Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli

The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.     

Isolation and characterization of melanin pigment from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> (telomorph of <Emphasis Type="Italic">Antromycopsis</Emphasis><Emphasis Type="Italic">macrocarpa</Emphasis>)

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. For more than 40 years, fungi have been known to produce pigments called melanins. Melanin pigment production by mushrooms was not intensively studied. The present study was carried out on isolation and characterization of melanin from an edible mushroom Pleurotus cystidiosus var. formosensis. The mushroom produced dark mucous mass of hyaline arthrospores on mycelium. The coremia exclusively produced dikaryotic arthrospores with the remnant of a clamp connection. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). The black coloured coremea (conidia) were produced by Antromycopsis macrocarpa (anamorph of P. cystidiosus) when cultured on potato dextrose agar medium. The agar plate was incubated at continuous light illumination for high amount of pigment (coremea) production. The slimy layer of the coremea was extracted and partially purified by alkaline and acid treatment. The black pigment was confirmed as melanin based on UV, IR and EPR spectra apart from chemical analysis. This is the first report on characterization of melanin obtained from Pleurotus cystidiosus var. formosensis.     

Ultrastructural study of highly enriched follicular dendritic cells reveals their morphology and the periodicity of immune complex binding

Follicular dendritic cells (FDCs) are immune accessory cells found in the follicles of secondary lymphoid organs where they promote B cell maturation in germinal centers (GCs) that develop following antigen exposure. Recently, we published a method for isolating functional murine FDCs in high purity. We reasoned that disruption of FDC reticula in vivo would alter FDC morphology. The present study was undertaken to determine the morphological features of isolated FDCs. FDC-M1 and immune complex (IC) labeling were used to identify FDCs in isolated preparations. Results at the light-microscopic level revealed that isolated FDCs trapped ICs, expressed FDC-M1 and cadherins, but generally appeared non-dendritic. However, at the ultrastructural level, the majority of FDCs exhibited dendrites and typical euchromatic nuclei that appeared as single, bilobed, or double nuclei. Based on morphology, four varieties of FDCs were distinguishable, possibly indicative of differences in maturity. Remarkably, ICs trapped by FDCs showed a distinctive periodic arrangement consistent with that known to induce immune responses by thymus independent-2 (TI-2) antigens that engage and cross-link multiple B cell receptors. The ability of FDCs to trap ICs and then display these T-cell-dependent antigens with repeating periodicity suggests that multiple B cell receptors are cross-linked by antigen on FDCs, thus promoting B cell stimulation and proliferation. Rapid proliferation is characteristic of the GC reaction, and the arrangement of T-dependent antigens in this periodic fashion may help to explain the profuse B cell proliferation in the GC microenvironment. This work was supported by National Institutes of Health Grant AI-17142. Electron microscopy and confocal microscopy were performed at the VCU Department of Neurobiology and Anatomy Microscopy Facility supported, in part, by funding from an NIH-NINDS Center core grant (5P30NS047463).     

Effect of unilateral motor cortex ablation on activity of choline acetyltransferase and levels of amino acid transmitter candidates in the spinal cord of adult monkeys

Evidence thatl-glutamate is a neurotransmitter of corticofugal fibers was sought by measuring changes in several biochemical markers of neurotransmitter function in discrete regions of spinal cord after ablation of sensorimotor cortex in monkeys. One and five weeks after unilateral cortical ablation, samples from six areas of spinal cord (ventral, lateral and dorsal regions of the left and right sides) were analysed for choline acetyltransferase (ChAT) activity and contents of amino acid transmitter candidates-glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), taurine (Tau) and -aminobutyric acid (GABA). During one to five weeks after unilateral cortical ablation of the monkey, prolonged hemiplegia in the contralateral side was observed. Histological examination of the spinal cord 5 weeks after unilateral (left) cortical ablation showed no apparent change in either control (ipsilateral, left) or affected (contralateral, right) sides of the cord as examined by the Klüver-Barrera method. The ChAT activity as a cholinergic marker was scarcely changed in any region of either left (control) or right (affected) side of the spinal cord at one and five weeks after unilateral (left side) ablation of the motor cortex. Amino acid levels in each region of the spinal cord were not significantly changed one week after unilateral ablation of the motor cortex. However, a significant decrease of Glu content was observed in the lateral column of the affected (right) side compared to the control (left) side of cervical and lumbar cord five weeks after cortical ablation of the left motor area. No concomitant alterations of other amino acids were detected. These data strongly suggest thatl-Glu is a neurotransmitter for corticofugal pyramidal tract fibers to anterior horn secondary neurons related to motor control activity in monkey spinal cord.     

Progression from papilloma to carcinoma is accompanied by changes in antibody response to papillomavirus proteins.

Cottontail rabbit papillomavirus induces benign tumors, papillomas, in rabbits which progress at a high frequency to malignant tumors, carcinomas. Cottontail rabbit papillomavirus therefore provides an experimental model for oncogenic human papillomaviruses. The nature of the antigens recognized by the host has not been identified at any stage of tumor development. Here, we characterized the humoral immune response to viral antigens in cottontail and domestic rabbits at the papilloma stage, in domestic rabbits at the carcinoma stage, and in animals in which papillomas had regressed. Antibodies to linear epitopes were identified by Western blotting (immunoblotting) with bacterial fusion proteins, and evidence for recognition of conformational epitopes was obtained by immunoprecipitation. An immune response to the early proteins E1, E2, E6, and E7 was detected only in a fraction of the animals, and all animals were negative for E4 and E5. The response to E6 and E7 peaked around 7 months and then decreased, while that to E1 and E2 remained level after an initial raise. The antibody response to structural proteins was low at the papilloma stage, and antibodies to L1 recognized predominantly conformational epitopes. As papillomas progressed to carcinomas, there was a drastic increase in the response to L1 and L2, suggesting a change in interaction between virus-infected host cells and the host's immune system.     

