S1R agonist modulates rat platelet eicosanoid synthesis and aggregation.

Sigma-1 receptor (S1R) is detected in different cell types and can regulate intracellular signaling pathways. S1R plays a role in the pathomechanism of diseases and the regulation of neurotransmitters. Fluvoxamine can bind to S1R and reduce the serotonin uptake of neurons and platelets. We therefore hypothesized that platelets express S1R, which can modify platelet function. The expression of the SIGMAR1 gene in rat platelets was examined with a reverse transcription polymerase chain reaction and a quantitative polymerase chain reaction. The receptor was also visualized by immunostaining and confocal laser scanning microscopy. The effect of S1R agonist PRE-084 on the eicosanoid synthesis of isolated rat platelets and ADP- and AA-induced platelet aggregation was examined. S1R was detected in rat platelets both at gene and protein levels. Pretreatment with PRE-084 of resting platelets induced elevation of eicosanoid synthesis. The rate of elevation in thromboxane B2 and prostaglandin D2 synthesis was similar, but the production of prostaglandin E2 was higher. The concentration-response curve showed a sigmoidal form. The most effective concentration of the agonist was 2 µM. PRE-084 increased the quantity of cyclooxygenase-1 as detected by ELISA. PRE-084 also elevated the ADP- and AA-induced platelet aggregation. S1R of platelets might regulate physiological or pathological functions.

Functionalization of gold nanoparticles with amino acid, beta-amyloid peptides and fragment.

Gold nanoparticles (Au NPs) were functionalized by cysteine (Cys), beta-amyloid peptides (Cys(0)Abeta(1-28), Cys(0)Abeta(1-40), Abeta(1-42)) and a pentapeptide fragment (Leu-Pro-Phe-Phe-Asp-OH (LPFFD-OH)). Optical absorption spectra of these systems were recorded and the plasmon resonance maximum values (lambda(max)) of the UV-vis spectra together with the transmission electron microscopy (TEM) images were also analysed. Both TEM images and the appearance of a new absorption band between approximately 720 and 750 nm in the visible spectra of the Au-cysteine and Au-LPFFD-OH systems most probably indicate that upon addition of these molecules to Au NPs-containing aqueous dispersions formation of aggregates is occurred. The wavelength shift between the two observed absorption bands in cysteine- and pentapeptide-modified Au NPs systems are Deltalambda=185 and 193 nm, respectively. These results suggest that the monodisperse spherical gold nanoparticles were arranged to chained structure due to the effect of these molecules. For confirmation of the binding of citrate and cysteine onto the plasmonic metal surface (1)H NMR measurements were also performed. (1)H NMR results may suggest that the citrate layer on the metal surface is replaced by cysteine leading to a formation of organic double layer structure. In the presence of beta-amyloid peptides the aggregation was not observed, especially in the Au-Cys(0)Abeta(1-40) and Au-Abeta(1-42) systems, however compared to the cysteine or LPFFD-OH-containing gold dispersion with Cys(0)Abeta(1-28) measurable less aggregation were occurred. The spectral parameters clearly suggest that Abeta(1-42) can attach or bind to the surface of gold nanoparticles via both the apolar and the N-donors containing side-chains of amino acids and no aggregation in the colloidal gold dispersion was observed.

When is high-Ca+ microdomain required for mitochondrial Ca+ uptake?

Ca(2+) release from IP(3)-sensitive stores in the endoplasmic reticulum (ER) induced by Ca(2+)-mobilizing agonists generates high-Ca(2+) microdomains between ER vesicles and neighbouring mitochondria. Here we present a model that describes when such microdomains are required and when submicromolar [Ca(2+)] is sufficient for mitochondrial Ca(2+) uptake. Mitochondrial Ca(2+) uptake rate in angiotensin II-stimulated H295R adrenocortical cells correlates with the proximity between ER vesicles and the mitochondrion, reflecting the uptake promoting effect of high-Ca(2+) peri-mitochondrial microdomains. Silencing or inhibition of p38 mitogen-activated protein kinase (MAPK) or inhibition of the novel isoforms of protein kinase C enhances mitochondrial Ca(2+) uptake and abolishes the positive correlation between Ca(2+) uptake and ER-mitochondrion proximity. Inhibition of protein phosphatases attenuates mitochondrial Ca(2+) uptake and also abolishes its positive correlation with ER-mitochondrion proximity. We postulate that during IP(3)-induced Ca(2+) release, Ca(2+) uptake is confined to ER-close mitochondria, because of the simultaneous activation of the protein kinases. Attenuation of Ca(2+) uptake prevents Ca(2+) overload of mitochondria and thus protects the cell against apoptosis. On the other hand, all the mitochondria accumulate Ca(2+) at a non-inhibited rate during physiological Ca(2+) influx through the plasma membrane. Membrane potential is higher in ER-distant mitochondria, providing a bigger driving force for Ca(2+) uptake. Our model explains why comparable mitochondrial Ca(2+) signals are formed in response to K(+) and angiotensin II (equipotent in respect to global cytosolic Ca(2+) signals), although only the latter generates high-Ca(2+) microdomains.

