Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets

Washed human platelets suspended in buffers containing either 1.8 mM Ca2+ and 0.49 mM Mg2+ or 1 mM EDTA were treated with human alpha-thrombin to induce secretion. Glycoprotein G, a major glycoprotein in alpha-granules, was quantitatively secreted from platelets activated in the EDTA-containing buffer but remained with the platelet in the presence of Ca2+ and Mg2+. Addition of Ca2+ to the platelets that were activated in the presence of EDTA caused glycoprotein G to bind to platelets. To determine if glycoprotein G is expressed on the membrane surface of the activated platelet, platelets were rapidly labeled by a method employing lactoperoxidase-catalyzed iodination. Although glycoprotein G was barely detected on the surface of unstimulated platelets, labveling 1 min after thrombin treatment showed that glycoprotein G rapidly became one of the prominent surface proteins. These findings show that an alpha-granule protein, glycoprotein G, is one of the major glycoproteins on the membrane surface of thrombin-activated platelets and that its binding is dependent on divalent cations.     

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the Dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.     

Cyanobacterial N2 fixation in presence of nitrogen fertilizers

Anabaena oryzae ARM 570 was examined for its growth (chlorophyll and protein), heterocyst frequency, nitrogenase (acetylene reduction) activity, ammonia excretion, and glutamine synthetase and nitrate reductase in response to two levels of urea-N vis-à-vis N2-N. Growth of cyanobacterium increased with duration of incubation. Reduction in heterocyst frequency (40%) was observed at 30 ppm of urea-N, whereas at 60 ppm of urea-N, filaments were completely devoid of heterocysts and no nitrogenase activity was observed. Maximum excretion of ammonia occurred at 30 ppm of urea-N, which was concomitant with minimum glutamine synthetase activity. These results suggested that A. oryzae could be effectively utilized in cyanobacterial biofertilizer programme even in the presence of combined nitrogen, for improving N-budget in rice cultivation.     

Alterations in Electron Transport Characteristics during Senescence of Cucumis Cotyledonary Leaves. Analysis of the Effects of Inhibitors

Cotyledonary leaves of Cucumis sativus cv. Poinsette exhibited senescence-induced losses in chlorophyll (Chl) and protein contents within three weeks since germination. Chl and protein concentrations in cotyledonary leaves approached maximum on 6th d after germination and they declined to 50 and 41 %, respectively, by the 20th day of growth. Activities of both photosystem (PS) 2 and PS1 decreased by 33 and 31 %, respectively, on the 20th day, compared to the control 6th day. Changes in sensitivity of PS2 to inhibitors like atrazine and dibromothymoquinone and sensitivity of PS1 to KCN accompanied the changes in PS2 and PS1 activities. Hence both the acceptor side of PS2 and the donor side of PS1 are affected by senescence-induced changes in cucumber cotyledonary leaves.     

Mercury ions inhibit photosynthetic electron transport at multiple sites in the cyanobacteriumSynechococcus 6301

Inhibition of electron transport activities in the spheroplasts ofSynechococcus 6301 by HgCl² is dependent on the concentration of mercury ions. The inhibition of whole chain electron transport activity occurs at low concentration of Hg²⁺ (6 ΜM@#@). This inhibition occurs mostly due to interaction of Hg²⁺ on plastocyanin. At an elevated concentration (24 ΜM@#@), mercury induces inhibition chiefly in photosystem II catalyzed electron transport. At this concentration it also alters both the absorption and emission characteristics of the phycocyanin. The photosystem I catalyzed electron transport was inhibited by 50% only at high concentrations (36 ΜM@#@) of HgCl₂. However, electron transport catalyzed by photosystems I and II from reduced duroquinone to methylviologen which involves intersystem electron transport is extremely sensitive to mercury (low concentration 6–9 ΜM) like that of whole chain assay indicating that the observed inhibition in whole chain electron transport at low concentrations is mostly contributed by the damage involving other intersystem electron transport carrier(s) like plastocyanin. Thus mercury ions depending on the concentration affects the electron transport at multiple sites in the spheroplasts ofSynechococcus.     

