Lipase catalyzed reactions of aliphatic and arylaliphatic carbonic acid esters

Symmetrical dialkyl carbonates and dibenzyl carbonates reacted with various nucleophiles in the presence of Candida antarctica lipase B in organic solvents. For example, reaction of dibutyl and dibenzyl carbonate with an alcohol gave a mixture of the mono- and disubstituted products. Aminolysis, however, afforded only the carbamates, without subsequent reaction to the ureum derivatives. The reaction rates were rather low compared with carboxylic esters; the reactivity increased in the order dimethyl相似文献   

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.     

Computational Investigations of the Chalcogen-Substituted Carboxylic Acids RC(=O)XH and RC(=X)OH and their Dimers [RC(=O)XH]2 and [RC(=X)OH]2 (X=S, Se, Te)

Semiempirical and ab initio theoretical methods have been used to investigate molecular structures of the chalcogen-substituted carboxylic acid isomers RC(=O)XH (chalcogenol acid) and RC(=X)OH (chalcogenon acid). A recent experimental report suggests that the chalcogenon isomers, although less stable at room temperature, predominate at low temperature in polar solvents and that there is only a small barrier to isomerization between the isomers. Theoretical calculations have been used to locate minimum energy structures of chalcogen-substituted carboxylic acid isomers and to calculate energy differences between pairs of isomers. Carboxylic acids are well known to dimerize, especially in the gas phase and in non-polar solvents. We have, therefore, also calculated energies of dimerization of the chalcogen-substituted acids by optimizing the geometries of the symmetric dimers. We note that the PM3 level of theory is only qualitatively correct for sulfur- and selenium-containing species but fails even qualitatively for the tellurium-containing compounds. Ab initio results confirm the experimental observations and provide good estimates of both isomerization and dimerization energies. We conclude that for many functional groups with tautomers RC(=X)YH and RC(=Y)XH, the more acidic tautomer is the one with the acid proton on the smaller, more electronegative atom, although in many cases this may not be the more stable tautomer.Electronic Supplementary Material available.     

In vivo neutralization of TNF-alpha promotes humoral autoimmunity by preventing the induction of CTL.

Neutralization of TNF-alpha in humans with rheumatoid arthritis or Crohn's disease has been associated with the development of humoral autoimmunity. To determine the effect of TNF-alpha neutralization on cell-mediated and humoral-mediated responses, we administered anti-TNF-alpha mAb to mice undergoing acute graft-vs-host disease (GVHD) using the parent-into-F(1) model. In vivo neutralization of TNF-alpha blocked the lymphocytopenic features characteristic of acute GVHD and induced a lupus-like chronic GVHD phenotype (lymphoproliferation and autoantibody production). These effects resulted from complete inhibition of detectable antihost CTL activity and required the presence of anti-TNF-alpha mAb for the first 4 days after parental cell transfer, indicating that TNF-alpha plays a critical role in the induction of CTL. Moreover, an in vivo blockade of TNF-alpha preferentially inhibited the production of IFN-gamma and blocked IFN-gamma-dependent up-regulation of Fas; however, cytokines such as IL-10, IL-6, or IL-4 were not inhibited. These results suggest that a therapeutic TNF-alpha blockade may promote humoral autoimmunity by selectively inhibiting the induction of a CTL response that would normally suppress autoreactive B cells.     

Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.

We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

Changes in the migration and adhesive activity of polymorphonuclear leukocytes as 1 of the characteristics of Gram-negative bacteria isolated from patients with suppurative lung destruction

The influence of Gram-negative bacteria on the migratory and adhesive activity of polymorphonuclear leukocytes (PMNL) in the peripheral blood of clinically normal donors has been studied by the specially developed method with the use of Boyden chambers. Pseudomonas and enterobacteria have been found to produce complex and various effects on the above-mentioned properties of PMNL. When incubated in fresh serum, Gram-negative bacteria are capable of enhancing the migratory activity of PMNL, this property being least pronounced in P. aeruginosa. The incubation of live bacteria from the authors' collection in the patients' sera or in sera obtained from normal donors and inactivated by heating induces no hemotaxis of PMNL, and P. aeruginosa strains even suppress it under such conditions. The isolated Gram-negative bacteria under study increase the number of highly adhesive PMNL in the population used in this investigation, but P. aeruginosa cultures do not produce such effect.     

