Structure of acidic polysaccharide from cell wall of Propionibacterium acnes strain C7

The structure of polysaccharide prepared by lysozyme digestion from the cell wall of Propionibacterium acnes strain C7 was examined. The polysaccharide fraction was composed of glucose, galactose, mannose, galactosamine, and diaminomannuronic acid in a molar ratio of 1:1:0.3:1:2. By Smith degradation of the polysaccharide, diaminouronic acid-containing fractions were obtained, and the configuration of diaminouronic acid was identified as 2,3-diacetamido-2,3-dideoxymannuronic acid [Man(NAc)2A] by means of 1H-NMR and 13C-NMR spectroscopic analyses. The results of analyses involving methylation and partial acid hydrolysis led to the conclusion that the polysaccharide has the repeating unit----6)Gal(alpha 1----4)Man(NAc)2A(beta 1----6)Glc(alpha 1----4)Man(NAc)2A (beta 1----3)GalNAc(beta 1--. In addition, a portion of the galactose residues were substituted at C-4 by alpha 1----2 linked mannotriose.     

Streptomyces hygroscopicus has two glutamine synthetase genes.

Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII). The glnB gene was cloned from S. hygroscopicus DNA by complementation in an Escherichia coli glutamine auxotrophic mutant (glnA). glnB was subcloned in Streptomyces plasmids by insertion into pIJ486 (pMSG3) and pIJ702 (pMSG5). Both constructions conferred resistance to the tripeptide form of phosphinothricin (bialaphos) and were able to complement a glutamine auxotrophic marker in S. coelicolor. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of S. lividans(pMSG5) revealed a highly overexpressed 40-kilodalton protein. When GS was purified from this strain, it was indistinguishable in apparent molecular mass from the 40-kilodalton protein. The nucleic acid sequence of the cloned region contained an open reading frame which encoded a protein whose size, amino acid composition, and N-terminal sequence corresponded to those of the purified GS. glnB had a high G + C content and codon usage typical of streptomycete genes. A comparison of its predicted amino acid sequence with the protein data bases revealed that it encoded a GSII-type enzyme which had previously been found only in various eucaryotes (47 to 50% identity) and nodulating bacteria such as Bradyrhizobium spp. (42% identity). glnB had only 13 to 18% identity with eubacterial GSI enzymes. Southern blot hybridization experiments showed that sequences similar to glnB were present in all of the five other Streptomyces species tested, as well as Frankia species. These results do not support the previous suggestion that GSII-type enzymes found in members of the family Rhizobiaceae represent a unique example of interkingdom gene transfer associated with symbiosis in the nodule. Instead they imply that the presence of more than one gene encoding GS may be more common among soil microorganisms than previously appreciated.     

Effects of YM264, a novel PAF antagonist, on puromycin aminonucleoside-induced nephropathy in the rat

We investigated the effects of YM264, WEB2086, methylprednisolone and ticlopidine on puromycin-induced nephropathy in the rat. Puromycin produces marked proteinuria, hypercholesterolemia, and hypoalbuminemia. The structurally differing PAF antagonists YM264 and WEB2086 inhibited proteinuria and improved hypercholesterolemia and hypoalbuminemia. Methylprednisolone also exhibited a beneficial effect on these variables. However, ticlopidine, a platelet inhibitor, showed no inhibitory effect on nephropathy. These results indicate that PAF may play a major role in puromycin-induced nephropathy in the rat, and that PAF antagonists may prove of therapeutic value in the treatment of nephropathy in humans.     

High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper

We report here an improved protocol for the preparation of full-lengthcDNA libraries that improves the previously reported method(Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics,137, 327–336), that allows long cDNAs to be cloned moreefficiently. One potential disadvantage of the original biotinylatedCAP trapper protocol is the exposure of mRNA to chemical andenzymatic attacks during the biotinylation of the cap structure,before the first-strand cDNA synthesis (and selection of full-lengthcDNA by biotinylated cap). Here, we show that the biotinylationof the cap structure is very specific and effective even ifbiotinylation is performed on the mRNA/cDNA hybrid producedby the first-strand cDNA synthesis reaction. Consequently, mRNAremains protected from chemical and enzymatic degradation duringthe overnight biotinylation step, thus making it possible toselect full-length cDNAs of longer average size. We herein reportthe efficiency and specificity of the new version of the protocolfor cap structure biotinylation and capture of full-length cDNA.     

