Cl- flux through a non-selective, stretch-sensitive conductance influences the outer hair cell motor of the guinea-pig. (57/630)

Outer hair cells underlie high frequency cochlear amplification in mammals. Fast somatic motility can be driven by voltage-dependent conformational changes in the motor protein, prestin, which resides exclusively within lateral plasma membrane of the cell. Yet, how a voltage-driven motor could contribute to high frequency amplification, despite the low-pass membrane filter of the cell, remains an enigma. The recent identification of prestin's Cl- sensitivity revealed an alternative mechanism in which intracellular Cl- fluctuations near prestin could influence the motor. We report the existence of a stretch-sensitive conductance within the lateral membrane that passes anions and cations and is gated at acoustic rates. The resultant intracellular Cl- oscillations near prestin may drive motor protein transitions, as evidenced by pronounced shifts in prestin's state-probability function along the voltage axis. The sensitivity of prestin's state probability to intracellular Cl- levels betokens a more complicated role for Cl- than a simple extrinsic voltage sensor. Instead, we suggest an allosteric modulation of prestin by Cl- and other anions. Finally, we hypothesize that prestin sensitivity to anion flux through the mechanically activated lateral membrane can provide a driving force that circumvents the membrane's low-pass filter, thus permitting amplification at high acoustic frequencies.  (+info)

Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. (58/630)

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.  (+info)

Potent inhibition of ribonuclease A by oligo(vinylsulfonic acid). (59/630)

Ribonuclease A (RNase A) can make multiple contacts with an RNA substrate. In particular, the enzymatic active site and adjacent subsites bind sequential phosphoryl groups in the RNA backbone through Coulombic interactions. Here, oligomers of vinylsulfonic acid (OVS) are shown to be potent inhibitors of RNase A that exploit these interactions. Inhibition is competitive with substrate and has Ki = 11 pm in assays at low salt concentration. The effect of salt concentration on inhibition indicates that nearly eight favorable Coulombic interactions occur in the RNase A.OVS complex. The phosphonic acid and sulfuric acid analogs of OVS are also potent inhibitors although slightly less effective. OVS is also shown to be a contaminant of MES and other buffers that contain sulfonylethyl groups. Oligomers greater than nine units in length can be isolated from commercial MES buffer. Inhibition by contaminating OVS is responsible for the apparent decrease in catalytic activity that has been observed in assays of RNase A at low salt concentration. Thus, OVS is both a useful inhibitor of RNase A and a potential bane to chemists and biochemists who use ethanesulfonic acid buffers.  (+info)

Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335. (60/630)

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70 degrees C, respectively. The activity was strongly enhanced in the presence of Cu(2+), Mn(2+), and Mg(2+) and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.  (+info)

Avasimibe induces CYP3A4 and multiple drug resistance protein 1 gene expression through activation of the pregnane X receptor. (61/630)

In vitro and clinical studies were conducted to characterize the potential of avasimibe, an acyl-CoA/cholesterol acyltransferase inhibitor to cause drug-drug interactions. Clinically, 3- and 6-fold increases in midazolam (CYP3A4 substrate) oral clearance were observed after 50 and 750 mg of avasimibe daily for 7 days, respectively. A 40% decrease in digoxin (P-glycoprotein substrate) area under the curve was observed with 750 mg of avasimibe daily for 10 days. In vitro studies were conducted to define the mechanisms of these interactions. Induction was observed in CYP3A4 activity and immunoreactive protein (EC50 of 200-400 nM) in primary human hepatocytes treated with avasimibe. Rifampin treatment yielded similar results. Microarray analysis revealed avasimibe (1 microM) increased CYP3A4 mRNA 20-fold, compared with a 23-fold increase with 50 microM rifampin. Avasimibe induced P-glycoprotein mRNA by about 2-fold and immunoreactive protein in a dose-dependent manner. Transient transfection assays showed that avasimibe is a potent activator of the human pregnane X receptor (hPXR) and more active than rifampin on an equimolar basis. Drug-drug interaction studies for CYP3A4 using pooled human hepatic microsomes and avasimibe at various concentrations, revealed IC50 values of 20.7, 1.6, and 3.1 microM using testosterone, midazolam, and felodipine as probe substrates, respectively. Our results indicate that avasimibe causes clinically significant drug-drug interactions through direct activation of hPXR and the subsequent induction of its target genes CYP3A4 and multiple drug resistance protein 1.  (+info)

Studies on estrification and sulphonation of riboflavin in the environment of highly concentrated sulphuric acid. (62/630)

The article presents investigations of riboflavin reactions in aqueous solutions of sulphuric acid. Analysis of UV/VIS, 1H NMR spectra and TLC indicates that at the beginning of the reaction ester of riboflavin were obtained and then sulphonation reaction took place. From the analysis of UV/VIS spectra the kinetics of the reaction was calculated, using own computer program.  (+info)

Identification of a rare sulfonic acid metabolite of andrographolide in rats. (63/630)

Andrographolide is widely used in clinic as an anti-inflammatory and antibiotic drug. In this paper, the metabolites of andrographolide in rats after single oral doses of 120 mg/kg were investigated. The structures of the metabolites were elucidated by high-resolution mass spectra, NMR spectroscopy including 1H NMR, 13C NMR, and two-dimensional NMR, through comparison to a synthetic standard. The main metabolite of andrographolide in rats was 14-deoxy-12(R)-sulfo andrographolide. In the proposed mechanism, the beta-carbon of alpha, beta-unsaturated carbonyl was attacked by sulfonic acid, to form the sulfonate compound. This was a rare metabolic reaction. It may be the main metabolic pathway of andrographolide in rats. The polarity of the sulfonate metabolite increased greatly and could be easily eliminated from body.  (+info)

Investigation of selective protein immobilization on charged protein array by wavelength interrogation-based SPR sensor. (64/630)

We have investigated the use of multilayer films of polyelectrolytes as selective surfaces to analyze protein interactions with a self-assembled SPR wavelength-shift sensor. Charged arrays were prepared by alternating adsorption of the charged polyelectrolytes, poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS). Multilayer formation was monitored with the SPR wavelength-shift sensor and a Spreeta SPR sensor. Protein immobilization on the charged surfaces, which was also analyzed by the SPR sensors, was dependent on the pI of the proteins. Tissue transglutaminase (tTGase) and beta-galactosidase (pIs, 5.1 and 5.3, respectively) were preferentially bound to the positively charged PDDA surface, whereas lysozyme (pI, 11.0) was selectively bound to the negatively charged PSS surface. Immobilization of tTGase on the PDDA surface was also dependent on the buffer pH. The interaction of tTGase with RhoA(V14), a constitutively active form of Rho, could be detected on the charged arrays with the wavelength-shift sensor. The arrays could be reutilized at least 5 times. Thus, it is likely that charged surfaces, assembled by the layer-by-layer method using polyelectrolytes, will prove useful for preparing selective protein arrays.  (+info)