Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid

The present results demonstrate that pyridoxal, pyridoxal 5′-phosphate (PLP) and pyridoxal 5′-diphospho-5′-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the ε-amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (K_i = 40 μM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK^*DSIR). Targeted lysine (K^*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K₅₃₂), Candida albicans (K₄₆₈), Saccharomyces cerevisiae (K₄₅₈) and Schizosaccharomyces pombe (K₅₀₅). In the human enzyme, K₅₃₂, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K₅₃₂–PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.     

Hereditary deafness: molecular genetics

This article outlines recent advances in explaining hereditary deafness in molecular terms, focusing on isolated (i.e. nonsyndromic) hearing loss. The number of genes identified (36 to date) is growing rapidly. However, difficulties inherent in genetic linkage analysis, coupled with the possible involvement of environmental causes, have so far prevented the characterization of the main genes causative or predisposing to the late-onset forms of deafness.     

Effective use of preparative chiral HPLC in a preclinical drug synthesis

Access to a key 3-aryl-delta-lactone intermediate in enantiopure form using preparative chiral chromatography allowed expedited preparation of an important drug discovery target. A preclinical drug discovery strategy that combines rapid route discovery with effective use of preparative chiral chromatography can result in significant savings of both time and labor.     

Potassium channel structures: do they conform?

Potassium channels are signalling elements vital to vertebrate neurotransmission, and cardiac and renal function. Two inherent qualities equip them for their role in the interconversion of chemical and electrical messages: high selectivity for potassium ions and the ability to open (gate) on cue. The crystal structure of KcsA, published in 1998, explained much about potassium selectivity and high ion flux. The enormous diversity of potassium channels (some hundreds of genes in humans) may have hampered similar progress in understanding gating processes. The recent determination of several representative structures has provided us with a valuable reference for discriminating between features that are utilized in gating across the potassium channel genre and features that determine responsiveness to family-specific gating cues.     

Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana

Salicylic acid (SA) is reported to protect plants from heat shock (HS), but insufficient is known about its role in thermotolerance or how this relates to SA signaling in pathogen resistance. We tested thermotolerance and expression of pathogenesis-related (PR) and HS proteins (HSPs) in Arabidopsis thaliana genotypes with modified SA signaling: plants with the SA hydroxylase NahG transgene, the nonexpresser of PR proteins (npr1) mutant, and the constitutive expressers of PR proteins (cpr1 and cpr5) mutants. At all growth stages from seeds to 3-week-old plants, we found evidence for SA-dependent signaling in basal thermotolerance (i.e. tolerance of HS without prior heat acclimation). Endogenous SA correlated with basal thermotolerance, with the SA-deficient NahG and SA-accumulating cpr5 genotypes having lowest and highest thermotolerance, respectively. SA promoted thermotolerance during the HS itself and subsequent recovery. Recovery from HS apparently involved an NPR1-dependent pathway but thermotolerance during HS did not. SA reduced electrolyte leakage, indicating that it induced membrane thermoprotection. PR-1 and Hsp17.6 were induced by SA or HS, indicating common factors in pathogen and HS responses. SA-induced Hsp17.6 expression had a different dose-response to PR-1 expression. HS-induced Hsp17.6 protein appeared more slowly in NahG. However, SA only partially induced HSPs. Hsp17.6 induction by HS was more substantial than by SA, and we found no SA effect on Hsp101 expression. All genotypes, including NahG and npr1, were capable of expression of HSPs and acquisition of HS tolerance by prior heat acclimation. Although SA promotes basal thermotolerance, it is not essential for acquired thermotolerance.     

Effect of administration of lovastatin on the development of late pulmonary effects after whole-lung irradiation in a murine model

Our group's work on late radiation effects has been governed by the hypothesis that the effects observed in normal tissues are a consequence of multicellular interactions through a network of mediators. Further, we believe that inflammation is a necessary component of this process. We therefore investigated whether the recruitment of mononuclear cells, observed during the pneumonitic period in the irradiated normal lung, is dependent on the expression of chemokines, notably Mcp1. Since statins have been shown to reduce chemokine expression and inflammatory cell recruitment, we specifically examined whether statins could be used to reduce monocyte recruitment. Mice received 15 Gy whole-lung irradiation; treated groups were administered lovastatin three times weekly starting either immediately or 8 weeks postirradiation. At subsequent intervals, animals were killed humanely, and cellular, mRNA and protein analyses were undertaken. Statin-treated animals demonstrated a statistically significant reduction in both macrophage and lymphocyte populations in the lung compared to radiation alone as well as improved rates of survival and decreased collagen content. In addition, ELISA measurements showed that radiation-induced increases in Mcp1 protein were reduced by statin treatment. Additional experiments are needed to assess whether statins offer a potential treatment for the amelioration of late effects in breast and lung cancer patients undergoing radiation therapy.     

Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

NPY and NPY Y1 receptor effects on noradrenaline overflow from the rat brain in vitro

Neurotransmitters and neuropeptides play important roles in the regulation of various neuroendocrine functions particularly feeding. The aim of this study was to investigate whether a functional interaction occurs among neuropeptide Y (NPY) at NPY Y₁ receptors and noradrenaline overflow, as this may contribute to the regulation of appetite. The release of endogenous noradrenaline and its metabolite 3,4-dihydroxyphenylglycol (DHPG) were examined from hypothalamic and medullary prisms using the technique of in vitro superfusion and high performance liquid chromatography (HPLC) with coulometric detection. Noradrenaline and DHPG overflow was investigated at rest, in response to NPY (0.1 μM) and in response to the NPY Y₁ receptor agonist, [Leu³¹,Pro³⁴]NPY (0.1 μM). Perfusion with NPY and [Leu³¹,Pro³⁴]NPY significantly reduced noradrenaline overflow from the hypothalamus and medulla. Perfusion with NPY and [Leu³¹,Pro³⁴]NPY was without significant effect on hypothalamic DHPG overflow, while medullary DHPG overflow was significantly reduced by NPY and [Leu³¹,Pro³⁴]NPY. Results from this study provide evidence of NPY Y₁ receptor-mediated inhibition of noradrenaline release in the hypothalamus and medulla, further illustrating a complex interaction between neurotransmitters and neuropeptides within the rat brain.