Blockage of human T lymphocyte Kv1.3 channels by Pi1, a novel class of scorpion toxin

Using the patch-clamp technique we determined that Pandinus imperator toxin Pi1, a recently described peptide toxin having four disulfide bridges instead of the usual three in scorpion toxins, blocked Kv1.3 channels of human T lymphocytes from the extracellular side with a 1:1 stoichiometry. Kv1.3 block was instantaneous and removable with toxin-free extracellular solution. The toxin did not influence activation or inactivation of the channels. We found that Pi1 blocked Kv1.3 with less affinity (K(d) = 11.4 nM) than the structurally related three disulfide bridge containing toxins Pi2 (50 pM) and Pi3 (0.5 nM). The fourth disulfide bridge in Pi1 had no influence on the channel binding ability of the toxin; the less effective block was due to differences in amino acid side chain properties at positions 11 and 35.     

Lipase-catalyzed enantiotope selective acetylation of 2-acyloxypropane-1,3-diols. Influence of the acyl moiety on the selectivity

Preparation and lipase-catalyzed enantiotope selective acetylation of the prochiral 2-acyloxypropane-1,3-diols (1a–h) including sulfonic ester (1a–c) and carboxylic ester (1d–h) series is described. A strong influence of the acyl moiety in these diols on the enantiotope selectivity of the porcine pancreatic lipase (PPL)-catalyzed reaction with vinyl acetate was observed. The best results were achieved with the 2-(4-methylbenzoyl)oxy- and cyclohexanecarbonyloxypropane-1,3-diols (1g and 1h) resulting in acetylated products (2g) of ≥98% e.e. in 77% yield and (2h) of 95% e.e. in 66% yield, respectively.     

Catalysis of serine oligopeptidases is controlled by a gating filter mechanism

Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. A recent crystal structure determination of prolyl oligopeptidase has suggested a way for substrate selection involving an unclosed seven-bladed β-propeller domain. We have engineered a disulfide bond between the first and seventh blades of the propeller, which resulted in the loss of enzymatic activity. These results provided direct evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins to prevent accidental proteolysis.     

DNA excision repair and DNA damage-induced apoptosis are linked to Poly(ADP-ribosyl)ation but have different requirements for p53

Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD(+) to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death.     

Metabolic Changes in Rat Brain After Prolonged Ethanol Consumption Measured by 1H and 31P MRS Experiments

SUMMARY 1. In vivo ¹H and ³¹P magnetic resonance spectroscopy techniques were applied to reveal biochemical changes in the rat brain caused by prolonged ethanol consumption.2. Three models of ethanol intoxication were used.3. ¹H MRS showed a significant decrease in the concentration of myo-inositol in the brain of rats fed with 20% ethanol for 8 weeks. This change is consistent with perturbances in astrocytes. On the other hand, N-acetyl aspartate and choline content did not differ from controls.4. ³¹P MRS did not reveal any significant changes in the high-energy phosphates or intracellular free Mg²⁺ content in the brain of rats after 14 weeks of 20% ethanol drinking. The intracellular pH was diminished.5. By means of a ³¹P saturation transfer technique, a significant decrease was observed for the pseudo first-order rate constant k _for of the creatine kinase reaction in the brain of rats administered 30% ethanol for 3 weeks using a gastric tube.6. The ¹H MRS results may indicate that myo-inositol loss, reflecting a disorder in astrocytes, might be one of the first changes associated with alcoholism, which could be detected in the brain by means of in vivo ¹H MRS.7. The results from ³¹P MRS experiments suggest that alcoholism is associated with decreased brain energy metabolism.8. ³¹P saturation transfer, which provides insight into the turnover of high-energy phosphates, could be a more suitable technique for studying the brain energetics in chronic pathological states than conventional ³¹P MRS.     

Expression patterns of two putative odorant-binding proteins in the olfactory organs of Drosophila melanogaster have different implications for their functions

The aqueous medium bathing the dendrites of olfactory neurons contains high concentrations of odorant-binding proteins (OBPs) whose role is still unclear. OBPs may facilitate interactions between odorants and their membrane-bound receptors, perhaps by increasing the water solubility of hydrophobic molecules. Alternatively, OBPs may be involved in the inactivation of odorants and other volatile molecules, preventing desensitization and/or protecting olfactory neurons from toxic chemicals. We report here novel features of the localization of two putative OBPs, PBPRP2 and PBPRP5, that have important and different implications for their role in olfaction. Unlike several other putative OBPs of Drosophila melanogaster that are only found in adult olfactory organs, PBPRP5 is also expressed in the larval olfactory organs, suggesting that it plays a common role in olfaction at both stages. In the adult, PBPRP5 expression is restricted to the sensillum lymph that bathes the olfactory dendrites of a subset of olfactory hairs, the basiconic sensilla. Since individual basiconic sensilla differ in olfactory specificity, PBPRP5 may be able to bind to and mediate olfactory responses to a wide range of odorants. In contrast, PBPRP2 is present in the space immediately below the antennal cuticle and in the outer cavity of approximately 30% of the double-walled coeloconic sensilla on the antennal surface. In neither case is PBPRP2 in contact with the dendritic membranes of olfactory neurons, making a carrier function unlikely for this protein. Instead, PBPRP2 may act as a sink, binding to odorants and other volatile chemicals and limiting their interactions with olfactory neurons.     

A simple and efficient method for PCR amplifiable DNA extraction from ancient bones

A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500–1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification.     

Linkage and LOH studies in 19 cylindromatosis families show no evidence of genetic heterogeneity and refine the CYLD locus on chromosome 16q12–q13

Familial cylindromatosis is an autosomal dominant predisposition to multiple neoplasms of the skin appendages. The susceptibility gene has previously been mapped to chromosome 16q12-q13 and has features of a recessive oncogene/tumour suppressor gene. We have now evaluated 19 families with this disease by a combination of genetic linkage analysis and loss of heterozygosity in cylindromas from affected individuals. All 15 informative families show linkage to this locus, providing no evidence for genetic heterogeneity. Recombinant mapping has placed the gene in an interval of approximately 1 Mb. There is no evidence, between families, of haplotype sharing that might be indicative of common founder mutations.