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     

Acute exposure of rhesus monkeys to DDT: Effect on carbohydrate metabolism

Four furocoumarins, two having a linear molecule, psoralen and 8-methylpsoralen and two having an angular molecule, angelicin and 4,5′-dimethylangelicin were tested for mutagenesis in Escherichia coli B wild type and in various strains deficient in known repair systems. The results indicate that both monoadducts and crosslinks are mutagenic. The mutagenic efficiency of the furocourmarins ranks in the following order 8-methylpsoralen > psoralen > angelicin > 4,5′-dimethylangelicin.     

Frequent recombination events generate diversity within the multi-copy variant antigen gene families of Plasmodium falciparum

The human malaria parasite Plasmodium falciparum utilises a mechanism of antigenic variation to avoid the antibody response of its human host and thereby generates a long-term, persistent infection. This process predominantly results from systematic changes in expression of the primary erythrocyte surface antigen, a parasite-produced protein called PfEMP1 that is encoded by a repertoire of over 60 var genes in the P. falciparum genome. var genes exhibit extensive sequence diversity, both within a single parasite's genome as well as between different parasite isolates, and thus provide a large repertoire of antigenic determinants to be alternately displayed over the course of an infection. Whilst significant work has recently been published documenting the extreme level of diversity displayed by var genes found in natural parasite populations, little work has been done regarding the mechanisms that lead to sequence diversification and heterogeneity within var genes. In the course of producing transgenic lines from the original NF54 parasite isolate, we cloned and characterised a parasite line, termed E5, which is closely related to but distinct from 3D7, the parasite used for the P. falciparum genome nucleotide sequencing project. Analysis of the E5 var gene repertoire, as well as that of the surrounding rif and stevor multi-copy gene families, identified examples of frequent recombination events within these gene families, including an example of a duplicative transposition which indicates that recombination events play a significant role in the generation of diversity within the antigen encoding genes of P. falciparum.     

Coupled adult-brood transport augments relocation in the Indian queenless ant Diacamma indicum

Colony relocation is an important aspect in the lives of social insects. In ants, the process of relocation is further complicated as brood, in addition to adults, have to be transported to the new nest. Here, we have investigated brood transport in the Indian ponerine ant Diacamma indicum, which uses tandem running—a primitive mode of recruitment—for the entire colony to relocate. We have found that there were no brood transport specialists and most of the brood was transported in the mandibles of followers that were being tandem run. Therefore, in a single tandem run, one adult and one brood item was effectively transported by tandem leaders augmenting the relocation process.     

Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.     

Sister-chromatid exchange analyses in rodent maternal, embryonic and extra-embryonic tissues: Transplacental and direct mutagen exposures

Sister-chromatid exchange (SCE) analyses were conducted in maternal, embryonic and extraembryonic tissues of pregnant rats and mice. The various tissues were substituted in vivo with 5-bromodeoxyuridine (BrdU) by implantation of a BrdU tablet in pregnant animals at mid-gestation. Following maternal exposure to 5–20 mg/kg cyclophosphamide, embryonic liver cells demonstrated dose-dependent SCE increases up to 10-fold that of control. Rat embryos revealed little intralitter variability for this transplacental effect. Maternal marrow and yolk sac cells examined in the rat also underwent significant increases in SCE, although to different extents. While marrow SCE frequencies were similar to those of embryo liver, yolk sac SCE frequencies were generally much lower.

SCE analyses were also conducted in rat yold sac cells substituted in vivo with BrdU and subsequently explanted to whole-embryo culture. In vitro exposure to cyclophosphamide at concentrations up to 100 μg/ml had no SCE-inducing effect. However, similar exposures to phosphoramide mustard, a presumed metabolite of cyclophosphamide, caused dose-dependent increases in SCE up to 8-fold higher than control at 2 μg/ml. Thus, cyclophosphamide appears to require maternal metabolic activation in order to cause an increased SCE frequency in yolk sac cells. The system described permits versatile SCE analyses which can help to define relative maternal and embryo tissue-specific sensitivities to chemical-induced genetic damage.     

The CBL–CIPK signaling module in plants: a mechanistic perspective

In a given environment, plants are constantly exposed to multitudes of stimuli. These stimuli are sensed and transduced to generate a diverse array of responses by several signal transduction pathways. Calcium (Ca²⁺) signaling is one such important pathway involved in transducing a large number of stimuli or signals in both animals and plants. Ca²⁺ engages a plethora of decoders to mediate signaling in plants. Among these groups of decoders, the sensor responder complex of calcineurin B‐like protein (CBL) and CBL‐interacting protein kinases (CIPKs) play a very significant role in transducing these signals. The signal transduction mechanism in most cases is phosphorylation events, but some structural role for the pair has also come to light recently. In this review, we discuss the structural nature of the sensor‐responder duo; their mechanism of substrate phosphorylation and also their structural role in modulating targets. Moreover, the mechanism of complex formation and mechanistic role of protein phosphatases with CBL–CIPK module has been mentioned. A comparison of CBL–CIPK with other decoders of Ca²⁺ signaling in plants also signifies the relatedness and diversity in signaling pathways. Further an attempt has been made to compare this aspect of Ca²⁺ signaling pathways in different plant species to develop a holistic understanding of conservation of stimulus–response‐coupling mediated by this Ca²⁺–CBL–CIPK module.     

Tobacco mosaic virus 126-kDa protein increases the susceptibility of Nicotiana tabacum to other viruses and its dosage affects virus-induced gene silencing

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein-green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.     

Dense molecular marking reveals genetic diversity in morphologically similar isolates of the nematophagous fungus Duddingtonia flagrans

Three morphologically similar isolates of Duddingtonia flagrans [(Duddington) R. C. Cooke] viz., Df-2550, Df-2507 and Df-BJ were subjected to RAPD (Randomly Amplified Polymorphic DNA) and SRFA (Selective Fragment Length Amplification) mode of DNA fingerprinting analysis to generate 233 different anonymous DNA markers. Mean number of alleles per primer/primer pair for RAPD and SRFA primers was 13.75 and 29.66 respectively. Phylogenetic analysis through bootstrapping of 1000 simulated replicates of the data set demonstrated that Df-2550 was ancestral in the group of three and did not align with Df-2507 and Df-BJ, which appeared to diversify recently and therefore remained at the end of the phylogenetic tree. Genomic islands were also identified by three SRFA primer pairs, where Df-2550 aligned with Df-BJ, which is reverse to the master consensus-grouping pattern. Scanning image of the amplicon profiles when represented graphically generated unique molecular signature for the isolates.