Effectiveness of a feedback signal in a computer mouse on upper extremity musculoskeletal symptoms: a randomised controlled trial with an 8-month follow-up.

OBJECTIVES: To study the effectiveness of using a computer mouse with a feedback signal for upper extremity musculoskeletal symptoms in office workers. METHODS: A randomised controlled trial with 8 months of follow-up was carried out. The intervention consisted of a computer mouse with a feedback signal. In total, 354 subjects were allocated to the intervention group or the control group. Measurements were performed with electronic questionnaires at baseline, after 4 months and after 8 months. Outcome variables were the prevalence and incidence of upper extremity musculoskeletal symptoms and disability in the upper extremities. The intervention process was evaluated by software registration. RESULTS: The use of the mouse with a feedback signal resulted in a significant decrease in duration of mouse usage over time. No differences were found in the number of mouse usage rest breaks. No differences were found in the prevalence (p = 0.29) or incidence (p = 0.832) of upper extremity musculoskeletal symptoms between the groups 8 months after baseline. The prevalence decreased from 49% at baseline to 44% after 8 months in the control group, while it remained at 36% in the intervention group. The incidence was 21% in the control group and 22% in the intervention group. Among the population with upper extremity musculoskeletal symptoms at baseline, the risk of experiencing symptoms after 8 months did not differ between the groups (p = 0.49). Minor disability was found in both groups. In the intervention group, a lower level of physical disability over time was reported than in the control group (p = 0.02). CONCLUSIONS: A feedback signal computer mouse does not affect the prevalence and incidence of upper extremity musculoskeletal symptoms, but it does lower disability scores. Given the high prevalence, studying preventive interventions for upper extremity musculoskeletal symptoms is of high importance. TRIAL REGISTRATION NUMBER: ISRCTN13222474.

Fibrinogen binding to the integrin alpha(IIb)beta(3) modulates store-mediated calcium entry in human platelets.

Effects of the occupation of integrin alpha(IIb)beta(3) by fibrinogen on Ca(++) signaling in fura-2-loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca(++)] concentrations ([Ca(++)](i)) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca(++) but not in the absence of external Ca(++) or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca(++) entry. Fibrinogen also inhibited store-mediated Ca(++) entry (SMCE) activated after Ca(++) store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to alpha(IIb)beta(3) was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca(++) chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60(src) to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin alpha(IIb)beta(3) inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60(src). This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected.

Quantification of Mycelium of Botrytis spp. and the Antagonist Ulocladium atrum in Necrotic Leaf Tissue of Cyclamen and Lily by Fluorescence Microscopy and Image Analysis.

ABSTRACT A technique was developed to localize and quantify the internal mycelial colonization of necrotic leaf tissue of cyclamen (Cyclamen persicum) or lily (Lilium) by pathogenic Botrytis spp. and the antagonist Ulocladium atrum. This technique allows investigation of competitive substrate colonization by both fungi, which is a key process for biological control of Botrytis spp. by U. atrum. A combination of differential fluorescent labeling and image analysis was applied on cryostat sections of necrotic leaf tissue. Botrytis mycelium was labeled specifically by indirect immunofluorescence using a monoclonal antibody specific for Botrytis spp. And an antimouse fluorescein conjugate. Wheat germ agglutinin conjugated to the fluorochrome TRITC was used to label mycelium of both fungi. Image analysis was used to measure the relative surface area of the cryostat section covered by fluorescing hyphae of Botrytis spp. and by fluorescing hyphae of both fungi. A mathematical conversion was derived and used to calculate the relative mycelial volume of each fungal species in the necrotic tissue based on the measured relative surface areas. Temporal aspects of substrate colonization were studied in a short time series. An analysis of components of variance provided insight into spatial colonization patterns for the fungal species involved and allowed the design of efficient sampling strategies for future experiments.

Estimation of lipid reserves in unstained living and dead nematodes by image analysis.

