Identification of seed storage protein markers for drought tolerance in mungbean

<p><strong>A set of 292 mungbean germplasm accessions including 62 popularly adapted local land races and two wild forms (<em>Vigna radiata</em> var. sublobata), important breeding lines and standard ruling varieties were screened for drought stress tolerance at seedling stage. Eight genotypes e.g., C. No. 35, OUM 14-1, OUM 49-2, Pusa 9072, OM 99-3, Banapur local B, Nipania munga, Kalamunga 1-A) have been identified to possess drought tolerance. Globulin seed storage protein profiling was carried out in 19 selected mungbean genotypes comprising eight drought tolerant, seven drought sensitive, two wild forms of mungbean (TCR 20 and TCR 213) and two standard checks (LGG 460 and T 2-1</strong><strong>)</strong><strong> to explore differentially expressed polypeptides. Seed protein profiles revealed 15 scorable polypeptide bands with molecular weights ranging from 10.0 to 102.2kD. A specific 12.8kD polypeptide band was present in all above drought tolerant test genotypes including the wild accession TCR 20. Such a polypeptide band may serve as useful biochemical marker for identification of drought tolerant genotypes in mungbean. </strong></p><p><strong> Key words: </strong>Genetic diversity, seed storage protein profile, wild and cultivated <em>Vigna radiata.</em><strong> </strong></p>

Neuroprotective effects of α-lipoic acid on the development of oxidative stress and astrogliosis in the brain of STZ-diabetic rats

The aim of this study was to examine whether the antioxidant alpha-lipoic acid protects neurons from diabetic-reperfusion injury. The streptozotocin (STZ) rat model was used to study the glial reactivity and prevention of gliosis by alpha-lipoic acid (alpha-LA) administration. The expression of glial fibrillary acidic protein (GFAP) was determined, as well as lipid peroxidation (LPO) and glu-tathione (GSH) levels in some brain tissues. We observed significant increasing of lipid peroxidation products in both hippocampus and cortex. Changesof polypeptide GFAP were observed in hippocampus and cortex. Both soluble and filamentous forms of GFAP featured the increase in hippocampus of rat with hyperthyreosis. In the filamentfractions, increase in the intensity of 49 kDa polypeptide band was found. In the same fraction of insoluble cytoskeleton proteins degraded HFKB polypeptides with molecular weight in the range of 46–41 kDa appeared. Markedincrease of degraded polypeptides was found in the soluble fraction of the brain stem. The intensity of the intact polypeptide – 49 kDa, as well as in the filament fraction, significantly increased. It is possible that increasing concentrations of soluble subunits glial filaments may be due to dissociation of own filaments during the reorganization of cytoskeleton structures. Given the results of Western blotting for filament fraction, increased content of soluble intact 49 kDa polypeptide is primarily the result of increased expression of HFKB and only partly due to redistribution of existing filament structures. Calculation and analysis of indicators showed high correlation between the increase in content and peroxidation products of HFKB.These results indicate the important role of oxidative stress in the induction of astroglial response under conditions of diabet encefalopathia. Administration of alpha-LA reduced the expression both of glial and neuronal markers. In addition, alpha-LA significantly prevented the increase in LPO levels found in diabetic rats. GSH levels increased by the administration of alpha-LA. This study suggests that alpha-LA prevents neural injury by inhibiting oxidative stress and suppressing reactive gliosis. All these changes were clearly counteracted by alpha-lipoic acid. The results of this study demonstrate that alpha-lipoic acid provides for protection to the GFAP, as a whole, from diabet -reperfusion injuries.

Вплив гіпертиреозу на процеси навчання та стан гліальних проміжних філаментів головного мозку щурів

The influence of hyperthyreosis on oxidative stress, state of glial intermediate filaments and memotry was investigated. Significant increasing of lipid peroxidation products into both hippocampus and cortex and change for the worse of memory was observed. Analysis of the behavioral reactions of rats in the test of passive avoidance conditioned reflex showed that the acquisition of skills of all groups of animals did not differ by time waiting period (latent period). Time saving memory test conditioned reflex of passive avoidance was excellent in the group of rats treated with thyroxine compared with controls. The change of polypeptide GFAP was observed in hippocampus and cortex. Both soluble and filamentous forms of GFAP increased in hippocampus of rat with hyperthyreosis. In filament fractions, increase in the intensity of 49 kDa polypeptide band was found. In the same fraction of insoluble cytoskeleton proteins degraded HFKB polypeptides with molecular weight in the region of 46–41 kDa appeared. Marked increase of degraded polypeptides was found in the soluble fraction of the brain stem. The intensity of the intact polypeptide (49 kDa), as well as in the filament fraction, significantly increased. It is possible that increasing concentrations of soluble subunits glial filaments may be due to dissociation of own filaments during the reorganization of cytoskeleton structures. Given the results of Western blotting for filament fraction, increased content of soluble intact 49 kDa polypeptide is primarily the result of increased expression of HFKB and only partly due to redistribution of existing filament structures. Calculation and analysis of indicators showed high correlation between the increase in content and peroxidation products of HFKB. These results indicate the important role of oxidative stress in the induction of astroglial reactive response under conditions of hyperthyroidism. This data shows the possibility of the glial cell cytoskeleton reconstruction under effect of thyroid hormones.

