The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer

We show here that under physiologically reasonable conditions, CGG repeats in RNA readily form hairpins. In contrast to its DNA counterpart that forms a complex mixture of hairpins and tetraplexes, r(CGG)₂₂ forms a single stable hairpin with no evidence for any other folded structure even at low pH. RNA with the sequence (CGG)₉AGG (CGG)₁₂AGG(CGG)₉₇, found in a fragile X syndrome pre-mutation allele, forms a number of different hairpins. The most prominent hairpin forms in the 3′ part of the repeat and involves the 97 uninterrupted CGG repeats. In contrast to the CUG-RNA hairpins formed by myotonic dystrophy type 1 repeats, we found no evidence that CGG-RNA hairpins activate PKR, the interferon-inducible protein kinase that is activated by a wide range of double-stranded RNAs. However, we do show that the CGG-RNA is digested, albeit inefficiently, by the human Dicer enzyme, a step central to the RNA interference effect on gene expression. These data provide clues to the basis of the toxic effect of CGG-RNA that is thought to occur in fragile X pre-mutation carriers. In addition, RNA hairpins may also account for the stalling of the 40S ribosomal subunit that is thought to contribute to the translation deficit in fragile X pre-mutation and full mutation alleles.     

Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, M _r6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml^–1 with a linear response for insulin from 10 g ml^–1 to 10 ng ml^–1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.     

Simple sequence repeat (SSR) markers linked to the Ligon lintless (Li(1)) mutant in cotton

Ligon lintless (Li(1)) is a monogenic, dominant mutant in cotton, whose expression results in extreme reductions in fiber length on mature seed. The objectives of this research were to compare fiber initiation between the Li(1) mutant and TM-1 to reveal the fiber initiation differences between normal and mutant phenotypes, to develop a linkage map of simple sequence repeat (SSR) markers with the Li(1) locus, and to identify the chromosomal location of the Li(1) locus. Comparative scanning electron microscopy studies of fiber development in a normal TM-1 genotype and the near-isogenic Li(1) mutant at 1 and 3 days postanthesis revealed little differences between the two during early stages of development, suggesting that Li(1) gene expression occurs later, probably during the elongation phase. Thirty-eight SSR loci were found to be polymorphic between TM-1 and Li(1) and were used for mapping in an F(2) population. Twenty-two SSR loci, along with Li(1), were located on eight linkage groups, covering a total genetic distance of 218.3 cM. Analysis of individual monosomic and monotelodisomic plants indicated that two SSR loci (MP4030 and MP673) from the Li(1) linkage group were located on chromosome 22.     

Detection of multiple active site domain motions in transient-state component time courses of the Clostridium symbiosum L-glutamate dehydrogenase-catalyzed oxidative deamination reaction

We present a multiwavelength, transient-state kinetic study of the oxidative deamination reaction catalyzed by Clostridium symbiosum glutamate dehydrogenase (csGDH) producing the real-time reaction courses of spectroscopically resolved kinetically competent intermediate complexes. The results show striking differences from a corresponding transient-state study of the same reaction by the structurally homologous enzyme from beef liver (blGDH). In addition to the highly blue-shifted alpha-iminoglutarate and highly red-shifted carbinolamine complexes observed in both reactions, the csGDH reaction appeared to show the release of free NADH at a very early and mechanistically unlikely point in the reaction. Using lactic acid dehydrogenase as a "reporter" for free NADH, we show that the early portion of this signal reflects previously unobserved spectrally unshifted enzyme-bound NADH complexes. We provide experimental evidence to show that such spectrally anomalous complexes must represent forms of the known alpha-imino and alpha-carbinolamine complexes in which the active site cleft is open. This evidence includes isothermal calorimetric measurements and pH-jump experiments that show the existence of differing two-state transitions in blGDH and csGDH and locate active site domain motions at differing points in the transient-state time courses of the two enzyme reactions. We prove the kinetic competence of a new and more highly detailed mechanism for the csGDH reaction that involves the alternation of open and closed enzyme complexes as integral steps. These findings, supported by the available X-ray crystal structure data, suggest the existence of a programmed time course of protein domain motions coordinated with the classically considered chemical time course. This new viewpoint may be presumed to be applicable to enzyme reactions other than those of the alpha-amino acid dehydrogenases.     