Genomic organization and chromosomal location of the human gene encoding the B-lymphocyte activation antigen B7

The human B lymphocyte activation antigen B7 provides regulatory signals for T lymphocytes as a consequence of binding to its ligands CD28 and CTLA-4. The cDNA for B7 has previously been isolated and predicted to encode a type I membrane protein. The predicted polypeptide has a secretory signal peptide followed by two contiguous Ig-like domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Here we report the exon-intron genomic organization of human B7 and the chromosomal location. The gene has six exons that span approximately 32 kilobases of DNA. Exon 1 is not translated and the second exon contains the initiation ATG codon and encodes a predicted signal peptide. This gene structure is characteristic for several eukaryotic genes with tissue-specific expression. The third and fourth exons correspond to two Ig-like domains whereas the fifth and sixth exons encode respectively the trans-membrane portion and the cytoplasmic tail. This close relationship between exons and functional domains is a characteristic feature of genes of the Ig superfamily. Cell surface expression of the B7 gene product has previously been mapped to human chromosome 12 by antibody reactivity with the B7-specific monoclonal antibody BB-1. We here demonstrate that theB7 gene is located to theq21-qter region of chromosome 3 by DNA blot analysis of human × rodent somatic cell hybrids.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83071-M83075, M83077. Address correspondence and offprint requests to: B. Dupont, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue (Room S709), New York, NY 10021, USA.     

Remedial effect of DL-α-lipoic acid against adriamycin induced testicular lipid peroxidation

Adriamycin (ADR), a cytotoxic antineoplastic drug is used in the treatment of various solid tumors. However, its efficacy continues to be challenged by significant toxicities including testicular toxicity. In the present study, the effect of lipoic acid, a "universal antioxidant" was investigated on ADR induced peroxidative damages in rat testis. Adult male albino rats of Wistar strain were administered ADR (1 mg/kg body weight, i.v.) once a week for 10 weeks. ADR injected rats showed a significant decline in the activities of enzymic antioxidants (catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase) and non-enzymic antioxidants (reduced glutathione, Vitamin A, Vitamin C and Vitamin E) with high malondialdehyde levels. The extent of testicular toxicity was evident from the decreased activities of testicular marker enzymes (sorbitol dehydrogenase and glucose-6-phosphate dehydrogenase). Treatment with lipoic acid (35 mg/kg body weight, i.p.) one day prior to ADR administration, maintained near normal activities of the enzymes and significantly reduced lipid peroxidation, thereby proving it to be an effective cytoprotectant.     

Effects of positional and geometrical isomerism on the biological activity of some novel oxazolidinones

Some novel oxazolidinone derivatives with benzotriazole as pendant have been synthesized and tested for antibacterial activity. Linearly attached benzotriazole derivative showed more potency compared to angular one in vitro. Out of E/Z-isomers of angularly attached derivatives E-isomer was found to be more potent than Z-isomer. Either less active or inactive molecules were obtained, when benzotriazole was replaced with benzimidazole, benzthiazole, or benzoxazole. Finally, thioacetamide analogue of linear compound gave a lead having activity similar to linezolid in vitro.     

Identification of additional sources of resistance to Puccinia striiformis f. sp. hordei (PSH) in a collection of barley genotypes adapted to the high input condition

A total of 336 barley genotypes consisting of released cultivars, advanced lines, differentials and local landraces from the ICARDA barley breeding programme were screened for seedling and adult‐plant resistances to barley stripe rust pathogen (Puccinia striiformis f. sp. hordei [PSH]). Seedling resistance tests were undertaken at Shimla, India by inoculating 336 barley genotypes with five prevalent PSH races [Q (5S0), 24 (0S0‐1), 57 (0S0), M (1S0) and G (4S0)] in India. Barley genotypes were also evaluated at the adult‐plant stage for stripe rust resistance at Durgapura (Rajasthan, India) in 2013 and 2014, and at Karnal (Haryana, India) in 2014 under artificial PSH infection in fields, using a mixture of the five races. Twelve barley genotypes (ARAMIR/COSSACK, Astrix, C8806, C9430, CLE 202, Gold, Gull, Isaria, Lechtaler, Piroline, Stirling, and Trumpf) were resistant to all five PSH races at the seedling and adult‐plant stages. Two of these genotypes, Astrix and Trumpf, were part of international differentials and reveal that five races were avirulent to genes Rps4 (yr4), rpsAst, rpsTr1 and rpsTr2. These genes were highly effective against PSH races prevalent in India. The virulence/avirulence formula reported in this study helped to determine the effectiveness of PSH resistance genes against Indian races. Forty‐five genotypes showed adult‐stage plant resistance (APR) in the field. The identified PSH resistant genotypes may possess novel resistance genes and might serve as potential donors of PSH resistance at seedling and APR in the future. Further research is needed to determine the nature of resistance genes through allelic studies and mapping of these genes.