Reactivity of glycoconjugate in membrane system II: Can a neutral glycolipid function as lectin receptor in the presence of gangliosides in plasma membrane?

To examine how surface Potential controls the reactivity of glycoconjugates at cell surface, the interaction of galactose-sPecific lectinse.g. peanut agglutinin,Ricinus cummunis agglutinin with liPosomes bearing asialo GM₁ were studied in the Presence of varying amount of ganglioside mixture, GM_n. The Presence of 5% GM_n causes comPlete slowing down of PreciPitin reaction and thereby make carbohydrate moiety of asialo GM₁ comPletely inaccessiblei.e. ‘cryPtic’. In contrast the Presence of 1–2% GM_n enhances the aPParent rate and amPlitude of the PreciPitin reaction as surface Potential becomes more negative. The relevance of the findings has been discussed in relation to the exPression and involvement of the cell-surface sialic acid residues during develoPment and differentiation.     

Localization of the gene for a third G protein β-subunit to mouse chromosome 6 near Raf-1

The large family of signal transducing proteins known as G proteins are heterotrimers that dissociate into an independent α-subunit and βγ-subunit complex after ligand binding or other stimulation. For Gα, at least 30 distinct sequences representing 10 different classes have been identified. On the other hand, cDNAs for only three Gβ-subunit genes have been isolated so far. All three of the Gβ genes have been chromosomally mapped in the human, but only two in the mouse. Using a human retinal cDNA for the third G protein β-subunit, we have mapped the corresponding gene, termed Gnb-3, to mouse Chromosome 6 with somatic cell hybrids and have positioned it distal to but near the marker Raf-1 by analysis of the progeny of three genetic crosses.     

Steroid binding activity is retained in a 16-kDa fragment of the steroid binding domain of rat glucocorticoid receptors

The steroid binding domain of the rat glucocorticoid receptor is considered as extending from amino acids 550 to 795. However, such a synthetic protein (i.e. amino acids 547-795; Mr approximately 31,000) has been reported to show very little affinity for the potent synthetic glucocorticoid dexamethasone. We now disclose that digestion of steroid-free rat glucocorticoid receptors with low concentrations of trypsin yields a single species, of Mr = 16,000, that is specifically labeled by dexamethasone 21-mesylate. This 16-kDa fragment retains high affinity binding for [3H]dexamethasone that is only approximately 23-fold lower than that seen with the intact 98-kDa receptor. Analysis of the protease digestion patterns obtained both with trypsin and with lysylendopeptidase C allowed us to deduce the proteolytic cleavage maps of the receptor with these enzymes. From these protease maps, the sequence of the 16-kDa fragment was identified as being threonine 537 to arginine 673. These results show that glucocorticoid receptor fragments smaller than 34 kDa do bind steroids and that the amino acids Thr537-Arg673 constitute a core sequence for ligand binding within the larger steroid binding domain. The much slower kinetics in generating the 16-kDa fragment from affinity-labeled receptors suggests that steroid binding causes a conformation change in the receptor near the cleavage sites.     

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A₂ activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A₂ activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A₂ activities and arachidonic acid release in cells pretreated with nominal Ca²⁺ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A₂ activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A₂ activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A₂ activity (ii) in addition to the presence of extracellular Ca²⁺, release of Ca²⁺ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A₂ activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A₂ activities does not appear to require new RNA or protein synthesis.Abbreviations A23187 calcium ionophore - AA arachidonic acid - PMSF phenylmethyl sulfonylfuoride - DFP diisopropyl-fluorophosphate - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - PBS phosphate buffered saline - HBPS Hank's buffered physiological saline - PLA₂ phospholipase A₂     

Antimicrobial activities of some antineoplastic and other withanolides

The antimicrobial activities of six withanolide compounds are compared.     

A sequential test for assessing observed agreement between raters

Assessing the agreement between two or more raters is an important topic in medical practice. Existing techniques, which deal with categorical data, are based on contingency tables. This is often an obstacle in practice as we have to wait for a long time to collect the appropriate sample size of subjects to construct the contingency table. In this paper, we introduce a nonparametric sequential test for assessing agreement, which can be applied as data accrues, does not require a contingency table, facilitating a rapid assessment of the agreement. The proposed test is based on the cumulative sum of the number of disagreements between the two raters and a suitable statistic representing the waiting time until the cumulative sum exceeds a predefined threshold. We treat the cases of testing two raters' agreement with respect to one or more characteristics and using two or more classification categories, the case where the two raters extremely disagree, and finally the case of testing more than two raters' agreement. The numerical investigation shows that the proposed test has excellent performance. Compared to the existing methods, the proposed method appears to require significantly smaller sample size with equivalent power. Moreover, the proposed method is easily generalizable and brings the problem of assessing the agreement between two or more raters and one or more characteristics under a unified framework, thus providing an easy to use tool to medical practitioners.     

Molecular characterization influencing metal resistance in the Cupriavidus/Ralstonia genomes

Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.     

Caenorhabditis elegans mutants resistant to attachment of Yersinia biofilms

The detailed composition and structure of the Caenorhabditis elegans surface are unknown. Previous genetic studies used antibody or lectin binding to identify srf genes that play roles in surface determination. Infection by Microbacterium nematophilum identified bus (bacterially unswollen) genes that also affect surface characteristics. We report that biofilms produced by Yersinia pestis and Y. pseudotuberculosis, which bind the C. elegans surface predominantly on the head, can be used to identify additional surface-determining genes. A screen for C. elegans mutants with a biofilm absent on the head (Bah) phenotype identified three novel genes: bah-1, bah-2, and bah-3. The bah-1 and bah-2 mutants have slightly fragile cuticles but are neither Srf nor Bus, suggesting that they are specific for surface components involved in biofilm attachment. A bah-3 mutant has normal cuticle integrity, but shows a stage-specific Srf phenotype. The screen produced alleles of five known surface genes: srf-2, srf-3, bus-4, bus-12, and bus-17. For the X-linked bus-17, a paternal effect was observed in biofilm assays.     

Characterization of a galactose specific adhesin of enteroaggregative Escherichia coli

A fimbrial adhesin was identified from an enteroaggregative Escherichia coli strain. The adhesin was purified to 740-fold by sequential chromatography on an affinity matrix and gel filtration column in the FPLC system. The homogeneity of the purified protein was established by analytical isoelectrofocussing (pI 7.25). The native adhesin appeared as a high-molecular-weight aggregative protein as revealed by gel filtration chromatography on Superose 12HR10/30 column. However, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the adhesin was found to be 18 kDa and this was further confirmed by gel filtration chromatography on Superose 6HR 10/30 column presence of 6 M guanidine hydrochloride. The N-terminal 15-amino-acid sequence of the adhesin did not show homology with any of the previously reported fimbrial adhesins. The purified adhesin showed adhesion to human erythrocytes in the presence of Ca(2+) (5 mM). The optimum temperature and pH for the hemadhesion activity was found to be 25 degrees C and 6.5, respectively. The inhibition study clearly suggested that the binding site of the adhesin could recognize galactose as the specific sugar. The fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside was quenched on binding to the adhesin and maximum reversal of fluorescence quenching was observed by competitive substitution titration with raffinose. The adhesin was found to contain one binding site per monomer for its specific sugar residue. The association constant and the free energy of binding were obtained as 3.98 x 10(5) M(-1) and -31.97 kJ/mol, respectively. The adherence of the bacteria to HEp-2 monolayer was inhibited in presence of galactose and this was further supported by a significant reduction in the bacterial adherence to the HEp-2 cells, pretreated with beta-D-galactosidase.