Rapid detection of 6-phosphogluconate dehydrogenase variants by electrophoresis on cellulose acetate gel

A procedure, suitable even under field conditions for the rapid detection of 6-phosphogluconate dehydrogenase electrophoretic variants, is described.Work supported by N.I.H.—grant no. ROI GM 13415.     

Biochemical aspects of the developing and ripening banana

The peel and pulp of the banana fruit and the pseudostem were examined for glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and aldolase activities and protein, phenolics, chlorophyll and starch. The peel-pulp ratio at various stages of fruit development on the plant and in detached fruits showing incipient ripening were used as an index of the physiological age of the fruit. The enzymes exhibited maximum activity at a stage corresponding to the initiation of the climacteric. GPT level at this stage was higher than that of GOT. An initial increase in the protein content was followed by a decline in both peel and pulp, the level reaching a minimum in climacteric fruits. Astringency, measured in terms of total phenolics, decreased with development; in mature fruits, peel contained 4–5 × as much phenolics as pulp. Chlorophyll in mature fruits was 10 × higher than in young fruits and decreased in ripe fruits. The onset of ripening was attended with a pronounced decrease in the starch. The various analyses were carried out also on the pseudostem removed from the plant soon after flower formation.     

Molecular analysis of the role of streptococcal pyrogenic exotoxin A (SPEA) in invasive soft-tissue infection resulting from Streptococcus pyogenes

Epidemiological studies strongly implicate the bacterial superantigen, streptococcal pyrogenic exotoxin A (SPEA), in the pathogenesis of necrotizing soft-tissue infection and toxic shock syndrome resulting from Streptococcus pyogenes. SPEA can act as a superantigen and cellular toxin ex vivo, but its role during invasive streptococcal infection is unclear. We have disrupted the wild-type spea gene in an M1 streptococcal isolate. Supernatants from toxin-negative mutant bacteria demonstrated a 50% reduction in pro-mitogenic activity in HLA DQ-positive murine splenocyte culture, and up to 20% reduction in activity in human PBMC culture. Mutant and wild-type bacteria were then compared in mouse models of bacteraemia and streptococcal muscle infection. Disruption of spea was not associated with attenuation of virulence in either model. Indeed, a paradoxical increase in mutant strain-induced mortality was seen after intravenous infection. Intramuscular infection with the SPEA-negative mutant led to increased bacteraemia at 24 h and a reduction in neutrophils at the site of primary muscle infection. Purified SPEA led to a dose-dependent increase in peritoneal neutrophils 6 h after administration. SPEA is not a critical virulence factor in invasive soft-tissue infection or bacteraemia caused by S. pyogenes, and it could have a protective role in murine immunity to pyogenic infection. The role of this toxin may be different in hosts with augmented superantigen responsiveness.     

A microtitre plate assay for measuring glycosidase activity

Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC(50) and K(i) values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors.     

Lipocalin signaling controls unicellular tube development in the Caenorhabditis elegans excretory system

Unicellular tubes or capillaries composed of individual cells with a hollow lumen perform important physiological functions including fluid or gas transport and exchange. These tubes are thought to build intracellular lumina by polarized trafficking of apical membrane components, but the molecular signals that promote luminal growth and luminal connectivity between cells are poorly understood. Here we show that the lipocalin LPR-1 is required for luminal connectivity between two unicellular tubes in the Caenorhabditis elegans excretory (renal) system, the excretory duct cell and pore cell. Lipocalins are a large family of secreted proteins that transport lipophilic cargos and participate in intercellular signaling. lpr-1 is required at a time of rapid luminal growth, it is expressed by the duct, pore and surrounding cells, and it can function cell non-autonomously. These results reveal a novel signaling mechanism that controls unicellular tube formation, and provide a genetic model system for dissecting lipocalin signaling pathways.     

A novel 99mTc-labeled testosterone derivative as a potential agent for targeting androgen receptors

With an insight that ligands possessing a N2S2 tetradentate array of donor atoms serve as ideal bifunctional chelating agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized conditions with 99mTc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radiolabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety with the testosterone receptor.     

Increasing Trends of Leptospirosis in Northern India: A Clinico-Epidemiological Study

Background

Leptospirosis, a zoonosis associated with potentially fatal consequences, has long been a grossly underreported disease in India. There is no accurate estimate of the problem of leptospirosis in non-endemic areas such as north India.

Methods/Principal Findings

In order to understand the clinical spectrum and risk factors associated with leptospirosis, we carried out a retrospective study in patients with acute febrile illness in north India over the last 5 years (January 2004 to December 2008). There was increased incidence of leptospirosis (11.7% in 2004 to 20.5% in 2008) as diagnosed by IgM ELISA and microscopic agglutination titer in paired acute and convalescent sera. The disease showed a peak during the rainy season (August and September). We followed up 86 cases of leptospirosis regarding their epidemiological pattern, clinical features, laboratory parameters, complications, therapy, and outcome. Mean age of patients was 32.6 years (2.5 years to 78 years) and males (57%) outnumbered females (43%). Infestation of dwellings with rats (53.7%), working in farm lands (44.2%), and contact with animals (62.1%) were commonly observed epidemiological risk factors. Outdoor workers including farmers (32.6%), labourers (11.6%), para-military personnel (2.3%), and sweepers (1.2%) were commonly affected. Modified Faine''s criteria could diagnose 76 cases (88.3%). Renal failure (60.5%), respiratory failure (20.9%), the neuroleptospirosis (11.6%), and disseminated intravascular coagulation (DIC) (11.6%) were the commonest complications. Five patients died, giving a case fatality rate of 5.9%.

Conclusions/Significance

There has been a rapid rise in the incidence of leptospirosis in north India. Severe complications such as renal failure, respiratory failure, neuroleptospirosis, and DIC are being seen with increasing frequency. Increased awareness among physicians, and early diagnosis and treatment, may reduce mortality due to leptospirosis.     

Molecular basis for an ancient partnership between prolyl isomerase Pin1 and phosphatase inhibitor-2

Pin1 is a prolyl isomerase that recognizes phosphorylated Ser/Thr-Pro sites, and phosphatase inhibitor-2 (I-2) is phosphorylated during mitosis at a PSpTP site that is expected to be a Pin1 substrate. However, we previously discovered I-2, but not phospho-I-2, bound to Pin1 as an allosteric modifier of Pin1 substrate specificity [Li, M., et al. (2008) Biochemistry 47, 292]. Here, we use binding assays and NMR spectroscopy to map the interactions on Pin1 and I-2 to elucidate the organization of this complex. Despite having sequences that are ～50% identical, human, Xenopus, and Drosophila I-2 proteins all exhibited identical, saturable binding to GST-Pin1 with K(0.5) values of 0.3 μM. The (1)H-(15)N heteronuclear single-quantum coherence spectra for both the WW domain and isomerase domain of Pin1 showed distinctive shifts upon addition of I-2. Conversely, as shown by NMR spectroscopy, specific regions of I-2 were affected by addition of Pin1. A single-residue I68A substitution in I-2 weakened binding to Pin1 by half and essentially eliminated binding to the isolated WW domain. On the other hand, truncation of I-2 to residue 152 had a minimal effect on binding to the WW domain but eliminated binding to the isomerase domain. Size exclusion chromatography revealed that wild-type I-2 and Pin1 formed a large (>300 kDa) complex and I-2(I68A) formed a complex of half the size that we propose are a heterotetramer and a heterodimer, respectively. Pin1 and I-2 are conserved among eukaryotes from yeast to humans, and we propose they make up an ancient partnership that provides a means for regulating Pin1 specificity and function.