Sequence and diversity of rabbit T-cell receptor gamma chain genes

The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes.The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases and have been assigned the accession numbers D38134-D38144 and D42090     

Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus

Streptomyces albidoflavus NRRL B-16746 secreted three types of chitinolytic enzymes: N -acetyl-glucosaminidase, chitobiosidase and endochitinase. Optimal activity for all three types of enzymes occurred at pH 4–6; however 55–74% of the chitobiosidase and endochitinase activity was detectable at pH 8–10. Chitobiosidase activity originated from two strongly acidic (pI < 3.0) proteins with molecular mass of 27 kDa and 34 kDa, while endochitinase activity originated from five major acidic proteins (pI 5.1, 5.3, 5.75, 5.8–5.9 and 6.4) with molecular mass of 59, 45, 38.5, 27 and 25.5 kDa. Purified chitobiosidases significantly reduced spore germination and germ tube elongation of Botrytis cinerea and Fusarium oxysporum. Chitinolytic enzymes with significant activity at pH 4–10 may be used, transgenically, to reduce the growth and/or development of a broad spectrum of insects and fungi that are major economic pests.     

Intracellular distribution of acridine derivatives in platelets and their suitability for cytoplasmic pH measurements

The site and mechanism of accumulation of acridine derivatives into platelets and their isolated organelles were investigated. In addition, their suitability as indicators of cytoplasmic pH was analysed. Direct microscopic observation showed that quinacrine and 9-aminoacridine are concentrated inside organelles in platelets. Using fractionation studies, the acridine derivatives were found to accumulate particularly in dense and α-granules. Uptake into these organelles is driven by a pH differential across their membrane (acidic inside). Because of their cellular distribution, acridine derivatives were found to be poor indicators of cytoplasmic pH. In contrast, a poorly permeant dicarboxylated fluorescein derivative, generated in situ by cytosolic enzymes, is shown to be a more reliable probe of intracellular pH. The results are compared with previous reports of the use of 9-aminoacridine as a cytoplasmic pH probe in platelets and of quinacrine as a selective dense-granule marker.     

Myelin development in infant brain

Myelin was isolated from subcortical areas of ten human brains, with ages ranging from 24 days to 350 days-of-age; samples were subsequently analyzed for lipid composition. Eight infants were victims of Sudden Infant Death Syndrome, and two infants were accident cases. Gray and white matter samples from each brain were also dissected and analyzed. Galactolipids were only 12% of the total lipids in white matter from brains of infants that were 24 days-of-age, a time when myelination was just starting in the subcortical areas. At 175 and 350 days of age, myelination was well underway and galactolipids measured 22% of the total lipids. Total phospholipids decreased (65% to 54%) in white matter during development, with the decrease mostly in phosphatidylcholine (23% to 15%). Even though there was little white matter present at early ages, myelin could be isolated. Surprisingly, the lipid composition of myelin, from the 24-day-infant brain was similar to that from adult brain. Galactolipids were 22% of the total lipids, cholesterol, 23%, and phospholipids, 52%. These results suggest that only subtle remodeling of myelin occurs in humans once myelination commences. All four major gangliosides were present in myelin during this first year of development. Interestingly, the yield of myelin from the 350-day-old infant subcortical white matter was similar to that from an adult. Thus major tracts in this area may have acquired most of the myelin by one year-after-birth. Since the control samples blend quite well into the developmental pattern obtained, it is believed there are no abnormalities in myelin lipids from SIDS infants.     

Cyanophycin granule polypeptide protease in a unicellular cyanobacterium

An assay was developed to measure the proteolysis of cyanophycin granule polypeptide in crude extracts of a unicellular cyanobacterium. The substrate was radioactively labeled cyanophycin granule polypeptide formed by an unicellular cyanobacterium grown in the presence of chloramphenicol. Substrate polypeptide displayed identical chemical properties with polypeptide isolated from non-chloramphenicol-treated cells. Solubilization of radioactivity as arginine indicated hydrolysis of polypeptide. Radioactively labeled aspartate and arginine from hydrolyzed polypeptide was related to nmol amino acid using a combination of paper chromatography, liquid scintillation analysis, and ninhydrin quantitation. Protease activity was found in extracts of nitrogen-limited cells harvested 16–24 h after a nitrogen source was added back. Optimal pH for protease activity was 8.0 and optimum temperature was 35°C. Protease activity in crude extracts followed Michaelis-Menten kinetics with a V _max of 92 nmol arginine per 15 min/mg protein and a K _m of 2.1×10³ nmol arginine. Protease activity was inhibited by arginine and by high concentrations of aspartate.     

Genetic and functional analyses of the primary in vitro CTL: Response of NZB lymphocytes toH-2-compatible cells

Spleen cells from NZB mice make an unexpected primary cytotoxic T lymphocyte (CTL) response to BALB/c cells in vitro. In this study, it is shown that this response is comprised of at least three independent components. These include a response to antigens recognized in association with H-2^d products, a response to Qa-1^b-associated antigens which is notH-2-restricted and a response directed toward antigens not associated with either H-2^d- or Qa-1^b-coded determinants. The last response appears to be the weakest of the three. In addition, cells from NZB F₁ mice which were either homozygous (Qa-1 ^a /Qa-1 ^a ) or heterozygous (Qa-1 ^a /Qa-1 ^b ) forQa-1 alleles, all responded to BALB/c cells. These data suggest that the NZB CTL response to BALB/c cells is not solely dependent on antigens coded for by genes in theH-2D-Tla region for either the sensitization or effector phases of the response. The ontogeny of the NZB anti-BALB/c CTL response coincides with that of a number of B-cell abnormalities but is shown in experiments with-suppressed NZB mice to be independent of B-cell dysfunction. Studies with (NZB x B10.D2)F₁ + B10.D2 mice demonstrated that the anti-BALB/cCTL response to antigens coded for outside ofQa-1 is governed by at least two genes. Finally, it is shown that another conventionallyH-2-restricted response, that to TNP-modified isologous cells, is neither significantly cross-reactive nor markedly elevated in NZB mice. — The foregoing observations suggest that some subsets of NZB T lymphocytes are intrinsically abnormal. The possibilities that the apparent hyperreactivity of NZB CTL precursors, evidenced in the response to BALB/c cells, is primary or results from the secondary effects of excess T-cell help are discussed.