During storage, non-feeding stages of entomopathogenic nematodes become visibly more transparent due to depletion of energy reserves. Optical density per unit area (OD per area) of infective juveniles of Steinernerna carpocapsae (All) and two Heterorhabditis isolates (UK211 and HF85) was measured with an image analysis system and compared with neutral lipid levels obtained by Oil Red O staining. Optical density (OD) measurements were compared with triglyceride levels of UK211 and HF85. Good correlations between OD per area and neutral lipids (0.90) and between OD and triglycerides (0.87) were found. Thus, OD reflects lipid levels and can be used as an indicator of lipid reserves in these nematodes. Heat-killing of nematodes had no significant effect on OD measurements, but length increased significantly. Storage in a triethanolamine in formaldehyde solution decreased the OD and OD/area by about 5% to 8%. An additional advantage of the image analysis method described is that repeated measurements can be performed on live nematodes. Key words: entomopathogenic nematode, Heterorhabditis, image analysis, neutral lipid, Oil Red O, optical density, Steinernema, triglyceride.

Kinetic and modelling studies of NAD+ and poly(ethylene glycol)-bound NAD+ in horse liver alcohol dehydrogenase.

Poly(ethylene glycol)-bound nicotinamide adenine dinucleotide (PEG-NAD+) has been successfully employed in the continuous production of L-amino acids from the corresponding alpha-keto acids by stereospecific reductive amination. Like many other dehydrogenases also horse liver alcohol dehydrogenase (HLADH) appears to be active with PEG-NAD+ as coenzyme, although the turnover number is three to four times lower. The possibilities were considered that the PEG-tail of a PEG-NAD+ bound to one active site of the HLADH dimer prevents the binding of another PEG-NAD+ to the second site, or that the PEG-tail causes destabilization of the active dimer. Both could be ruled out by kinetic studies. Neither can the observed lower intrinsic reactivity of PEG-NAD+ account for the diminished activity of the enzyme. Molecular dynamics studies, on the other hand, show that the pulling action of the polymer chain shifts the NAD position in the active site in the outside direction, causing small but significant changes in the enzyme/coenzyme interactions of a sufficient extent to explain the experimental results.

Purification and Characterization of an l-Amino Amidase from Mycobacterium neoaurum ATCC 25795.

An l-amino amidase from Mycobacterium neoaurum ATCC 25795 responsible for the enantioselective resolution of dl-alpha-methyl valine amide was purified and characterized. The purification procedure included ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography, which resulted in a homogeneous preparation of the enzyme with a native molecular mass of 136 kDa and a subunit molecular mass of 40 kDa. The purified enzyme displayed the highest activity at 50 degrees C and at pH 8.0 and 9.5. The enzyme was strongly inhibited by the metal-chelating agent 1,10-phenanthroline, the disulfide-reducing agent dithiothreitol, and the cysteine proteinase inhibitor iodoacetamide. The purified amino amidase showed a unique l-enantioselective activity towards a broad range of both alpha-H- and alpha-alkyl-substituted amino acid amides, with the highest activity towards the cyclic amino acid amide dl-proline amide. No activity was measured with dl-mandelic acid amide nor with the dipeptide l-phenylalanine-l-leucine. The highest catalytic efficiency (k(cat)/K(m) ratio) was measured with dl-alpha-allyl alanine amide, dl-alpha-methyl phenylalanine amide, and dl-alpha-methyl leucine amide.

Purification and Characterization of an l-Aminopeptidase from Pseudomonas putida ATCC 12633.

An l-aminopeptidase of Pseudomonas putida, used in an industrial process for the hydrolysis of d,l-amino acid amide racemates, was purified to homogeneity. The highly l-enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and alpha-H amino acid amides, e.g., l-phenylglycine amide.

New developments in the synthesis of natural and unnatural amino acids.

Amino acids play an important role in biochemistry and chemistry. They are the building blocks of proteins and play an essential role in the regulation of the metabolism of living organisms. In general, it can be stated that microbial processes (fermentation) are the industrial production methods of choice for large-scale production of naturally occurring proteinogenic L-alpha-H-amino acids, while for the production of synthetic D- and/or L-alpha-H-amino acids, several other methods are highly competitive. At DSM, several routes, i.e., (chemoenzymatic) synthesis, towards L-alpha-H and D-alpha-H-amino acids have been elaborated since the midseventies. A general process for the synthesis of natural as well as synthetic optically pure amino acids has been developed, using an enzymatic kinetic resolution step on racemic amino acid amides as the key step. In this case, both enantiomers of the alpha-H-amino acids are prepared in one single step. This process has been commercialized since 1988. More recent developments using L- or D-amino peptidases in combination with amino acid amide racemases and an asymmetric transformation concept are discussed.

