A new mechanism of strain transfer in polycrystals.

At the grain boundaries of plastically deforming polycrystals, strain transfer mechanisms can accommodate the shear strain carried by slip bands and mechanical twins to prevent stress build-ups and damage. So far, only the accommodation obtained through slip (and twinning) alone has been considered in the mechanism known as slip (and twin) transfer. Here, a strain transfer mechanism that also requires the rotation of the crystal lattice is demonstrated. A region of accumulated slip develops perpendicular to the active slip plane in the impinged grain. The slip gradients enable a localized lattice rotation that accommodates the shear strain in the incoming band, preventing the build-up of interfacial stresses. The mechanism operates preferentially at the boundaries between highly misoriented grains. Facilitating strain transfer at these interfaces opens up new possibilities to improve the mechanical properties of polycrystals, as discussed.

Simultaneous determination of zidovudine and lamivudine in human serum using HPLC with tandem mass spectrometry.

A method employing high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS) has been developed and validated for the simultaneous determination of clinically relevant levels of zidovudine (AZT) and lamivudine (3TC) in human serum. The method incorporates a fully automated ultrafiltration sample preparation step that replaces the solid-phase extraction step typically used for HPLC with UV detection. The calibration range of the dual-analyte LC-MS/MS method is 2.5-2,500 and 2.5-5,000 ng ml-1 for AZT and 3TC, respectively, using 0.25 ml of human serum. The lower limit of quantification was 2.5 ng ml-1 for each analyte, with a chromatographic run time of approximately 6 min. Overall accuracy, expressed as bias, and inter- and intra-assay precision are < +/- 7 and < 10% for AZT, and < +/- 5 and < 12.1% for 3TC over the full concentration ranges. A cross-validation study demonstrated that the LC-MS/MS method afforded equivalent results to established methods consisting of a radioimmuno-assay for AZT and an HPLC-UV method for 3TC. Moreover, the LC-MS/MS was more sensitive, allowed markedly higher-throughput, and required smaller sample volumes (for 3TC only). The validated method has been used to support post-marketing clinical studies for Combivir a combination tablet containing AZT and 3TC.

Effect of spermine on the translocation of Mg2+-dependent phosphatidate phosphohydrolase.

Adipose cytosol treated with spermine showed an aggregation of a cytosolic component which was isolated by centrifugation at 16,000 X g for 20 min. The resultant pellet contained 10% of protein, 40% of lipid and over 75-97% of Mg2+-dependent phosphatidate phosphohydrolase and CTP:phosphocholine cytidylyltransferase activities present in the original cytosol. The specific activities of these enzymes increased 4-fold by the spermine treatment. Characterization of lipids in this component indicated the presence of mainly phospholipids. These studies suggest that the interaction between spermine, the cytosolic component and microsomal membranes may be involved in the translocation of Mg2+-dependent phosphatidate phosphohydrolase.

Isolation and characterization of multiple forms of Mg2+-dependent phosphatidate phosphohydrolase from rat adipose cytosol.

In the present studies, we have made several unique observations. First, we have shown that cytosolic phosphatidate phosphohydrolase from adipose tissue subjected to butyl-agarose chromatography was resolved into four different components. These components, designated as passthrough (PT), D150, D250 and E, were present in the proportions of 51:7:24:16, respectively, in the rat adipose cytosol. Comparison of the properties of these components revealed some similar properties, and also several differences. These components showed the same pH optimum, required Mg2+ for activity and were inhibited by N-ethylmaleimide, indicating a requirement of active sulfhydryl groups for activity. These components differed from one another with respect to hydrophobicity, sedimentation behavior, Stokes diameter, Km values, thermolability and susceptibility to proteinase treatment. Second, we have shown that each component of this system was associated with lipids which were found to be essential for the catalytic activity. Perturbation of this association by organic solvent or by adding excess amounts of exogenous lipids resulted in the loss of enzyme activity. Finally, we analyzed lipid composition of individual components. These studies suggest that the multi-component system of Mg2+-dependent phosphatidate phosphohydrolase may be a part of the cytomembrane network.

Glycerolipid biosynthesis in rat adipose tissue. Influence of age and cell size on substrate utilization.

The conversion of [14C]-labeled compounds such as acetate, glucose, pyruvate and palmitate into CO2, glyceride-glycerol, glyceride fatty acids and total lipids was monitored in the average and matching adipocyte (with respect to size) preparations from young (6-9 wk) and old (age 56-60 wk) male Sprague-Dawley rats. The average cell size populations from young and old rats were 46 +/- 3 and 83 +/- 11 microns in diameter, respectively. The incorporation of [14C]acetate, pyruvate and glucose into fatty acids was significantly reduced in the adipocytes from older rats, irrespective of their sizes. The production of CO2 and glyceride-glycerol did not change significantly as a function of either cell size or animal age. Palmitate incorporation into lipids was similar in the average cell population derived from old and young rats, but it was considerably lower in the smaller adipocytes (46-50 microns diameter) from old animals. Irrespective of the cell size, triacylglycerol formation from sn-glycerol-3-phosphate was also significantly diminished in the adipocytes from older animals compared to younger ones as evidenced by decreases in activities of several enzymes, including sn-glycerol-3-phosphate acyltransferase, Mg2+-dependent phosphatidate phosphohydrolase and diacylglycerol acyltransferase. However, triacylglycerol formation from monoacylglycerol did not change as a function of either cell size or age. These measurements of the metabolic and enzymic activities provide evidence that the synthesis of fatty acids from various precursors and triacylglycerol formation from sn-glycerol-3-phosphate are significantly reduced in adipocytes from older animals and that such changes occur independently of adipocyte size.

