WRKY transcription factors: evolution,regulation, and functional diversity in plants

Protoplasma - The recent advancements in sequencing technologies and informatic tools promoted a paradigm shift to decipher the hidden biological mysteries and transformed the biological issues...     

Inhibition of Development of Myxococcus xanthus by Eukaryotic Protein Kinase Inhibitors

Myxococcus xanthus is a social bacterium that lives in the soil and undergoes spectacular development to form multicellular fruiting bodies. It contains a large family of eukaryote-like serine/threonine protein kinases. We found that a number of inhibitors for eukaryotic protein serine, threonine, and tyrosine kinases could inhibit the development and sporulation of M. xanthus to various degrees. These results suggest that serine/threonine and tyrosine phosphorylation may be involved in development of M. xanthus. None of the inhibitors tested had any effect on vegetative growth of M. xanthus. Most of them seemed to act during the early stages of development. However, the expression of a very early development-specific gene, Ω4521, was not significantly affected by the inhibitors. The patterns of protein phosphorylation during development were also not significantly altered by the inhibitors, suggesting that the targets of the inhibitors are minor or unstable phosphoproteins but play key roles in fruiting-body formation in M. xanthus.     

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes

Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (approximately 60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (approximately 3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells. When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites. CF remained firmly bound after prolonged perfusion with 0.1-0.2 M KCl or NaCl. It was displaced by perfusion with buffers of high ionic strength (0.4-0.5 M KCl) or pH (less than 3.0 or greater than 10.0). CF also bound (clustered at approximately 60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix. The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations.     

Tissue-specific expression and dimerization of the endoplasmic reticulum oxidoreductase Ero1beta

Endoplasmic reticulum oxidoreductases (Eros) are essential for the formation of disulfide bonds. Understanding disulfide bond catalysis in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. Mammals express two related Ero proteins, Ero1alpha and Ero1beta. Ero1beta is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1beta can form homodimers and mixed heterodimers with Ero1alpha, in addition to Ero-PDI dimers. Ero-Ero dimers require the Ero active site, occur in vivo, and can be modeled onto the Ero1p crystal structure. Our data indicate that the Ero1beta protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1beta occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1beta expression is high, but is inversely correlated with PDI and PDIp levels, demonstrating that cell-specific differences exist in the regulation of oxidative protein folding in vivo.     

Cytohistologic correlation of cirrhosis and hepatocellular carcinoma. Pitfall in diagnosis?

OBJECTIVE: To compare the diagnostic criteria for cirrhosis and hepatocellular carcinoma (HCC) noted on liver fine needle aspirates (FNAs) and their corresponding liver needle core biopsies (NCBs). STUDY DESIGN: We reviewed FNA slides from 15 cases of cirrhosis and 6 cases of HCC and their corresponding NCBs. We compared a variety of specific nonarchitectural criteria, including small cell dysplasia (SCD) and large cell dysplasia (LCD), for distinguishing cirrhosis from HCC. RESULTS: FNA smears diagnostically correlated with NCBs. The cytologic criterion with the greatest correlation in predicting HCC on FNA was SCD. This was not noted in all the core biopsies, probably due to sampling error. LCD was seen more frequently in cirrhosis than HCC on both cytology and histology and therefore was not a criterion useful in establishing a diagnosis of malignancy. The remaining cytologic criteria had good correlations but did not aid in diagnosing HCC. CONCLUSION: FNA has good cytohistologic correlation with NCB for both cirrhosis and HCC. There is an association of SCD with HCC; however, LCD is not a reliable "precancerous" change as it is commonly seen in cirrhosis and HCC. Therefore, the presence of SCD on FNA should be reported and is an indication for close clinical follow-up to exclude HCC.     

CD45 variant alleles: possibly increased frequency of a novel exon 4 CD45 polymorphism in HIV seropositive Ugandans

The CD45 (leucocyte common) antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor signalling in lymphocytes, and expression of different CD45 isoforms is associated with distinct functions. Here we describe a novel polymorphism in exon 4 (A54G) of the gene encoding CD45 (PTPRC) that results in an amino acid substitution of Thr-19 to Ala in exon 4. The 54G allele was identified in African Ugandan populations and was found with a suggestive but not statistically significant increase in frequency amongst HIV-seropositive Ugandans. This suggests that the 54G variant and CD45 splicing abnormalities might be associated with HIV infection.     

Cytosolic NADPH may regulate differences in basal Nox oxidase-derived superoxide generation in bovine coronary and pulmonary arteries

Because systems controlled by basal NAD(P)H oxidase activity appear to contribute to differences in responses of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to hypoxia, we characterized the Nox oxidases activities present in these vascular segments and how cytosolic NAD(P)H redox systems could be controlling oxidase activity. BPA generated approximately 60-80% more lucigenin (5 microM) chemiluminescence detectable superoxide than BCA. Apocynin (10 microM), a NAD(P)H oxidase inhibitor, and 6-aminonicotinamide (1 mM), a pentose phosphate inhibitor (PPP), both attenuated (approximately by 50-70%) superoxide detected in BPA and BCA. There was no significant difference in the expression of Nox2 or Nox4 mRNA or protein detected by Western blot analysis. NADPH and NADH increased superoxide in homogenates and isolated microsomal membrane fractions in a manner consistent with BPA and BCA having similar levels of oxidase activity. BPA had 4.2-fold higher levels of NADPH than BCA. The activity and protein levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting PPP enzyme generating cytosolic NADPH, were 1.5-fold higher in BPA than BCA. Thus BPA differ from BCA in that they have higher levels of G6PD activity, NADPH, and superoxide. Because both arteries have similar levels of Nox expression and activity, elevated levels of cytosolic NADPH may contribute to increased superoxide in BPA.     

Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis

Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.     

Copper affinity precipitation as an initial step in protein purification

Summary The affinity of copper ions for histidine residues can be utilized to carry out fractional precipitation of proteins. Using this approach it is possible to purify concanavalin A by a simple two step procedure. The fold purification obtained was 4.5 fold.     

Studies on Cytokinins in Watermelon Seeds

Extracts of immature seeds of watermelon contain three cytokinins which are adsorbed on Dowex 50W ion exchange resin. The Rf of the fastest moving factor is similar to that of zeatin. The slowest moving factor is insoluble in n-butanol and shows some resemblance to zeatin ribotide. The chromatographic properties of the second factor are, however, different from those of any of the known cytokinins and is believed to be new to literature.