Tenuazonic Acid, a Toxin Produced by Alternaria alternata

Fifty-seven of 87 isolates of Alternaria alternata (Fr) Keissler grown on autoclaved, moist corn-rice substrate and fed to rats were lethal. The major toxin produced was isolated and characterized as tenuazonic acid. Twenty of 23 toxigenic Alternaria isolates examined produced tenuazonic acid. No tenuazonic acid could be detected in either of the field samples of sorghum or blackeyed peas, which were heavily invaded by Alternaria.     

Sequencing, distribution, and inactivation of the dipeptidase A gene (pepDA) from Lactobacillus helveticus CNRZ32.

Previously, the gene for a general dipeptidase (pepDA) was isolated from a gene bank of Lactobacillus helveticus CNRZ32. The pepDA gene consists of a 1,422-bp open reading frame which could encode a polypeptide of 53.5 kDa. No significant identity was found between the deduced amino acid sequence of the pepDA product and the sequence for other polypeptides reported in GenBank. Southern hybridization studies with a pepDA probe indicated that the nucleotide sequence for pepDA is not well conserved among a variety of lactic acid bacteria. Growth studies indicated that a pepDA deletion had no detectable effect on growth rate or acid production by L. helveticus CNRZ32 in milk. Furthermore, no difference in total cellular dipeptidase activity was detected between the mutant and wild-type strains during logarithmic growth in MRS medium.     

alpha-actinin-2 couples to cardiac Kv1.5 channels, regulating current density and channel localization in HEK cells

Voltage-gated K(+) (Kv) channels are particularly important in the physiology of excitable cells in the heart and the brain. PSD-95 is known to cluster Shaker channels and NMDA receptors and the latter is known to couple through alpha-actinin-2 to the post-synaptic cytoskeleton [Wyszynski et al. (1997) Nature 385, 439-442], but the mechanisms by which Kv channels are linked to the actin cytoskeleton and clustered at specific sites in the heart are unknown. Here we provide evidence that Kv1.5 channels, widely expressed in the cardiovascular system, bind with alpha-actinin-2. Human Kv1.5 interacts via its N-terminus/core region and can be immunoprecipitated with alpha-actinin-2 both after in vitro translation and from HEK cells expressing both proteins. The ion channels and alpha-actinin-2 co-localize at the membrane in HEK cells, where disruption of the actin cytoskeleton and antisense constructs to alpha-actinin-2 modulate the ion and gating current density.     

Two divergent cellular src genes are expressed in Xenopus laevis.

Genomic and cDNA clones of the X. laevis src gene have been isolated and characterized by hybridization and DNA sequence analyses. The haploid genome of X. laevis contains two src genes, which can be distinguished from one another by virtue of sequence divergence in the 3' untranslated regions. Both of the genes are functional as indicated by the fact that oocytes contain RNAs transcribed from each of the genes. The two genes each encode an RNA which is 3.3 kb in length, or twice the length required to encode the 60,000 dalton src protein (pp60). Sequence analysis of the cDNA clones revealed that nearly all of the non-coding sequence is located at the 3' end. The availability of sequence data from cDNA clones has also made it possible for the first time to identify with certainty the carboxyl terminal sequence of a cellular pp60 molecule.     

Chronic Inflammation Alters Production and Release of Glutathione and Related Thiols in Human U373 Astroglial Cells

Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD.     

Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL

The regulatory-targeting subunit (RGL), also called GM) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/RGL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of RGL. To investigate the function of the phosphatase, RGL knockout mice were generated. Animals lacking RGL show no obvious defects. The RGL protein is absent from the skeletal and cardiac muscle of null mutants and present at approximately 50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by approximately 50% in the RGL-deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant RGL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by approximately 90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and RGL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that RGL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/RGL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.     

Absence of cytosine methylation at C-C-G-G and G-C-G-C sites in the rDNA coding regions and intervening sequences of Drosophila and the rDNA of other insects.

Cytosine residues in C-G dinucleotides are frequently methylated in eukaryote DNA. In DNA of the dinoflagellate C. cohnii, the sequence C-MeC-G-G apparently renders Hpa II (C-C-G-G) incapable of digesting whole cell DNA in general, and rDNA in particular. Msp I, which also recognizes C-C-G-G but cleaves irrespective of methylation, degrades C. cohnii DNA and produces rDNA segments of 10.2 to 1.4 kb. We have applied this Hpa II/Msp I test to unfractionated DNA, and to rDNA and the rDNA intervening sequence of Drosophila virilis embryos and adults. There is no evidence of C-MeC-G-G sequences in either developmental stage of this species. Absence of G-MeC-G-C from coding and intervening sequences of rDNA was shown in comparisons of Hha I (G-C-G-C) cleavage patterns of unfractionated DNA and cloned (unmodified) segments of rDNA. Comparisons of Hpa II and Msp I cleavage products of DNA from the house fly, the flesh fly and a bumblebee also revealed no internal cytosine methylation in the sequence C-C-G-G. Because amounts of McC in C-G dinucleotides vary greatly among species, from apparent nonexistence to substantial proportions, no inference may yet be drawn about the role of such base modifications in DNA.