Cell growth and morphology of Dictyostelium discoideum in space environment.

Two strains of cellular slime mold Dictyostelium discoideum, a radiation-sensitive mutant and the parental wild-type strain, were used to investigate the effects of microgravity and/or cosmic radiation on their morphology through the whole life span from spores to fruiting bodies for about 7 days in space shuttle of NASA. We found almost no effect of space environment on amoeba cell growth in both strains. It was also observed that almost the same number and shape of fruiting bodies in space compared to the control experiments on earth. These results suggest that there is little effect of microgravity and space radiation on germination, cell aggregation, cell differentiation and cell morphology in the cellular slime mold.     

Space radiation enhancement linked to geomagnetic disturbances.

Space radiation dosimetry measurements have been made on board the Space Shuttle. A newly developed active detector called "Real-time Radiation Monitoring Device (RRMD)" was used (Doke et al., 1995; Hayashi et al., 1995). The RRMD results indicate that low Linear Energy Transfer (LET) particles steadily penetrate around the South Atlantic Anomaly (SAA) without clear enhancement of dose equivalent and some daily periodic enhancements of dose equivalent due to high LET particles are seen at the lower geomagnetic cutoff regions (Doke et al., 1996). We also have been analyzing the space weather during the experiment, and found that the anomalous high-energy particle enhancement was linked to geomagnetic disturbance due to the high speed solar wind from a coronal hole. Additional analysis and other experiments are necessary for clarification of these phenomena. If a penetration of high-energy particles into the low altitude occurs by common geomagnetic disturbances, the prediction of geomagnetic activity becomes more important in the next Space Station's era.     

Effects of space flight on the histological characteristics of the aortic depressor nerve in the adult rat: electron microscopic analysis.

The effects of microgravity on the histological characteristics of the aortic depressor nerve, which is the afferent of the aortic baroreflex arc, were determined in 10 female adult rats. The rats were assigned for nursing neonates in the Space Shuttle Columbia or in the animal facility on the ground (NASA Neurolab, STS-90), and were housed for 16 days under microgravity in space (microg, n=5) or under one force of gravity on Earth (one-g, n=5). In the Schwann cell unit in which the axons of unmyelinated fibers are surrounded by one Schwann cell, the average number of axons per unit in the microg group was 2.1 +/- 1.6 (mean +/- SD, n=312) and significantly less than that in the one-g group (3.0 +/- 2.9, n=397, p<0.05). The proportion of unmyelinated fibers in the aortic depressor nerve in the microg group was 64.5 +/- 4.4% and significantly less than that in the one-g group (74.0 +/- 7.3%, p<0.05). These results show that there is a decrease in the number of high-threshold unmyelinated fibers in the aortic depressor nerve in adult rats flown on the Shuttle Orbiter, suggesting that the aortic baroreflex is depressed under microgravity during space flight.     

Organization of variable region segments of the human immunoglobulin heavy chain: duplication of the D5 cluster within the locus and interchromosomal translocation of variable region segments.

We have studied the organization of variable region (V) genes of the human immunoglobulin heavy chain (H) by cosmid cloning. We isolated two independent immunoglobulin D5 clusters (D5-a and D5-b) from cosmid libraries of the human genome. Restriction maps of these two regions showed that downstream 15 kb portions of the 55 kb overlap were different although upstream 40 kb portions were almost identical. Four more D segments, (DM, DXP, DA and DK) were found around the D5 segment in the conserved region of each cluster. Nucleotide sequences of the corresponding D segments from each cluster were almost identical and they encoded potentially functional D regions. Analysis using human-rodent somatic cell hybrids demonstrated that both clusters were located in the immunoglobulin heavy chain (H) locus on chromosome 14, suggesting that the D5-a and D5-b regions evolved by internal duplication within this locus. We also isolated a 60 kb DNA region carrying four VH segments, designated as VH-F region, which was located on chromosome 16. Nucleotide sequences of the four VH segments were determined. Two of them encoded potentially functional VH segments, and the other two were pseudogenes. Some more VH segments were found to be located outside chromosome 14, by Southern blot hybridization of human-rodent hybrid cell DNAs. These results provide further evidence that the human VH locus has undergone recent reorganization.