involvement of protein sulfhydryls in the trigger reaction of rhodotorucine A, a farnesyl peptide mating pheromone of Rhodosporidium toruloides.

The involvement of protein sulfhydryls for the signaling of rhodotorucine A, a mating pheromone produced by mating type A cells of Rhodosporidium toruloides, was investigated by the use of sulfhydryl compounds. The sulfhydryl-blocking reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent) strongly inhibited both the biological effect of the pheromone on the recipient cell and the hydrolysis of the pheromone, which is catalyzed by the mating type-specific surface endopeptidase of the recipient cell. Conversely, the two reactions were markedly enhanced by the presence of the reducing reagent dithiothreitol. The inhibitory effect of DTNB on the pheromone response of the recipient cell was specific to an initial stage of the differentiation; once it had initiated, the reagent had no effect on its progression. The results suggested that dithiothreitol enhances and DTNB impairs the efficiency with which the pheromone triggers sexual d differentiation. The reaction of DTNB with cellular protein sulfhydryls was highly restricted to those at the exterior surface of the membrane due to the impermeability of the reagent through the membrane. Phosphorylation of endogenous proteins, which is modulated by the pheromone added to an in vitro phosphorylation system, was also blocked by DTNB. The results showed that sulfhydryl groups are involved in the pheromone hydrolysis by the surface endopeptidase of the recipient cell and that pheromone metabolism is indispensable for the signaling reaction. We suggest that the modulation of protein phosphorylation of membrane proteins by the pheromone is an initial transmembrane response coupled to pheromone metabolism.     

Characterization of pathogenic constituents of Cryptococcus neoformans strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 X 10(5) or 1 X 10(7) viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; a cell surface structure for responsible for resistance to intra- or extracellular killing and induction of the granulomatous lesion; a growth rate suitable for interacting with phagocytic elimination.     

Mating pheromone-induced alteration of cell surface proteins in the heterobasidiomycetous yeast Tremella mesenterica.

Mating pheromone-induced alteration of the cell surface proteins of haploid cells, presumed to play crucial roles in the specific cell-cell interactions during sexual conjugation of Tremella mesenterica , was investigated. Exposed surface proteins were revealed by lactoperoxidase-catalyzed iodination in combination with polyacrylamide gel electrophoresis and autoradiography. From comparison of the molecular species of 125I-labeled surface proteins of the vegetative and the gamete (mating pheromone-treated) cells of the two compatible mating types (ab and AB), it was suggested that a striking change in cell surface structure occurs during the differentiation; although labeled protein species of the vegetative cells of the two mating types were indistinguishable, several new species, both mating type specific and nonspecific, appeared in the gamete cells. Turnover of the labeled proteins of the vegetative cells was negligible, whereas that of the gamete cells was rapid with release of low-molecular-weight labeled proteins in the medium. A role for the labeled surface proteins of the gamete cells in the cell-cell interactions during sexual conjugation was suggested by the following: the surface changes were induced by mating pheromone; the labeled proteins were preferentially localized on the surface of the mating tube; the labeled species appeared sequentially during the differentiation; and mating type-specific species were present in both mating types.     

Ultrastructure of neurofibrillary tangles in Alzheimer's disease

The ultrastructure of neurofibrillary tangles (NFT) was examined by electron microscopy. The fibrils of NFT seemed to consists of about eight protofilaments consisting of globular subunits; these protofilaments were helically wound in a longitudinal direction. The fibrils of NFT had hollow structures at their centers surrounded by the eight globular subunits. The subunits were tightly connected in the narrow parts of the fibril, but more loosely connected in the wider parts. From these findings, it seemed that the fibrils of NFT consist of a twisted tubule having periodical constrictions and is made up of eight helically wound protofilaments, forming globular subunits.