Effects of phalloidin on K+-dependent, Ca2+-independent neurotransmitter efflux and K+-facilitated, Ca2+-dependent neurotransmitter release.

Phalloidin tightly binds to actin and converts soluble actin into depolymerization-resistant actin filaments. Phalloidin promotes the potassium-dependent, calcium-independent efflux of γ-amino butyric acid and nore-pinephrine from synaptosomes but inhibits the potassium-facilitated, calcium-dependent release of these neurotransmitters. This suggests that an actomyosin system is involved in synaptic transmission.     

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity-purified anti-P₂ and anti-myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P₂ protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P₂ protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high-molecular-weight proteins were also detected with anti-myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high-molecular-weight P₂ cross-reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.     

Structural diversity of the classical H-2 genes: K, D, and L.

Twenty-three class I DNA sequences, representing alleles of the H-2K, D, and L loci, were analyzed to assess patterns of nucleotide and amino acid diversity. Comparisons of the allelic and nonallelic sequences revealed locus specificity in regions encoding the leader peptides and the carboxyl-terminal segments of the Ag presenting molecules. Analyses focusing on the sequences that determine the Ag binding domains revealed weak or insignificant allelic associations, a finding that is in sharp contrast to previously observed relationships among the homologous human sequences. The amino acid positions exhibiting high diversity in the encoded glycoproteins in both mice and humans are localized primarily to the Ag binding site. In the mouse, diverse amino acids were positioned similarly in the K and D/L glycoproteins, although in humans, the A and B glycoproteins exhibit distinctive differences in their locations within the Ag binding site. The absence of locus specificity among the sequences that determine the Ag binding domains of the mouse is consistent with the hypothesis that ectopic gene conversion leads to interlocus exchange of class I sequences. Comparable interlocus exchanges among human class I genes have not played a similar role in shaping human A and B sequences. The basis of this difference between mice and humans is not clear. The nature of amino acid substitutions distinguishing class I loci in mice and humans are comparable, and the role of natural selection in determining diversity appears to be similar in the two species.     

PCR assay for detection and differentiation of K88ab1, K88ab2, K88ac, and K88ad fimbrial adhesins inE. coli strains isolated from diarrheic piglets

Primers were designed and prepared and conditions were determined for PCR detection and differentiation of enterotoxigenic E. coli bacterial strains isolated from diarrheic pigs. Primers K88/1 and K88/2 are 25 bp oligomers that correspond to a region of genes encoding one of serological variants of the K88 antigen (K88ab(1), K88ab(2), K88ac or K88ad). A positive result of PCR is an amplificate of 792 bp in size for K88ab and K88ad variant or 786 bp for K88ac variant. The individual serological variants of genes of the K88 antigen could be differentiated by cutting the obtained PCR amplificates by restriction endonucleases. The PCR analysis of 674 E. coli strains isolated from diarrheic pigs showed that 184 strains were K88 positive. By using restriction endonucleases the K88-positive strains were in 4 cases classified as K88ab variant, 180 as K88ac variant and none contained gene for the K88ad variant. Ninety-five % coincidence with serological examination using K88ab, K88ac and K88ad specific antibodies was shown.     

Indicain, a dimeric serine protease from Morus indica cv. K2

A high molecular mass serine protease has been purified to homogeneity from the latex of Morus indica cv. K2 by the combination of techniques of ammonium sulfate precipitation, hydrophobic interaction chromatography, and size-exclusion chromatography. The protein is a dimer with a molecular mass of 134.5 kDa and with two monomeric subunits of 67.2 kDa and 67.3 (MALDI-TOF), held by weak bonds susceptible to disruption on exposure to heat and very low pH. Isoelectric point of the enzyme is pH 4.8. The pH and temperature optima for caseinolytic activity were 8.5 and 80 degrees C, respectively. The extinction coefficient (epsilon280(1%)) of the enzyme was estimated as 41.24 and the molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residues. The enzyme activity was inhibited by phenylmethylsulfonylflouride, chymostatin and mercuric chloride indicating the enzyme to be a serine protease. The enzyme is fairly stable and similar to subtilases in its stability toward pH, strong denaturants, temperature, and organic solvents. Polyclonal antibodies specific to enzyme and immunodiffusion studies reveal that the enzyme has unique antigenic determinants. The enzyme has activity towards broad range of substrates comparable to those of subtilisin like proteases. The N-terminal residues of indicain (T-T-N-S-W-D-F-I-G-F-P) exhibited considerable similarity to those of other known plant subtilases, especially with cucumisin, a well-characterized plant subtilase. This is the first report of purification and characterization of a subtilisin like dimeric serine protease from the latex of M. indica cv. K2. Owing to these unique properties the reported enzyme would find applications in food and pharma industry.