Glycerolipid biosynthesis in rat adipose tissue 12. Properties of Mg2+-dependent and -independent phosphatidate phosphohydrolase.

The properties of Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities were investigated in different subcellular fractions in rat adipose tissue. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. The Mg2+-dependent phosphatidate phosphohydrolase was inhibited in the presence of N-methyl- or N-ethylmaleimide, whereas the Mg2+-independent activity was unaffected by these agents. The Mg2+-dependent phosphatidate phosphohydrolase was more sensitive to proteolysis and to high temperature (55 degrees C) compared to the Mg2+-independent enzyme. The Mg2+-dependent phosphatidate phosphohydrolase activity was reduced significantly during aging without any appreciable effects on the Mg2+-independent phosphatidate phosphohydrolase activity. These studies demonstrate that, in addition to Mg2+-dependency, these two forms of phosphatidate phosphohydrolases differ in several respects irrespective of their location in the adipose cell.

Photosynthetic response of seagrasses to ultraviolet-a radiation and the influence of visible light intensity.

Inhibition of photosynthesis by ultraviolet-A radiation (UV-A, 315-380 nanometers) was examined in three marine angiosperms: Halophila engelmannii Aschers, Halodule wrightii Aschers, and Syringodium filiforme Kütz. Sensitivity to UV-A and photosensitization to UV-A by photosynthetically active radiation (PAR, 380-700 nanometers) were characterized.Net photosynthesis by Halodule and Syringodium was unaffected by UV-A irradiation in the absence of PAR. Irradiation of Syringodium by a combined beam of UV-A and PAR resulted in photosynthetic inhibition. The depression of net photosynthesis was found to be a function of PAR intensity at a fixed level of UV-A irradiation. Inhibition of photosynthesis in Halodule by the combined beam was minimal and suggests adaptation to environmental irradiation levels.Halophila was the only species examined, subject to photosynthetic inhibition by UV-A in the absence of PAR. Irradiation with PAR intensities characteristic to Halophila in the natural system as the combined beam, appeared to negate the inhibition. Increasing the PAR component of the combined beam above environmental norms resulted in photosynthetic inhibition greater than that observed for UV-A alone.

Inhibition of seagrass photosynthesis by ultraviolet-B radiation.

Effects of ultraviolet-B radiation on the photosynthesis of seagrasses (Halophila engelmanni Aschers, Halodule wrightii Aschers, and Syringodium filiforme Kütz) were examined. The intrinsic tolerance of each seagrass to ultraviolet-B, the presence and effectiveness of photorepair mechanisms to ultraviolet-B-induced photosynthetic inhibition, and the role of epiphytic growth as a shield from ultraviolet-B were investigated.Halodule was found to possess the greatest photosynthetic tolerance for ultraviolet-B. Photosynthesis in Syringodium was slightly more sensitive to ultraviolet-B while Halophila showed relatively little photosynthetic tolerance. Evidence for a photorepair mechanism was found only in Halodule. This mechanism effectively attenuated photosynthetic inhibition induced by ultraviolet-B dose rates and dosages in excess of natural conditions. Syringodium appeared to rely primarily on a thick epidermal cell layer to reduce photosynthetic damage. Halophila seemed to have no morphological or photorepair capabilities to deal with ultraviolet-B. This species appeared to rely on epiphytic and detrital shielding and the shade provided by other seagrasses to reduce ultraviolet-B irradiation to tolerable levels. The presence of epiphytes on leaf surfaces was found to reduce the extent of photosynthetic inhibition from ultraviolet-B exposure in all species.Observations obtained in this study seem to suggest the possibility of anthocyanin and/or other flavonoid synthesis as an adaptation to long term ultraviolet-B irradiation by these species. In addition, Halophila appears to obtain an increased photosynthetic tolerance to ultraviolet-B as an indirect benefit of chloroplast clumping to avoid photo-oxidation by intense levels of photosynthetically active radiation.

Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves.

Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPH-phosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90%) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH(2)PO(4) and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).

Protein synthesis in the cotyledons of Pisum sativum L. Protein factors involved in the binding of phenylalanyl-transfer ribonucleic acid to ribosomes.

1. Proteinaceous factors contained in a 0.5m-KCl extract of ribosomes from pea cotyledons form a ternary complex at 0 degrees C with [(14)C]phenylalanyl-tRNA and poly(U). The complex is measured by its quantitative retention on Millipore filters. 2. Complex-assembly is optimal at 5mm-Mg(2+) and is independent of GTP and ribosomes. 3. The addition of ribosomes is required to stabilize the complex at 34 degrees C. The complex binds to a puromycin-sensitive site on the ribosome. 4. Soluble factors from the 250000g supernatant of pea cotyledon form a Millipore-retainable complex dependent on GTP and ribosomes. 5. Complex-formation by soluble factors has a Mg(2+) optimum of 10-12mm and forms a puromycin-insensitive complex with ribosomes. 6. The function of the ribosomal protein factors and the supernatant fraction in initiation of protein synthesis is discussed.