Effects of solution polarity and viscosity on peptide deamidation.

Deamidation kinetics were measured for a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0-50% w/w) and poly(vinyl pyrrolidone) (PVP, 0-20% w/w) at 37 degrees C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo-first order deamidation rate constants, k(obs), decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities >13 cp. Values of k(obs) also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side-chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k(obs). Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.     

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. Significance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria.     

Iron plaque enhances phosphorus uptake by rice (Oryza sativa) growing under varying phosphorus and iron concentrations

Both solution culture and pot experiments were performed to investigate (a) the effects of external Fe (II) concentrations and forms on the formation of iron plaque on the roots of rice (Oryza sativa) and subsequent P adsorption on iron plaque and shoot P concentrations and (b) the effects of soil moisture regimes on the formation of iron plaque and P adsorption on root surfaces and P accumulation in shoots. The results showed that iron plaque was significantly increased with increasing Fe²⁺ concentrations in the solution culture. The amounts of P adsorbed on the iron plaque were increased significantly with external Fe²⁺ concentrations. Although shoot P concentration was not significantly affected by Fe²⁺ treatment after incubation for 2 days, it was significantly increased in the Fe‐treated plants compared with Fe‐deprived ones after incubation for 4 days. Soil culture experiment showed that the formation of iron plaque on root surfaces was promoted by exogenous iron, with greater amount of iron plaque being formed by addition of ferric hydroxide than of ferric oxide. Phosphorus adsorption on iron plaque also increased with the addition of iron oxides, and increasing soil P increased the amounts of P associated with the iron plaque and shoot P concentration. The amounts of iron plaque were almost sixfold higher under flooding condition than under field capacity condition. Plants pretreated under flooding condition generally had higher shoot P concentrations when they were transplanted to solutions with varying P levels, and this was most pronounced in the treatment with highest solution P concentration. The results suggest that iron plaque acts as a nutrient reservoir for phosphorus in the rhizosphere and helps enhance P acquisition by rice.     

BJ-HCC-20, a potential novel cancer-testis antigen.

In an effort to identify novel Cancer-Testis genes, we analyzed the sequence in the q26-28 region of human X chromosome by several on-line tools. The candidate sequences were then confirmed by experiments. We have obtained a novel Cancer-Testis gene, BJ-HCC-20. In vivo, it was found to have two isoforms. In samples of liver, colon, gastric and lung cancer tested, the expression frequency of BJ-HCC-20 is 25%, 17%, 21% and 15%, respectively. Full-length cDNAs of both BJ-HCC-20 isoforms were isolated and their gene structures and promoter regions were characterized. BJ-HCC-20 might have implications in theoretical and practical tumor biology.     

Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules

Mammalian cells express a spectrum of tubulin isotypes whose relationship to the diversity of microtubule function is unknown. To examine whether different isotypes are segregated into functionally distinct microtubules, we generated immune sera capable of discriminating among the various naturally occurring beta-tubulin isotypes. Cloned fusion proteins encoding each isotype were used first to tolerogenize animals against shared epitopes, and then as immunogens to elicit a specific response. In experiments using these sera, we show that there is neither complete nor partial segregation of beta-tubulin isotypes: both interphase cytoskeletal and mitotic spindle microtubules are mixed copolymers of all expressed beta-tubulin isotypes. Indeed, a highly divergent isotype normally expressed only in certain hematopoietic cells is also indiscriminately assembled into all microtubules both in their normal context and when transfected into HeLa cells.     

Metabolism of glucose by fetus and placenta of sheep. The effects of normal fluctuations in uterine blood flow

The metabolism by the fetus and placenta of [2-3H, U-14C]glucose infused into fetal sheep has been studied. Uptake of glucose from the fetus by the placenta and transfer to the ewe, as well as placental metabolism of glucose to fructose and lactate have been quantified. About two-thirds of the glucose removed from the fetal circulation was taken up by placenta. Less than 15% of this passed back into the maternal circulation, the remainder was converted, at roughly equivalent rates, into lactate and fructose, most of which was transferred back to the fetus. It seems likely that little of this glucose is oxidised by the placenta. This data indicates that there are substrate cycles between the placenta and fetus, one possible function of which is to limit fetal glucose loss back to the mother; lactate and fructose have limited placental permeability. At uterine blood flow rates in the middle of the normal range net glucose uptake by the placenta from the maternal circulation was about 7-fold higher than that from the fetus. About 20% of this was transported to the fetus, 50% was oxidised and much of the remainder converted to lactate and transferred back to the ewe. Labelling patterns in fructose and lactate make it unlikely that this placental pool of glucose mixes freely with that derived from uptake from the fetus. Net movement of glucose across the placenta is markedly influenced by fluctuations in uterine blood flow over the normal range of 500-3000 ml/min. At low flow rates there is net output of glucose from the fetus to the placenta, and in some instances from the placenta to the ewe, i.e. there is evidence of net utero-placental production of glucose to the ewe separate from output by the fetus. There is a close linear relationship between uterine glucose supply (maternal arterial concentration x uterine blood flow) and net balance across the placenta. As uterine supply of glucose falls there is increased uptake by the placenta of glucose from the fetal circulation and corresponding enhanced recycling of fructose and lactate to the fetus. This production of fructose and lactate by the placenta may function to reduce glucose loss from the fetus to the ewe. Hence at high rates of placental uptake of glucose from the fetus placental production of lactate and particularly fructose may approach saturation and allow significant backflow of glucose from the fetus to the ewe. Under these conditions glucose uptake may in part sustain placental oxygen consumption.     

