Substitutions in a flexible loop of horse liver alcohol dehydrogenase hinder the conformational change and unmask hydrogen transfer.

When horse liver alcohol dehydrogenase binds coenzyme, a rotation of about 10 degrees brings the catalytic domain closer to the coenzyme binding domain and closes the active site cleft. The conformational change requires that a flexible loop containing residues 293-298 in the coenzyme binding domain rearranges so that the coenzyme and some amino acid residues from the catalytic domain can be accommodated. The change appears to control the rate of dissociation of the coenzyme and to be necessary for installation of the proton relay system. In this study, directed mutagenesis produced the activated Gly293Ala/Pro295Thr enzyme. X-ray crystallography shows that the conformations of both free and complexed forms of the mutated enzyme and wild-type apoenzyme are very similar. Binding of NAD(+) and 2,2, 2-trifluoroethanol do not cause the conformational change, but the nicotinamide ribose moiety and alcohol are not in a fixed position. Although the Gly293Ala and Pro295Thr substitutions do not disturb the apoenzyme structure, molecular modeling shows that the new side chains cannot be accommodated in the closed native holoenzyme complex without steric alterations. The mutated enzyme may be active in the "open" conformation. The turnover numbers with ethanol and acetaldehyde increase 1.5- and 5.5-fold, respectively, and dissociation constants for coenzymes and other kinetic constants increase 40-2,000-fold compared to those of the native enzyme. Substrate deuterium isotope effects on the steady state V or V/K(m) parameters of 4-6 with ethanol or benzyl alcohol indicate that hydrogen transfer is a major rate-limiting step in catalysis. Steady state oxidation of benzyl alcohol is most rapid above a pK of about 9 for V and V/K(m) and is 2-fold faster in D(2)O than in H(2)O. The results are consistent with hydride transfer from a ground state zinc alkoxide that forms a low-barrier hydrogen bond with the hydroxyl group of Ser48.     

Spectral Repeat Finder (SRF): identification of repetitive sequences using Fourier transformation

MOTIVATION: Repetitive DNA sequences, besides having a variety of regulatory functions, are one of the principal causes of genomic instability. Understanding their origin and evolution is of fundamental importance for genome studies. The identification of repeats and their units helps in deducing the intra-genomic dynamics as an important feature of comparative genomics. A major difficulty in identification of repeats arises from the fact that the repeat units can be either exact or imperfect, in tandem or dispersed, and of unspecified length. RESULTS: The Spectral Repeat Finder program circumvents these problems by using a discrete Fourier transformation to identify significant periodicities present in a sequence. The specific regions of the sequence that contribute to a given periodicity are located through a sliding window analysis, and an exact search method is then used to find the repetitive units. Efficient and complete detection of repeats is provided together with interactive and detailed visualization of the spectral analysis of input sequence. We demonstrate the utility of our method with various examples that contain previously unannotated repeats. A Web server has been developed for convenient access to the automated program. AVAILABILITY: The Web server is available at http://www.imtech.res.in/raghava/srf and http://www2.imtech.res.in/raghava/srf     

Quantitation of tadalafil in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionization

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Solvent specified conformation in poly(alpha-L-glutamic acid) thin films

The ability to control conformational properties of polypeptides in their films is of considerable interest for many possible applications of these materials. By rational choice of the solvent system for film fabrication, control over the conformation of the main chain, the intermolecular hydrogen bonding in the side chain is easily achieved in poly(alpha-L-glutamic acid) (PLGA) thin films. The spectral data from circular dichromism (CD), FT-IR, and solid state (13)C NMR spectroscopies suggest that the beta-sheet conformation is dominant in PLGA films cast from trifluoroacetic acid (TFA) solution, whereas the right-handed alpha-helix is dominant in those cast from pyridine or DMF solution. In comparison with films cast from TFA solutions, the films fabricated from pyridine or DMF solutions exhibit strong intermolecular hydrogen bondings between -COOH groups and have a more ordered arrangement of side chains. Moreover, the extent of alpha-helix conformation of the PLGA backbone in films cast from pyridine or DMF solution is several times higher than that observed in the PLGA powder precipitated from aqueous solution at pH 4. All spectroscopic studies indicate clearly that the solvents (used for casting these films) play a crucial role in directing the organization of PLGA in these thin films.     

Noninvasive identification of the total peripheral resistance baroreflex

We propose two identification algorithms for quantitating the total peripheral resistance (TPR) baroreflex, an important contributor to short-term arterial blood pressure (ABP) regulation. Each algorithm analyzes beat-to-beat fluctuations in ABP and cardiac output, which may both be obtained noninvasively in humans. For a theoretical evaluation, we applied both algorithms to a realistic cardiovascular model. The results contrasted with only one of the algorithms proving to be reliable. This algorithm was able to track changes in the static gains of both the arterial and cardiopulmonary TPR baroreflex. We then applied both algorithms to a preliminary set of human data and obtained contrasting results much like those obtained from the cardiovascular model, thereby making the theoretical evaluation results more meaningful. This study suggests that, with experimental testing, the reliable identification algorithm may provide a powerful, noninvasive means for quantitating the TPR baroreflex. This study also provides an example of the role that models can play in the development and initial evaluation of algorithms aimed at quantitating important physiological mechanisms.     

Cartilage interstitial fluid load support in unconfined compression

Under physiological conditions of loading, articular cartilage is subjected to both compressive strains, normal to the articular surface, and tensile strains, tangential to the articular surface. Previous studies have shown that articular cartilage exhibits a much higher modulus in tension than in compression, and theoretical analyses have suggested that this tension–compression nonlinearity enhances the magnitude of interstitial fluid pressurization during loading in unconfined compression, above a theoretical threshold of 33% of the average applied stress. The first hypothesis of this experimental study is that the peak fluid load support in unconfined compression is significantly greater than the 33% theoretical limit predicted for porous permeable tissues modeled with equal moduli in tension and compression. The second hypothesis is that the peak fluid load support is higher at the articular surface side of the tissue samples than near the deep zone, because the disparity between the tensile and compressive moduli is greater at the surface zone. Ten human cartilage samples from six patellofemoral joints, and 10 bovine cartilage specimens from three calf patellofemoral joints were tested in unconfined compression. The peak fluid load support was measured at 79±11% and 69±15% at the articular surface and deep zone of human cartilage, respectively, and at 94±4% and 71±8% at the articular surface and deep zone of bovine calf cartilage, respectively. Statistical analyses confirmed both hypotheses of this study. These experimental results suggest that the tension–compression nonlinearity of cartilage is an essential functional property of the tissue which makes interstitial fluid pressurization the dominant mechanism of load support in articular cartilage.     

Lymph node topography of the head and neck in New Zealand White rabbits

Investigations of the lymphogenic metastatic spread of VX2 carcinomas in New Zealand White rabbits require an exact knowledge of the topography of cervical and facial lymph nodes. The topography of neck lymph nodes was evaluated from 16 rabbits macroscopically, histologically and by lymphographic investigations, and the possibility of their surgical removal (neck dissection) was examined. The upper aerodigestive tract and the ear of New Zealand White rabbits drain via four consistent groups of 12-18 lymph nodes. Except for the paratracheal lymph node, they are all easily accessible to surgery. The data presented in this study encourage the use of induced VX2 carcinomas in New Zealand White rabbits as an animal model to study the lymphogenic metastatic spread of squamous cell carcinomas of the head and neck. Such investigations could lead to an improvement of surgical and pharmaceutical treatment of this tumour entity.