Changes in free amino acid synthesis in the perfused liver of an air-breathing walking catfish, Clarias batrachus infused with ammonium chloride: a strategy to adapt under hyperammonia stress

The changes in the free amino acid (FAA) levels, the rate of efflux of FAAs from the perfused liver, and the activity of some enzymes related to amino acid metabolism such as glutamate dehydrogenase (GDH, both reductive amination and oxidative deamination), glutamine synthetase (GS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were studied in the liver of a freshwater air-breathing teleost, the walking catfish, Clarias batrachus, perfused with 5 and 10 mM NH(4)Cl. The level of the various non-essential FAAs increased significantly, with a total increase of about 150%, which was accompanied by a significant increase of both ammonia and urea-N in the perfused liver both with 5 and 10 mM NH(4)Cl. The rate of efflux of these non-essential FAAs from the perfused liver also increased significantly with a total increase of about 115% and 160% at 5 and 10 mM NH(4)Cl, respectively. The activity of the mentioned amino acid metabolism-related enzymes in the perfused liver also got stimulated, except for GDH in the ammonia forming direction and ALT, under a higher ammonia load. The activity (both tissue and specific) of GDH in the glutamate forming direction increased maximally, followed by AST and GS in a decreasing order. Owing to these physiological adaptive strategies related to amino acid metabolism along with the presence of a functional and regulatory urea cycle (reported earlier), it is believed that this catfish is able to survive in very high ambient ammonia or in the air or in the mud during habitat drying.     

Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contraction and the AMP-activated protein kinase activator 5-aminoimidazole-4-carboxamide-1-beta -D-ribofuranoside

Alterations in the concentration of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, have been linked to the regulation of fatty acid oxidation in skeletal muscle. During contraction decreases in muscle malonyl-CoA concentration have been related to activation of AMP-activated protein kinase (AMPK), which phosphorylates and inhibits acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in malonyl-CoA formation. We report here that the activity of malonyl-CoA decarboxylase (MCD) is increased in contracting muscle. Using either immunopurified enzyme or enzyme partially purified by (NH(4))(2)SO(4) precipitation, 2-3-fold increases in the V(max) of MCD and a 40% decrease in its K(m) for malonyl-CoA (190 versus 119 micrometer) were observed in rat gastrocnemius muscle after 5 min of contraction, induced by electrical stimulation of the sciatic nerve. The increase in MCD activity was markedly diminished when immunopurified enzyme was treated with protein phosphatase 2A or when phosphatase inhibitors were omitted from the homogenizing solution and assay mixture. Incubation of extensor digitorum longus muscle for 1 h with 2 mm 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, a cell-permeable activator of AMPK, increased MCD activity 2-fold. Here, too, addition of protein phosphatase 2A to the immunopellets reversed the increase of MCD activity. The results strongly suggest that activation of AMPK during muscle contraction leads to phosphorylation of MCD and an increase in its activity. They also suggest a dual control of malonyl-CoA concentration by ACC and MCD, via AMPK, during exercise.     

Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis

BACKGROUND: Plant-derived flavonoids, which occur abundantly in our daily dietary intake, possess antitumor, antibacterial, and free radical scavenging properties. They form active constituents of a number of herbal and traditional medicines. Several flavonoids have been shown to exert their action by interacting with DNA topoisomerases and promoting site-specific DNA cleavage. Therefore, flavonoids are potential candidates in drug design. We report here that, although the flavonoids luteolin and quercetin are potent antileishmanial agents, luteolin has great promise for acting as a lead compound in the chemotherapy of leishmaniasis, a major concern in developing countries. MATERIALS AND METHODS: Kinetoplast DNA (kDNA) minicircle cleavage in drug-treated parasites was measured by electrophoresis of the total cellular DNA, followed by Southern hybridization using 32P labeled kDNA as a probe. Cell cycle progression and apoptosis were measured by flow cytometry using propidium iodide and fluorescein isothiocyanate (FITC)-labeled Annexin V. RESULTS: Luteolin and quercetin inhibited the growth of Leishmania donovani promastigotes and amastigotes in vitro, inhibited DNA synthesis in promastigotes, and promoted topoisomerase-II-mediated linearization of kDNA minicircles. The IC50 values of luteolin and quercetin were 12.5 microM and 45.5 microM, respectively. These compounds arrest cell cycle progression in L. donovani promastigotes, leading to apoptosis. Luteolin has no effect on normal human T-cell blasts. Both luteolin and quercetin reduced splenic parasite burden in animal models. CONCLUSION: Luteolin and quercetin are effective antileishmanial agents. Quercetin has nonspecific effects on normal human T cells, but luteolin appears nontoxic. So, luteolin can be a strong candidate for antileishmanial drug design.     

Genomic diversity among Vibrio cholerae O139 strains isolated in Bangladesh and India between 1992 and 1998

In order to assess the extent of genomic diversity among Vibrio cholerae O139 strains, restriction fragment length polymorphisms in two genetic loci, rrn and ctx, were studied. Analysis of 144 strains isolated from different regions of Bangladesh and India between 1992 and 1998 revealed the presence of at least six distinct ribotypes (B-I through B-VI) of which three were new ribotypes, and one of these was represented by a nontoxigenic O139 strain. Strains of ribotypes B-I through B-V shared 11 different CTX genotypes (A through K). Antimicrobial resistance patterns of the strains varied independently of their ribotypes and CTX genotypes. Results of this study suggest that V. cholerae O139 is undergoing rapid genetic changes leading to the origination of new variants, and temporal changes in antimicrobial resistance patterns may be contributing to the selection of different variants.