Optimization of the separation of the Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides.

The separation by reversed-phase high-performance liquid chromatography of Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides was studied with respect to pH, organic modifier type and concentration and reversed-phase packing material. Drylab G was used to deduce optimum conditions. On the basis of the observed discrepancies between the computer predictions and experimental results, the gradient operation procedure with volatile buffers was improved. By repetitive chromatography on a 250 x 22 mm I.D. reversed-phase column, fourteen diastereomeric pairs were obtained in at least 97% purity and 60% yield, in amounts of 10-100 mg.

Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-fiber membrane reactor.

The aptitude of a hollow-fiber membrane reactor to determine lipase kinetics was investigated using the hydrolysis of triacetin catalyzed by lipase from Canadida cylindracea as a model system. The binding of the lipase to the membrane appears not to be very specific (surface adsorption), and probably its conformation is hardly altered by immobilization, resulting in an activity comparable to that of the enzyme in its native form. The reaction kinetics defined on the membrane surface area were found to obey Michaelis-Menten kinetics. The specific activity of the lipase in the membrane reactor was found to be significantly higher than in an emulsion reactor. The activity and stability of the enzyme immobilized on a hydrophilic membrane surface seem not to be influenced significantly by the choice of the membrane material. The hollow-fiber membrane reactor is a suitable tool to assess lipase kinetics in a fast and convenient way.

Peptides from chiral C alpha,alpha-disubstituted glycines. Synthesis and characterization, conformational energy computations and solution conformational analysis of C alpha-methyl, C alpha-isopropylglycine [(alpha Me)Val] derivatives and model peptides.

Conformational energy computations on Ac-L-(alpha Me)Val-NHMe indicate that turns and right-handed helical structures are particularly stable conformations for this chiral C alpha-methyl, C alpha-alkylglycyl residue. We have synthesized and characterized a variety of L-(alpha Me)Val derivatives and peptides (to the pentamer level). The results of the solution conformational analysis, performed using infrared absorption, 1H nuclear magnetic resonance, and circular dichroism, are in general agreement with those obtained from the theoretical investigation, in the sense that the L-(alpha Me)Val residue turns out to be a strong beta-turn and right-handed helix former. A comparison is also made with the conclusions extracted from published work on peptides rich in other C alpha-methyl, C alpha-alkylglycyl residues.

Peptides from chiral C alpha,alpha-disubstituted glycines. Crystallographic characterization of conformation of C alpha-methyl, C alpha-isopropylglycine [(alpha Me)Val] in simple derivatives and model peptides.

The molecular and crystal structures of one derivative and three model peptides (to the pentapeptide level) of the chiral C alpha,alpha-disubstituted glycine C alpha-methyl, C alpha-isopropylglycine [(alpha Me)Val] have been determined by X-ray diffraction. The derivative is mClAc-L-(alpha Me)Val-OH, and the peptides are Z-L-(alpha Me)Val-(L-Ala)2-OMe monohydrate, Z-Aib-L-(alpha Me)Val-(Aib)2-OtBu, and Ac-(Aib)2-L-(alpha Me)Val-(Aib)2OtBu acetonitrile solvate. The tripeptide adopts a type-I beta-turn conformation stabilized by a 1----4N--H...O = C intramolecular H-bond. The tetra- and pentapeptides are folded in regular right-handed 3(10)-helices. All four L-(alpha Me)Val residues prefer phi, psi angles in the right-handed helical region of the conformational map. The results indicate that: (i) the (alpha Me)Val residue is a strong type-I/III beta-turn and helix former, and (ii) the relationship between (alpha Me)Val chirality and helix screw sense is the same as that of C alpha-monosubstituted protein amino-acids. The implications for the use of the (alpha Me)Val residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.

A conformation-activity study of the [L-(alpha Me)Phe]3 analog of the formyl methionyl tripeptide chemoattractant.