The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor.

The leukemogenic membrane glycoprotein of Friend spleen focus-forming virus (SFFV) has an apparent Mr of 55,000 (gp55), is encoded by a recombinant env gene, and occurs on cell surfaces and in intracellular organelles. There is evidence that the amino-terminal region of gp55 forms a dualtropic-specific domain that is connected to the remainder of the glycoprotein by a proline-rich linker (C. Machida, R. Bestwick, B. Boswell, and D. Kabat, Virology 144:158-172, 1985). Using the colinear form of a cloned polycythemic strain of SFFV proviral DNA, we constructed seven in-phase env mutants by insertion of linkers and by a deletion. The mutagenized SFFVs were transfected into fibroblasts and were rescued by superinfection with a helper murine leukemia virus. Four of the mutants cause erythroblastosis. These include one with a 6-base-pair (bp) insert in the ecotropic-related sequence near the 3' end of the gene, two with a 12- or 18-bp insert in the region that encodes the proline-rich linker, and one with a 6-bp insert in the dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific sequences that are highly conserved among strains of SFFV. A pathogenic revertant (RI-rev) was isolated from one mouse that developed erythroblastosis 3 weeks after infection with RI. RI-rev contains a second-site env mutation that affects the same domain as the primary mutation does and that increases the size of the encoded glycoprotein. All pathogenic SFFVs encode glycoproteins that are expressed on cell surfaces, whereas the nonpathogenic glycoproteins are exclusively intracellular. The pathogenic SFFVs also specifically cause a weak interference to superinfection by dualtropic MuLVs. These results are compatible with the multidomain model for the structure of gp55 and suggest that processing of gp55 to plasma membranes is required for pathogenesis. The amino-terminal region of gp55 binds to dualtropic murine leukemia virus receptors, and this interaction is preserved in the SFFV mutants that cause erythroblastosis.     

Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity.

The role of Epstein-Barr virus (EBV) early antigen diffuse component (EA-D) and its relationship with EBV DNA polymerase in EBV genome-carrying cells are unclear, EBV-specified DNA polymerase was purified in a sequential manner from Raji cells treated with phorbol-12,13-dibutyrate and n-butyrate by phosphocellulose, DEAE-cellulose, double-stranded DNA-cellulose, and blue Sepharose column chromatography. Four polypeptides with molecular masses of 110,000, 100,000, 55,000, and 49,000 daltons were found to be associated with EBV-specified DNA polymerase activity. A monoclonal antibody which could neutralize the EBV DNA polymerase activity was prepared and found to recognize 55,000- and 49,000-dalton polypeptides. An EA-D monoclonal antibody, R3 (G. R. Pearson, V. Vorman, B. Chase, T. Sculley, M. Hummel, and E. Kieff, J. Virol. 47:183-201, 1983), was also able to recognize these same two polypeptides associated with EBV DNA polymerase activity. It was concluded that EBV EA-D polypeptides, as identified by R3 monoclonal antibody, are critical components of EBV DNA polymerase.     

Xanthophylls and abscisic Acid biosynthesis in water-stressed bean leaves

Experiments were designed to obtain evidence about the possible role of xanthophylls as abscisic acid (ABA) precursors in water-stressed leaves of Phaseolus vularis L. Leaves were exposed to ¹⁴CO₂ and the specific activities of several major leaf xanthophylls and stress-induced ABA were determined after a chase in ¹²CO₂ for varying periods of time. The ABA specific radioactivities were about 30 to 70% of that of lutein and violaxanthin regardless of the chase period. The specific activity of neoxanthin, however, was only about 15% of that of ABA. The effects of fluridone on xanthophyll and ABA levels and the extent of labeling of both from ¹⁴CO₂ were determined. Fluridone did not inhibit the accumulation of ABA when leaves were stressed once, although subsequent stresses in the presence of fluridone did lead to a reduced ABA accumulation. The incorporation of ¹⁴C from ¹⁴CO₂ into ABA and the xanthophylls was inhibited by fluridone and to about the same extent. The incorporation of ¹⁸O into ABA from violaxanthin which had been labeled in situ by means of the violaxanthin cycle was measured. The results indicated that a portion of the ABA accumulated during stress was formed from violaxanthin which had been labeled with ¹⁸O. The results of these experiments are consistent with a preformed xanthophyll(s) as the major ABA precursor in water-stressed bean leaves.