Human 76p

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. γ-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and γ-tubulin are associated in the γ-tubulin complexes. (3) γ-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human γ-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.

Detection of Leishmania lipophosphoglycan binding proteins in the gut of the sandfly vector

Binding to the midgut microvillar surface in the sandfly Phlebotomus papatasi is a prerequisite for successful development of Leishmania major within the gut of the vector. This paper describes a method for detecting microvillar-associated proteins which act as ligands for the parasite surface glycoconjugate lipophosphoglycan (LPG). Adhesion of LPG to midgut proteins was visualized by probing midgut extracts with LPG using a Western ligand blotting technique. Procyclic L. major LPG bound to a microvillar polypeptide band of 65 kDa (estimated in the non-reduced state) and bound variably to several lower molecular weight bands, probably degradation products or subunits of the primary binding polypeptides. Specificity of binding was confirmed by co-incubating biotinylated LPG with an LPG-specific mAb which resulted in a great reduction in binding.

Secretory carbonic anhydrase isoenzyme (CA VI) in human serum

Carbonic anhydrase VI (CA VI) is a secretory isoenzyme that, by analogy to α-amylase, is produced in the salivary glands and delivered into saliva. To determine whether CA VI is transferred into the circulation and is detectable in human serum, we collected blood samples from four healthy subjects at 3-h intervals throughout a 24-h period and measured concentrations of CA VI by a specific time-resolved immunofluorometric assay. All serum samples contained CA VI, the concentrations being ∼22 times lower in serum than in the corresponding saliva samples. The presence of CA VI in serum was confirmed by Western blotting, which under reducing conditions identified a 42-kDa polypeptide band corresponding to the monomeric CA VI. The described time-resolved immunofluorometric assay for CA VI might be useful to identify or exclude diseases of the salivary glands in the differential diagnosis of patients whose serum amylase concentrations are increased.

G-protein from Medicago sativa: functional association to photoreceptors

G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5′-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form.

Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes

A guanosine 5′-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.

Identification of the intestinal Na-phosphate cotransporter

Na-dependent phosphate uptake in intestinal brush-border membrane vesicles was sensitive to arginine group-specific reagents in a substrate-sensitive manner. Four different arginine group-specific reagents were tested. All four reagents irreversibly inhibited Na-dependent phosphate uptake with the concentration for 50% inhibition, K0.5, varying between 150 and 40 microM. Maximum inhibition approached 80%. Addition of substrates during exposure to these reagents resulted in protection of Na-phosphate cotransport only in the presence of Na and phosphate. Na-phosphate cotransporter labeling at or near the phosphate site was accomplished using a pretreatment step with phenylglyoxal and substrates followed by a fluorescent phenylglyoxal-labeling step. Fluorescein isothiocyanate-phenylglyoxal (FITC-PG) specifically labeled a 130-kDa polypeptide on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a substrate-sensitive manner, consistent with its effect on Na-phosphate cotransport. n-Acetylimidazole (NAI) inhibited Na-phosphate cotransport in a Na+- but not K+ -sensitive manner. NAI or fluorescein n-acetylimidazole (FNAI) inhibited Na-dependent phosphate uptake with a K0.5 for inhibition of 38 microM. Maximum inhibition of Na-phosphate cotransport was 75%. On SDS-PAGE, FNAI labeled five polypeptide bands in a Na-sensitive manner including the 130-kDa polypeptide band labeled by FITC-PG. Of these five bands only the 130-kDa polypeptide lost substrate protectability against FITC-PG inhibition of Na-phosphate cotransport and FITC-PG labeling on prior exposure to NAI in the absence of Na+. On this basis the 130-kDa polypeptide is tentatively identified as the intestinal Na-phosphate cotransporter and this polypeptide band contains both the Na substrate site and the phosphate substrate site.

Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.