A computational investigation of kinetoplastid trans-splicing

Trans-splicing is an unusual process in which two separate RNA strands are spliced together to yield a mature mRNA. We present a novel computational approach which has an overall accuracy of 82% and can predict 92% of known trans-splicing sites. We have applied our method to chromosomes 1 and 3 of Leishmania major, with high-confidence predictions for 85% and 88% of annotated genes respectively. We suggest some extensions of our method to other systems.     

Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro

It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.     

Gamma-secretase is a functional component of phagosomes

Gamma-secretase is a high molecular mass protein complex that catalyzes the intramembrane cleavage of its protein substrates. Two proteins involved in phagocytosis, CD44 and the low density lipoprotein receptor-related protein, are gamma-secretase substrates, suggesting that this complex might regulate some aspects of phagocytosis. Our results indicate that the four components of gamma-secretase, viz. presenilin, nicastrin, APH-1, and PEN-2, are present and enriched on phagosome membranes from both murine macrophages and Drosophila S2 phagocytes. The gamma-secretase components form high molecular mass complexes in lipid microdomains of the phagosome membrane with the topology expected for the functional enzyme. In contrast to the majority of the phagosome proteins studied so far, which appear to associate transiently with this organelle, gamma-secretase resides on newly formed phagosomes and remains associated throughout their maturation into phagolysosomes. Finally, our results indicate that interferon-gamma stimulates gamma-secretase-dependent cleavages on phagosomes and that gamma-secretase activity may be involved in the phagocytic response of macrophages to inflammatory cytokines.     

Functional identification of SLC5A8, a tumor suppressor down-regulated in colon cancer, as a Na(+)-coupled transporter for short-chain fatty acids

SLC5A8, a tumor suppressor gene down-regulated in human colon cancer, codes for a transporter in the Na(+)/glucose cotransporter gene family, but the definitive functional identity of the transporter protein is not known. Since this gene is expressed abundantly in the colon where short-chain fatty acids are generated by bacterial fermentation, we tested the hypothesis that it codes for a Na(+)-coupled transporter for these fatty acids. The coding region of SLC5A8 mRNA was amplified from human intestine and expressed heterologously in Xenopus laevis oocytes. Transport function was monitored by uptake of radiolabeled substrates and by substrate-induced currents under voltage-clamp conditions. Uptake of short-chain fatty acids (lactate, pyruvate, acetate, propionate, and butyrate) in oocytes expressing SLC5A8 was severalfold higher than in uninjected oocytes. Exposure of SLC5A8-expressing oocytes to these fatty acids induced inward currents under voltage-clamp conditions in a Na(+)-dependent manner. These currents were saturable and the substrate concentrations needed for half-maximal induction of the current were in the range of 0.08-2.5 mm. The substrate-induced currents decreased as the carbon chain length of the substrates increased. The Na(+)-activation kinetics indicated involvement of more than one Na(+) ion in the activation process. Direct measurements of substrate (propionate) and charge transfer showed that three positive charges are transferred into oocytes per substrate molecule. These studies establish the functional identity of SLC5A8 as a Na(+)-coupled transporter for short-chain fatty acids.     

Vacuum foam drying for preservation of LaSota virus: Effect of additives

The purpose of this research was to apply vacuum foam drying (VFD) for processing of LaSota virus and to screen formulation additives for its stability. The aqueous dispersion of harvest containing sucrose or trehalose in combination with additive (monosaccharides, polymers, N-Z-amine) was prepared. The diluted dispersions in vials were vacuum concentrated, foamed to form a continuous structure, and vacuum dried. The products were evaluated for foam characteristics, residual moisture, virus titer, x-ray diffraction pattern, and stability profile. The foamability increased with solid content in solutions. The foamability of sucrose was enhanced with incorporation of N-Z-amine (10% and 15% wt/vol) and polyvinyl pyrrolidone (PVP K30, 3% wt/vol). The fructose- or galactose-containing mixtures were deposited irregularly on the vial surface. The virus titer increased with disaccharides in the formulation. Sucrose provided better protection than trehalose. Unlike lyophilization, N-Z-amine with sucrose protected the virus from Millard’s Browning. Amino acids do not have a catalytic effect on hydrolysis of sucrose during VFD. Monosaccharides were ineffective. A synergistic effect of PVP K30 or polyethylene glycol 6000 (3% wt/vol) with N-Z-amine provided the maximum virus titer (6.97 and 7.15, respectively). This formulation retained the desired virus potency at 5°, 25°, and 40°C. The diffraction pattern revealed that a threshold concentration of N-Z-amine was required for inhibiting crystallization of sucrose during VFD. VFD was successfully applied to produce a solid LaSota formulation. The products were amorphous and did not devitrify on storage. Published: July 21, 2006     

Eicosapentaenoic and docosahexaenoic acids enriched polyunsaturated fatty acids from the coastal marine fish of Bay of Bengal and their therapeutic value

Eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) enriched polyunsaturated fatty acids (PUFA) significantly present in marine fish oil emerge as preventive agents for combating many health problems specially in chronic or metabolic disorders. The fish in the coastal area of Bay of Bengal has remained unexplored with respect to EPA/DHA enriched PUFA content in its oils, although it may be a potential source in harnessing the health benefit. In this study, seven varieties of the coastal fish were analysed for the content of EPA/DHA. The one locally known as lotte, (Harpadon nehereus) though has low content of total lipids, was found to have high EPA/DHA in its oil. The phospholipids rich fraction was extracted from the total fish oil. The EPA/DHA enriched PUFA was isolated to investigate the potential use for health benefits. EPA/DHA is found to act as protective agent against mercury poisoning studied in cell culture as well as in animal mode. It is found to be highly preventive in diabetes. The lotte is available in the coastal area of Bay of Bengal adjoining West Bengal, India in large scale and it is the first report showing EPA/DHA enriched PUFA in these fish oil that can be availed to harness in important health benefits.     

