Pigeon liver phosphoprotein phosphatase: an effective activator of pyruvate dehydrogenase in tissue homogenates

A fluoride-insensitive, non-metal-requiring pyruvate dehydrogenase phosphatase has been purified 730-fold from pigeon liver acetone powder and proven to be a convenient reagent for studies of pyruvate dehydrogenase complex and its activation (phosphorylation) state in brain and other tissues. This phosphatase is a cytoplasmic enzyme (Mr = 80,000), and fits the functional definition of a type 1 phosphoprotein phosphatase. The pigeon liver phosphatase can be used to activate pyruvate dehydrogenase complex in vitro in brain and other crude tissue homogenates. Addition of the cytoplasmic pigeon liver phosphatase to a homogenate from rat or mouse brain frozen in situ activated pyruvate dehydrogenase to levels comparable to that found in ischemic brain. The fluoride insensitivity of this phosphatase was used to develop a convenient technique for stopping the pyruvate dehydrogenase activation state in situ in cultured skin fibroblasts and then fully activating the complex in vitro in 5 min. The use of this phosphatase as a reagent can facilitate the study of pyruvate dehydrogenase activation defects in mammalian tissues including cultured cells in normal and disease states.     

Selection of the most appropriate life cycle inventory dataset: new selection proxy methodology and case study application

The International Journal of Life Cycle Assessment - Using generic or country-specific life cycle inventory (LCI) datasets for life cycle assessments (LCAs) with no site-specific LCI datasets can...     

Discovery and in vitro/in vivo studies of tetrazole derivatives as Kv1.5 blockers

A novel class of tetrazole-derived Kv1.5 blockers is disclosed. In in vitro studies, several compounds had IC₅₀s ranging from 180 to 550 nM. In vivo studies indicated that compounds 2f and 2j increased right atrial ERP about 40% without affecting ventricular ERP.     

Cross linking of polyglutamine domains catalyzed by tissue transglutaminase is greatly favored with pathological-length repeats: does transglutaminase activity play a role in (CAG)(n)/Q(n)-expansion diseases?

Protein aggregates are a hallmark of Huntington's disease (HD) and other inherited neurodegenerative diseases caused by an elongated (CAG)(n) repeat in the genome and to a corresponding increase in the size of the Q(n) domain in the expressed protein. When the protein associated with HD (huntingtin) contains <35 Q repeats disease does not occur. However, an n>/=40 leads to disease. Some investigators have proposed that aggregates in the nuclei of affected cells are toxic, but other workers have suggested that the aggregates may be neutral or even protective. Whether or not they are toxic, an understanding of the processes whereby the aggregates develop may shed light on the neuropathological processes involved in the (CAG)(n)/Q(n)-expansion disorders. Q(n) domains have a tendency to non-covalently self align as 'polar zippers' rendering them less soluble, but evidence that such polar zippers occur in the aggregates in intact HD brain has so far been limited. The human brain contains at least three Ca(2+)-dependent enzymes (transglutaminases, TGases) that catalyze protein cross-linking reactions, namely TGase 1, TGase 2 (tissue transglutaminase, tTGase) and TGase 3. Q(n) aggregates have been found by several groups to be excellent substrates of tTGase. Moreover, the activity toward the Q(n) domains increases greatly as n is increased to 40 or beyond. tTGase mRNA and total TGase activity are elevated in HD brain. Moreover, some evidence suggests that Ca(2+) homeostasis is disrupted in HD brain. We propose that the combination of increased huntingtin (or huntingtin fragment containing the Q(n) domain) in the nucleus, increased the ability of the Q(n) domains to act as substrate, increased Ca(2+) levels and increased inherent TGase activity all contribute to increased cross-linking of proteins in HD brain. At first the proteasome machinery can recognize and degrade the cross-linked proteins, but over time the proteasome machinery may be overwhelmed and protein aggregates will accumulate.     

Investigation of the interactions of polyhedral borane anions with serum albumins

The retention of polyhedral borane anions within tumor cells has been attributed to the possible formation of covalent bonds with nucleophilic protein substituents. In an effort to identify the nature of possible interactions between polyhedral borane anions and proteins, three polyhedral borane anions, [B(20)H(18)](2-), [B(20)H(17)OH](4-), and [B(20)H(17)SH](4-), were allowed to react with either bovine or human serum albumin. Reaction products were analyzed with matrix assisted laser desorption ionization (MALDI) mass spectrometry and gel electrophoresis. Evidence of disulfide bond formation was observed with the [B(20)H(17)SH](4-) anion, whereas no evidence of covalent binding was observed with the [B(20)H(18)](2-) and [B(20)H(17)OH](4-) ions. The potential for disulfide bond formation was confirmed by examining the reactions of the [B(20)H(17)SH](4-) ion with both DTNB and reduced glutathione. An understanding of the nature of the binding will provide a basis for the design and synthesis of boron-containing compounds for application in boron neutron capture therapy.     

A sensitive fluorometric assay for tissue transglutaminase

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).     

The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes

A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast. Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 microM. No activity was detected against Gram-negative bacteria, with the exception of some E. coli strains. Growth inhibitory activity was detected against some species of filamentous fungi. Defensin was not active against yeast. The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively. Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes. Native peptide levels were quantitated in both hemolymph and midgut tissues. The polytene chromosome position of the defensin locus was mapped by in situ hybridization.     

The effect of dexamethasone on disruption of ovarian steroid levels and receptors in female rats

This study was conducted to investigate if the injection of a single dose of dexamethasone may cause disruption of adult female rat gonadal function in terms of plasma and ovarian level of both androgen and estrogen, ovarian morphology, and changes in localization of androgen, estrogen and glucocorticoid receptors. Adult female Long Evans rats (n=50, 250-300 g) were used. At day 0 rats received subcutaneously 1 ml of saline (n=25; control group) or dexamethasone at 0.1 mg/kg (n=25, treated group). Rats were sacrificed in groups of five on days 10, 15, 20, 25 and 30 after injection. Blood samples and one ovary were collected to analyze dexamethasone, 17beta-estradiol (E2), testosterone (T) and androstenedione (A4) concentrations by amplified EIA. The remaining ovary was removed and processed for histopathology and immunocytochemistry. Differences between individual means were analyzed by Pairwise t-test and Bonferroni post test to asses whether values presented statistical significance. Increased E2, T and A4 levels were observed both in plasma and ovary samples in treated group when comparing with control (p< 0.01) at all days post-injection even when dexamethasone was undetectable. Ovarian morphology of treated group showed features compatible with female infertility. Inmmunolocalization of androgen and estrogen receptors showed that both were negative in treated group while controls showed highest positivity (AR +++, ER ++). Glucocorticoid receptor showed higher positivity in dexamethasone treated rats (GR ++) than in controls (GR +). Obtained results showed clear evidence that a single dose of dexamethasone may disrupt gonadal function in rats, and that possibly leads to infertility.