Exposure to nuclear magnetic resonance imaging procedure attenuates morphine-induced analgesia in mice

Adult male mice exposed to a Nuclear Magnetic Resonance Imaging (NMRI) procedure during the mid-dark period and injected with morphine (10 mg/kg) failed to exhibit the normal nocturnally enhanced morphine analgesia response to a thermal stimulus that was displayed by mice exposed to a sham imaging procedure and treated with morphine (p less than .01). When tested during the mid-light period, animals exposed to the NMRI procedure and given morphine displayed attenuated analgesia levels relative to sham exposed mice (p less than .01) treated with morphine. However, the morphine induced analgesia was not totally abolished since the imaged mice still exhibited analgesia relative to saline treated mice (p less than .01). These results suggest that the magnetic and/or radio-frequency fields associated with the NMRI procedure alter both day- and night-time responses to morphine. These results may reflect magnetic field induced alterations in neuronal calcium binding and/or alterations in nocturnal pineal gland activity.     

Frequency of Erwinia carotovora in the Alyth Burn in eastern Scotland and the sources of the bacterium

Contamination of the Alyth Burn by Erwinia carotovora was monitored monthly over 2 years at nine sites spread over a distance of ca 20 km. The bacterium was detected only once in the upper reaches of the river where it flows in uninhabited moorland but frequency of detection and contamination level tended to increase progressively as the river flowed through the middle reaches mostly in grassland to the lower reaches in arable land where the bacterium was almost always present. Erwinia populations rose from < 10² cells/1 before May to frequently > 10³ but < 10⁴ cells/l thereafter at sites in the arable land zone. A similar pattern was found in the grassland zone except that erwinia numbers were lower. Erwinia numbers at one site in the arable land zone were positively and negatively correlated with the river water temperature and flow rate respectively when there was a 1 month lag between the environmental data and the population recorded. More than 80% of isolates tested were E. carotovora subsp. carotovora.
Water from field drains in arable fields, especially those recently planted with potatoes, was frequently contaminated by E.c. carotovora , with numbers and a temporal pattern similar to those of the Alyth Burn. Drainage water from non-arable fields was rarely contaminated. Infected and rotting potatoes deposited in rivers temporarily contaminated the water. Survival of E. carotovora in dilute phosphate buffer was greater at pH 5˙7 than at pH 7˙7 and they survived for at least 10 d in river water.     

Characterization of polymorphic microsatellite markers for the Carnaby's cockatoo (Calyptorhynchus latirostris) and related black cockatoo species

Microsatellite loci were isolated from Carnaby's black cockatoo (Calyptorhynchus latirostris: Aves), a highly valued, endangered, and endemic species of bird from Western Australia. This study describes three dinucleotide and one tetranucleotide microsatellite loci for which the primers produced clear and polymorphic amplification patterns with between two and nine alleles and moderate levels of variability. Two additional dinucleotide markers which were monomorphic in the Carnaby's cockatoo were able to amplify and were polymorphic in two other species of black cockatoo, greatly increasing the utility of these markers.     

Efficacy of phototherapy in non-haemolytic hyperbilirubinaemia.

Clinical experience of phototherapy for non-haemolytic hyperbilirubinaemia in 3999 infants in Kandang Kerbau Hospital, Singapore, is documented. Phototherapy was most effective in extremely preterm infants with very low birth weight (gestation less than or equal to 32 weeks, birth weight less than or equal to 1500 g) and least effective in full term infants with very low birth weight (gestation greater than or equal to 37 weeks, birth weight less than or equal to 1500 g) and large preterm infants (gestation less than 37 weeks, birth weight greater than 2270 g). Overall, phototherapy was effective in almost all the infants, with a failure rate of only 2.00/1000 infants. No characteristic features common to all the failures could be detected. The bilirubin rebound was usually mild; repeat phototherapy was required in only 30 infants (7.50/1000), with the response to the second exposure comparable to that to the first. No infant required a third exposure. All the infants tolerated phototherapy well, none developing any illness that could be attributed to the treatment. This clinical experience shows that phototherapy for the treatment of nonhaemolytic hyperbilirubinaemia is effective and safe.