Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays

Efficient construction of BAC-based human artificial chromosomes (HACs) requires optimization of each key functional unit as well as development of techniques for the rapid and reliable manipulation of high-molecular weight BAC vectors. Here, we have created synthetic chromosome 17-derived alpha-satellite arrays, based on the 16-monomer repeat length typical of natural D17Z1 arrays, in which the consensus CENP-B box elements are either completely absent (0/16 monomers) or increased in density (16/16 monomers) compared to D17Z1 alpha-satellite (5/16 monomers). Using these vectors, we show that the presence of CENP-B box elements is a requirement for efficient de novo centromere formation and that increasing the density of CENP-B box elements may enhance the efficiency of de novo centromere formation. Furthermore, we have developed a novel, high-throughput methodology that permits the rapid conversion of any genomic BAC target into a HAC vector by transposon-mediated modification with synthetic alpha-satellite arrays and other key functional units. Taken together, these approaches offer the potential to significantly advance the utility of BAC-based HACs for functional annotation of the genome and for applications in gene transfer.     

Effects of altitude, ethnicity-religion, geographical distance, and occupation on adult anthropometric characters of eastern Himalayan populations

With a view to estimating the effect of altitude on body dimensions vis-à-vis ethnicity-religion, geographical distance, and occupation, a comprehensive multivariate statistical analysis was performed on data pertaining to 16 anthropometric characters collected from 1,103 individuals (643 males and 460 females) belonging to two ethnic groups-Sherpa and Lepcha. Samples were drawn from several locations in the eastern Himalayan region-Darjeeling and Kalimpong in West Bengal (India), and Nepal, situated at low (1,000-2,000 meters) and high (above 3,500 meters) altitudes. The individuals sampled practice different occupations and follow different religions. Significant age and sex effects were observed. The data were age-adjusted, and sexes were treated separately. A test of equality of mean vectors indicated heterogeneity among population groups. Almost all characters were found to contribute significantly to the ability to discriminate between the groups. The overall probability of correctly classifying an individual based on body dimensions into the group in which she or he actually belongs was high (between 0.64 and 0.77). Shape and size factors could be identified that explained about 50% of the total variance and yielded a reasonable separation of the groups. Results of four different types of multivariate statistical analyses were in agreement, and showed that altitude is most highly associated with body dimensions.     

Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells.

Upon exposure to lysates or supernatants of necrotic transformed cell lines, human dendritic cells (DCs) undergo maturation. In contrast, DCs exposed to apoptotic transformed cell lines or necrotic lysates of primary cells remain immature. Analysis of supernatants of necrotic transformed cell lines showed them to be enriched in the heat shock proteins (hsp)70 and gp96, in contrast to supernatants of primary cells. Likewise, cells from a variety of primary human tumors contained considerably higher levels of hsp than their normal autologous tissue counterparts. Of the majority of human tumors enriched in hsps (hsp70 and/or gp96), their corresponding lysates matured DCs. The maturation effect of tumor cell lysates was abrogated by treatment with boiling, proteinase K, and geldanamycin, an inhibitor of hsps, suggesting that hsps rather than endotoxin or DNA were the responsible factors. Supporting this idea, highly purified, endotoxin-depleted hsp70, induced DC maturation similar to that seen with standard maturation stimuli LPS and monocyte conditioned medium. These results suggest that the maturation activity inherent within tumor cells and lines is mediated at least in part by hsps. The release of hsps in vivo as a result of cell injury should promote immunity through the maturation of resident DCs.     

Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD₅₀ = 40 μM tellurite, 40 min exposure). The organism reduced 70% of 40 μM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status. The level of ROS and protein carbonyl contents increased while the cellular reduction potential substantially decreased following tellurite exposure. Cellular thiols levels initially increased (within 30 min) of tellurite exposure but decreased at later time points. At proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes (superoxide dismutase and thioredoxin reductase), and protein folding chaperones (DnaK, EF‐Ts, and PPIase) displayed increased abundance in tellurite‐stressed cells. However, remarkably decreased levels of key metabolic enzymes (aconitase, transketolase, 3‐hydroxy acyl‐CoA dehydrogenase, acyl‐CoA dehydrogenase, electron transfer flavoprotein alpha, and beta) involved in carbon and energy metabolism were observed upon tellurite stress. The results demonstrate that depletion of reduction potential in intensive tellurite reduction with impaired energy metabolism lead to tellurite toxicity in D. radiodurans.     

Auxin activity of 3-methyleneoxindole in wheat

A product of the enzymatic oxidation of indole-3-acetic acid, 3-methyleneoxindole, is at least 50-fold more effective than indole-3-acetic acid in stimulating the growth of wheat (Triticum vulgare, red variety) coleoptiles. Ethylenediaminetetra-acetic acid can antagonize the growth-stimulating properties of the parent compound, indole-3-acetic acid, presumably by chelating Mn²⁺, which is required for the enzymatic oxidation of indole-3-acetic acid. The growth stimulating effect of 3-methyleneoxindole, a product of the blocked reaction, on the other hand, is still evident in the presence of ethylenedia-minetetraacetic acid. In the presence of 2-mercaptoethanol, indole-3-acetic acid fails to stimulate the elongation of wheat coleoptiles. The property of binding to sulfhydryl compounds including 2-mercaptoethanol is unique to 3-methyleneoxindole among indole-3-acetic acid and its oxidation products. These findings suggest that 3-methyleneoxindole is an obligatory intermediate in indole-3-acetic acid induced elongation of wheat coleoptiles.     

Characterization of the phospholipid requirement of a rat liver beta-glucosidase.

The lipid requirement of membrane-bound rat liver beta-glucosidase was investigated using 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. The enzyme was solubilized and delipidated by sequential extraction of a crude lysosomal fraction from rat liver lysosomes with sodium cholate and ice-cold butan-1-ol. Neither saturated nor unsaturated phosphatidylcholine activated this enzyme. In contrast, acidic phospholipids like phosphatidylglycerol (PtdGro) and phosphatidylserine (PtdSer) were effective activators. For the PtdGro series, fatty acid composition was important, with the shorter chain or unsaturated fatty acid-containing PtdGro species being the best activators. Heat-stable factor (HSF) from Gaucher spleen by itself (1-2 micrograms) had no effect on enzyme activity. However, the same amount of HSF when combined with 10 micrograms of PtdSer markedly stimulated beta-glucosidase activity. In the presence of HSF, di-9-cis-octadecenoyl-PtdGro (1 microgram) or -PtdSer (5 micrograms) provided maximum protection of beta-glucosidase against heat (60 degrees C) inactivation. In the absence of phospholipids, HSF had no effect on the rate of inactivation of the enzyme by the suicide inhibitor conduritol B epoxide (t0.5, 12 +/- 0.5 min); the maximum rate of inactivation was achieved in the presence of a mixture of PtdGro (2.5-5 micrograms) and HSF (t0.5, 2.8 min). The combination of PtdSer (10 micrograms) and HSF (1.3 micrograms) lowered the Km for 4-methylumbelliferyl-beta-D-glucopyranoside from 24 to 2.7 mM. Inhibition of the enzyme by the glucocerebrosidase substrate analogues N-hexyl-O-glucosylsphingosine and glucosylsphingosine was influenced by the activator substances. The inclusion of PtdSer and HSF in the beta-glucosidase assay medium lowered the Ki of N-hexyl-O-glucosylsphingosine 20-fold. The same combination of activators decreased the I0.5 of the enzyme for glucosylsphingosine from 89.4 to 7.6 microM. A study of log (Vmax./Km) versus pH indicated that the PtdSer-HSF pair creates the active site of beta-glucosidase, making apparent three ionizable groups on the enzyme with pK values in the range 4.5-5.1.