CHO-L-Met-L-Leu-L-(alpha Me)Phe-OMe, an analog of the formyl methionyl tripeptide chemoattractant CHO-L-Met-L-Leu-L-Phe-OH, was synthesized by solution methods and fully characterized. This compound was prepared to determine the effect of the incorporation of a conformationally restricted C alpha,alpha-disubstituted alpha-amino acid residue at position 3 on the relation of three-dimensional structure to biological activity. The peptide was tested for its ability to induce granule enzyme secretion from rabbit peritoneal polymorphonuclear leukocytes. In parallel, a conformational analysis was performed in the crystal state by x-ray diffraction and in organic solution by infrared absorption and 1H nuclear magnetic resonance. These biological and conformational data are discussed in relation to those of the prototype tripeptide and its methyl ester.

NAD+ and NAD+ analogues in horse liver alcohol dehydrogenase. Relationship between reactivity and conformation simulated with molecular mechanics.

In the present study we show that the enzymatic activity of the coenzyme nicotinamide adenine dinucleotide (NAD+) and its analogues (C(O)NH2 replaced by C(S)NH2, C(O)CH3, C(O)H and CN) with horse liver alcohol dehydrogenase (LADH) (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) can be rationalized by their conformation in the active site determined with molecular mechanics (AMBER, assisted model building with energy refinement). In order to establish the relation between the hydride transfer rate and the conformation of the NAD+ and its analogues, kinetic experiments with the poor substrate isopropanol were carried out. It appears that the enzymatic activity can be readily explained by the geometry of the pyridinium ring, in particular the magnitude of the 'out-of-plane' rotation of the carboxamide side chain (or analogues). The latter is nicely illustrated in the case of 3-cyanopyridine adenine dinucleotide which lacks any 'out-of-plane' rotation and concomitantly exhibits no significant enzymatic activity.

New developments in the chemo-enzymatic production of amino acids.

Recent progress in the chemo-enzymatic production of amino acids is reviewed. Both recently developed commercial processes and potentially important new developments are discussed. Emphasis is placed on the use of acylases, aminopeptidases and hydantoinases. The discovery of D-specific enzymes in combination with racemases is an exciting and promising new area. Also, a goal-orientated approach towards the selective generation of these novel enzyme activities using in vivo protein engineering techniques is highlighted. The interest in dipeptide sweeteners has triggered a major research effort towards the production of L-phenylalanine and D-alanine. A number of methods for the production of these amino acids is briefly discussed. Finally, chemo-enzymatic methods for the synthesis of enantiomerically pure alpha-alkyl-alpha-amino acids are reviewed.

Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligodeoxynucleotides.

A new synthesis route for long phosphate-methylated oligodeoxynucleotides is described, which were used as antisense inhibitors of the DNA replication. Phosphate-methylated oligomers hybridize more strongly with natural DNA than their natural analogues, due to the absence of electrostatic interstrand repulsions. Compared with phosphate-ethylated and methyl phosphonate systems, phosphate-methylated systems are preferable as antisense DNA, which was concluded from the high Tm values and sharp melting transitions of duplexes of phosphate-methylated and natural DNA. By using the Sanger dideoxy technique, it was shown that a complementary phosphate-methylated 18-mer can effectively and site-specifically block the DNA replication process at room temperature.

Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase.

The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy-minimization procedures. Starting geometries were taken from X-ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co-workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essential interactions between NAD+ analogue, enzyme, Me2SO and Zn2+ without the necessity of additional X-ray data. The results presented here demonstrate that the reactivity of NAD+ derivatives as reported in literature can be qualitatively related to the position of the pyridine moiety in the active site.

Vanadium containing bromoperoxidase: An example of an oxidoreductase with high operational stability in aqueous and organic media.

The conversion is described of phenolsulphonephtalein (phenol red) to 3,3',5,5'-tetrabromophenolsulphonephthalein (bromophenol blue) by bromoper-oxidase from the brown alga Ascophyllum nodosum. This reaction provides a convenient assay for the detection of bromoperoxidase activity in vitro. Bromoperoxidase was shown to be stable under turnover conditions for three weeks at room temperature, catalyzing the bromination of phenol red into bromophenol blue. When stored at room temperature in organic sol vents such as acetone, methanol, ethanol [present up to 60% (v/v)], and 1-propanol [40% (v/v)], bromoperoxidase was stable for more than one month. As far as we know this is the first example of an oxidoreductase which displays such great stability. This enhances the applicability of the enzyme in organic synthesis.

Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite.

1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.