Salt-stress induced alterations in protein profile and protease activity in the mangrove Bruguiera parviflora

Two-month-old seedlings of Bruguiera parvifora were treated with varying levels of NaCl (100, 200 and 400 mM) under hydroponic culture. Total proteins were extracted from leaves of control and NaCl treated plants after 7, 14, 30 and 45 d of treatment and analysed by SDS-PAGE. As visualized from SDS-PAGE, the intensity of several protein bands of molecular weight 17, 23, 32, 33 and 34 kDa decreased as a result of NaCl treatment. The degree of decrease of these protein bands seemed to be roughly proportional to the external NaCl concentration. The most obvious change concerned a 23 kDa-polypeptide (SSP-23), which disappeared after 45 d treatment in 400 mM NaCl. Moreover, the SSP-23 protein, which disappeared in B. parviflora under salinity stress, reappeared when these salinized seedlings were desalinized. These observations suggest the possible involvement of these polypeptides for osmotic adjustment under salt stress. NaCl stress also caused an increase in the activity of both acid and alkaline protease. The increasing activity of proteases functions as a signal of salt stress in B. parviflora, which induces the reduction of protein level.     

Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry

We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 microM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34(cdc2)/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34(cdc2)/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0-7.5 Gy. The observed dose-effect relationship for the percentage of cells with aberrant chromosome 1 (Y) was explained by using both a linear [Y=(2.77+/-0.230)D+0.90+/-0.431, r(2)=0.966] and a nonlinear power [Y=(5.70+/-0.46)D((0.61+/-0.05)), r(2)=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.     

Protective effect of supplemental low intensity white light on ultraviolet-B exposure-induced impairment in cyanobacterium <Emphasis Type="Italic">Spirulina platensis</Emphasis>: formation of air vacuoles as a possible protective measure

Nitric oxide (NO) is a diffusible, very reactive gas that is involved in the regulation of many processes in plants. Several enzymatic sources of NO production have been identified in recent years. Nitrate reductase (NR) is one of them and it has been shown that this well-known plant protein, apart from its role in nitrate reduction and assimilation, can also catalyse the reduction of nitrite to NO. This reaction can produce large amounts of NO, or at least more than is needed for signalling, as some escape of NO to the outside medium can be detected after NR activation. A role for NO and NR in stomata functioning in response to abscisic acid has also been proposed. The question that remains is whether this NR-derived NO is a signalling molecule or the mere product of an enzymatic side reaction like the products generated by the oxygenase activity of RuBisCO.     

Application of InChI to curate, index, and query 3-D structures

The HIV structural database (HIVSDB) is a comprehensive collection of the structures of HIV protease, both of unliganded enzyme and of its inhibitor complexes. It contains abstracts and crystallographic data such as inhibitor and protein coordinates for 248 data sets, of which only 141 are from the Protein Data Bank (PDB). Efficient annotation, indexing, and querying of the inhibitor data is crucial for their effective use for technological and industrial applications. The application of IUPAC International Chemical Identifier (InChI) to index, curate, and query inhibitor structures HIVSDB is described.     

Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective single-centre study

Psoriasis and psoriatic arthritis are inflammatory diseases that respond well to anti-tumour necrosis factor-α therapy. To evaluate the effects of anti-tumour necrosis factor-α treatment on expression of adhesion molecules and angiogenesis in psoriatic lesional skin and synovial tissue, we performed a prospective single-centre study with infliximab therapy combined with stable methotrexate therapy. Eleven patients with both active psoriasis and psoriatic arthritis received infusions of infliximab (3 mg/kg) at baseline, and at weeks 2, 6, 14 and 22 in an open-label study. In addition, patients continued to receive stable methotrexate therapy in dosages ranging from 5 to 20 mg/week. Clinical assessments, including Psoriasis Area and Severity Index (PASI) and Disease Activity Score (DAS), were performed at baseline and every 2 weeks afterward. In addition, skin biopsies from a target psoriatic plaque and synovial tissue biopsies from a target joint were taken before treatment and at week 4. Immunohistochemical analysis was performed to detect the number of blood vessels, the expression of adhesion molecules and the presence of vascular growth factors. Stained sections were evaluated by digital image analysis. At week 16, the mean PASI was reduced from 12.3 ± 2.4 at baseline to 1.8 ± 0.4 (P ≤ 0.02). The mean DAS was reduced from 6.0 ± 0.5 to 3.6 ± 0.6 (P ≤ 0.02). We found some fluctuations in DAS response as compared with the change in PASI, with the latter exhibiting a steady decrease over time. After 4 weeks the cell infiltrate was reduced in both skin and synovium. There was a significant reduction in the number of blood vessels in dermis and synovium at week 4. A significant reduction in the expression of α_vβ₃ integrin, a marker of neovascularization, was also found in both skin and synovium at week 4. In addition, a significant reduction in the expression of adhesion molecules was observed in both skin and synovium at week 4. We also observed a trend toward reduced expression of vascular endothelial growth factor in both skin and synovium. In conclusion, low-dose infliximab treatment leads to decreased neoangiogenesis and deactivation of the endothelium, resulting in decreased cell infiltration and clinical improvement in psoriasis and psoriatic arthritis.