Old fold in a new X‐ray diffraction dataset? Low‐resolution molecular replacement using representative structural templates can provide phase information

The advent of structural genomics has led to a dramatic increase in the number of structures deposited in the Protein Data Bank. The number of new folds, however, still remains a very small fraction of the total number of deposited structures. Recent data on the progress of the structural genomics initiative reveals that more than 85% of target proteins that progress to the stage of data collection and structure determination have a known fold. Enzymes, which tend to exploit reaction space while adopting a common stable scaffold, contribute significantly to this observation. Herein, we evaluate a method to examine the "old fold in a new dataset" scenario likely to be encountered in the structural genomics pipeline. We demonstrate that a fold detection strategy based on secondary structure signatures followed by molecular replacement using a minimalist model can be effectively used to solve the phase problem in X-ray crystallography without further recourse to heavy atom derivatives or multiple anomalous dispersion techniques. Three common folds-the triosephosphate isomerase (TIM), adenine nucleotide alpha hydrolase-like (HUP), and RNA recognition motif (RRM)-were examined using this approach. The results presented herein also provide an estimate of the extent of phase information that can be derived from a single domain in a large multidomain structure.     

Identification of an antimicrobial entity from the cyanobacterium Fischerella sp. isolated from bark of Azadirachta indica (Neem) tree

The active principle in a methanolic extract of the laboratory-grown cyanobacterium, Fischerella sp. isolated from Neem (Azadirachta indica) tree bark was active against Mycobacterium tuberculosis, Enterobacter aerogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli as well as three multi-drug resistant E. coli strains in in vitro assays. Based on MS, UV, IR ¹H NMR analyses the active principle is proposed to be hapalindole T having the empirical formula C₂₁H₂₃N₂ClSO and a molecular weight of 386 with the melting point range 179–182 ^°C. The estimated production of Hapalindole T from the cyanobacterium is 1.25 mg g⁻¹ lyophilized biomass. It is suggested that cyanobacteria colonizing specialized niches such as tree bark could be an antibacterial drug resource.     

Identification and characterization of a sperm motility promoting glycoprotein from buffalo blood serum

Early investigators reported the occurrence of unidentified protein factors in biological fluids that may regulate sperm motility essential for fertility potential. This study reports for the first time purification of a forward motility stimulating protein (FMSF-I), to apparent homogeneity, from a biological fluid (buffalo blood serum) and its characterization. FMSF-I is the major motility protein of buffalo serum: a rich source of the factor. FMSF showed high protein specificity and affinity for activating forward motility of goat cauda epididymal spermatozoa. The motility promoter at 0.5 microM level showed maximal activity when nearly 60%-70% of spermatozoa expressed forward motility. It is a 66 kDa monomeric acidic protein rich in aspartate, glutamate, and leucine with isoelectric point of 3.7. FMSF: a Mg2+ -dependent protein binds to concanavalin A-agarose and the glycoprotein nature of FMSF has been confirmed by PAS staining. The factor lost activity completely when treated with alpha-mannosidase showing that the sugar part of the protein is essential for its biological activity. FMSF has no species specificity for its motility-activating potential. Sperm surface has specific receptors of FMSF, which is strongly immunogenic. The factor is present in testis and epididymis although liver is its richest source. Motility promoting efficacy of FMSF is markedly higher than the well-known non-protein motility activators: theophylline and bicarbonate or their combination. FMSF is a physiological activator of sperm motility and as a slaughterhouse byproduct it has potentiality for solving some of the problems of animal breeding, conservation of endangered species, and human infertility: a global social problem.     

Functional dichotomy in CD40 reciprocally regulates effector T cell functions

Activation of T cells requires signals through Ag-specific TCR and costimulatory molecules such as CD40L. Although the use of defined tumor Ags for the induction of protective T cells met with limited success, the CD40-CD40L interaction that was proposed to induce antitumor T cells did not prevent tumor growth completely. Using a model for prostate tumor, a leading cause of tumor-induced mortality in men, we show that the failure is due to a novel functional dichotomy of CD40 whereby it self-limits its antitumor functions by inducing IL-10. IL-10 prevents the CD40-induced CTL and TNF-alpha and IL-12 production, Th1 skewing, and tumor regression. Priming mice with tumor lysate-pulsed IL-10-deficient dendritic cells (DCs) or wild-type DC plus anti-IL-10 Ab establishes antitumor memory T cells that can transfer the protection into syngenic nude mice. Infusion of Ag-pulsed IL-10-deficient but not wild-type DCs back into syngenic mice results in successful therapeutic autovaccination. Thus, we demonstrate the IL-10-sensitive antitumor T cell memory formulating a novel prophylactic and therapeutic principle.