Confirmation of a gene for multiple sclerosis (MS) to chromosome region 19q13.3

To identify the chromosomal localizations of the multiple sclerosis (MS) genes, we conducted a genomewide linkage analysis using eighteen affected families. A MS gene is linked to markers located in the 19q13.3 region (multipoint lod-score = 2.1). Apolipoprotein E (ApoE) gene, located in this region, is an excellent candidate gene for MS because the ApoEe4 allele is acting as a severity allele in the disease.     

The catalytic mechanism of glucose 6-phosphate dehydrogenases: assignment and 1H NMR spectroscopy pH titration of the catalytic histidine residue in the 109 kDa Leuconostoc mesenteroides enzyme

The chemical shifts of the C(epsilon1) and C(delta2) protons of His-240 from the 109 kDa Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) were assigned by comparing 1H and 13C spectra of the wild-type and mutant G6PDs containing the His-240 to asparagine mutation (H240N). Unambiguous assignment of the His-240 1H(epsilon1) resonance was obtained from comparing 13C-1H heteronuclear multiple quantum coherence NMR spectra of wild-type and H240N G6PDs that were selectively labeled with 13C(epsilon1) histidine. The results from NOESY experiments with wild-type and H240N variants were consistent with these assignments and the three-dimensional structure of G6PD. pH titrations show that His-240 has a pK(a) of 6.4. This value is, within experimental error, identical to the value of 6.3 derived from the pH dependence of kcat [Viola, R. E. (1984) Arch. Biochem. Biophys. 228, 415-424], suggesting that the pK(a) of His-240 is unperturbed in the apoenzyme despite being part of a His-Asp catalytic dyad. The results obtained for this 109 kDa enzyme indicate that 1H NMR spectroscopy in combination with heteronuclear methods can be a useful tool for functional analysis of large proteins.     

Grass group I pollen allergens (beta-expansins) lack proteinase activity and do not cause wall loosening via proteolysis.

Group I grass pollen allergens make up a subgroup of the beta-expansin family of cell wall loosening proteins in plants. A recent study reported that recombinant Phl p 1, the group I allergen from timothy grass pollen, was associated with papain-like proteinase activity and suggested that expansins loosen the plant cell wall via proteolysis. We tested this idea with three experimental approaches. First, we evaluated three purified native group I allergens from timothy grass, ryegrass and maize (Phl p 1, Lol p 1, Zea m 1) using five proteinase assays with a variety of substrates. The proteins had substantial wall loosening activity, but no detectable proteolytic activity. Thus we cannot confirm proteolytic activity in the pollen allergen class of beta-expansins. Second, we tested the ability of proteinases to induce cell wall extension in vitro. Tests included cysteine proteinases, serine proteinases, aspartic proteinases, metallo proteinases, and aggressive proteinase mixtures, none of which induced wall extension in vitro. Thus, wall proteins are unlikely to be important load-bearing components of the plant cell wall. Third, we tested the sensitivity of beta-expansin activity and native wall extension activity to proteinase inhibitors. The results show that a wide range of proteinase inhibitors (phenylmethanesulfonyl fluoride, N-ethylmaleimide, iodoacetic acid, Pefabloc SC, and others) inhibited neither activity. From these three sets of results we conclude proteolysis is not a likely mechanism of plant cell wall loosening and that the pollen allergen class of beta-expansins do not loosen cell walls via a proteolytic mechanism.     

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.      

Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O- sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.      

Water uptake by growing cells: an assessment of the controlling roles of wall relaxation, solute uptake, and hydraulic conductance

Growing plant cells increase in volume principally by water uptake into the vacuole. There are only three general mechanisms by which a cell can modulate the process of water uptake: (a) by relaxing wall stress to reduce cell turgor pressure (thereby reducing cell water potential), (b) by modifying the solute content of the cell or its surroundings (likewise affecting water potential), and (c) by changing the hydraulic conductance of the water uptake pathway (this works only for cells remote from water potential equilibrium). Recent studies supporting each of these potential mechanisms are reviewed and critically assessed. The importance of solute uptake and hydraulic conductance is advocated by some recent studies, but the evidence is indirect and conclusions remain controversial. For most growing plant cells with substantial turgor pressure, it appears that reduction in cell turgor pressure, as a consequence of wall relaxation, serves as the major initiator and control point for plant cell enlargement. Two views of wall relaxation as a viscoelastic or a chemorheological process are compared and distinguished.     

Translation of mRNA associated with monosomes and residual polysomes following disaggregation of brain polysomes by LSD and hyperthermia

Intravenous administration of LSD to young adult rabbits induces a transient disaggregation of brain polysomes and a relocalization of mRNA from polysomes to monosomes. To analyze the spectrum of mRNA molecules which were associated with either the residual polysomes or the translationally inactive monosome complex, these two fractions were isolated on sucrose gradients and translated in a reticulocyte cell-free system. Analysis of [³⁵S]methionine labeled translation products by one and two dimensional gel electrophoresis revealed that a full spectrum of mRNA molecules was relocalized from polysomes to monosomes following drug induced polysome disaggregation. The only exception was the mRNA coding for the LSD-induced 74K protein which was associated with the residual polysome fraction and not with the monosome complex. This brain protein is similar in molecular weight to one of the major heat shock proteins which are induced in tissue culture cells following elevation of ambient temperature and disaggregation of existing polysomes. The mRNA coding for the 74K brain protein was not observed in polysomes isolated following blockage of LSD-induced hyperthermia but it was noted when hyperthermia was induced by elevation of ambient temperature. The mRNA species coding for the 74K protein was polyadenylated.     

洛南盆地1995-1999年野外地点发现的石制品

本文研究了1995-1999年期间在洛南盆地南洛河及其支流两侧阶地发现的50处旷野类型旧石器地点的1751件石制品.这些野外地点分布在第2级阶地者41处,第3级阶地4处,第4级阶地3处,另外还有2处分布于较高的第5级古老的阶地上.石制品的统计分析显示洛南盆地野外地点是以大中型石片和第二次加工修理的大型石片及砾石工具为代表的、两面加工技术发达的旧石器时代早期文化.     

Analysis of peg formation in cucumber seedlings grown on clinostats and in a microgravity (space) environment.

In young cucumber seedlings, the peg is a polar out-growth of tissue that functions by snagging the seed coat, thereby freeing the cotyledons. Previous studies have indicated that peg formation is gravity dependent. In this study we analyzed peg formation in cucumber seedlings (Cucumis sativus L. cv Burpee Hybrid II) grown under conditions of normal gravity, microgravity, and simulated microgravity (clinostat rotation). Seeds were germinated on the ground, in clinostats and on board the space shuttle (STS 95) for 1-2 days, frozen and subsequently examined for their stage of development, degree of hook formation, number of pegs formed, and peg morphology. The frequency of peg formation in space grown seedlings was found to be nearly identical to that of clinostat grown seedlings and to differ from that of seedlings germinated under normal gravity only in a minority of cases; approximately 6% of the seedlings formed two pegs and nearly 2% of the seedlings lacked pegs, whereas such abnormalities did not occur in ground controls. The degree of hook formation was found to be less pronounced for space grown seedlings, compared to clinostat grown seedlings, indicating a greater degree of decoupling between peg formation and hook formation in space. Nonetheless, in all seedlings having single pegs and a hook, the peg was found to be positioned correctly on the inside of the hook, showing that there is coordinate development even in microgravity environments. Peg morphologies were altered in space grown samples, with the pegs having a blunt appearance and many pegs showing alterations in expansion, with the peg extending out over the edges of the seed coat and downwards. These phenotypes were not observed in clinostat or ground grown seedlings.