Effects of sex hormones on ECG parameters and expression of cardiac ion channels in dogs.

AIM: The aim of the study was to examine the effects of testosterone and oestrogen on the ECG parameters and expression of cardiac ion channels in male and female dogs, and to compare the dofetilide-induced lengthening of QTc interval in control, castrated and hormone-treated animals. METHODS: ECG records were taken from male and female anaesthetized dogs (n = 10 in each group) before castration, after castration, and following inverted hormone substitution. The animals were challenged with dofetilide at each stage of the experiment. Finally, the hearts were excised and expression of ion channels was studied using Western blot technique. RESULTS: Heart rate was decreased and PQ interval increased by deprivation of sex hormones in both genders (orchiectomy or ovarectomy), while inverted hormonal substitution restored control values. Orchiectomy significantly increased the duration of QT and QTc intervals, QTc-dispersion and the dofetilide-induced lengthening of QTc, while testosterone treatment of castrated females had opposite effects. Intraventricular conduction (QRS duration) was independent of the endocrine status of the animals. Ovarectomy or oestrogen treatment of castrated males failed to alter significantly these parameters except for QTc-dispersion. Expression of ion channel proteins responsible for mediation of I(K1) and I(to) currents (Kir2.1 and Kv4.3, respectively), was significantly higher in the testosterone-treated castrated females and normal males than in the oestrogen-treated castrated males and normal females. CONCLUSION: Repolarization of canine ventricular myocardium is significantly modified by testosterone, but not oestrogen, in both genders. This effect is likely due to augmentation of expression of K(+)-channel proteins, and thus may provide protection against arrhythmias via increasing the repolarization reserve.

Evaluation of rat and rabbit sera lipoproteins in experimentally induced hyperlipidemia by analytical ultracentrifugation.

Animals of various species are widely used as models with which to study atherosclerosis and the lipoprotein metabolism. The objective of this study was to investigate the lipoprotein profiles in Wistar rats and New Zealand white rabbits with experimentally induced hyperlipidemia by means of ultracentrifugation. The Schlieren curves were utilized to compare suckling and adult rat sera to determine whether aging causes alterations in lipoprotein profiles. A striking feature of the data is the high concentration of low-density lipoproteins (LDL), (>5.2 mmol/l cholesterol) in the 2-week old rat serum pool which was greatly decreased in the 3-weeks rat serum pool (<1.3 mmol/l cholesterol). Additional experiments were performed to permit a direct comparison of the amounts of lipoprotein present in rat sera in experimental hyperlipidemia post-Triton WR 1339 administration. Rapid changes in concentrations in very low-density lipoproteins (VLDL), LDL and high-density lipoproteins (HDL) were observed after Triton injection. The administration of Triton WR 1339 to fasted rats resulted in an elevation of serum cholesterol levels. Triton physically alters VLDL, rendering them refractive to the action of lipolytic enzymes in the blood and tissues, preventing or delaying their removal from the blood. Whereas the VLDL concentration was increased markedly, those of LDL and HDL were decreased at 20 h after Triton treatment. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate of LDL aliquots, to prepare radioactive-labeled lipoproteins and to study induced hyperlipidemia in rabbits. Analytical ultracentrifugation was applied to investigate the LDL flotation peaks before and after cholesterol feeding of rabbits. Modified forms of LDL were detected in the plasma of rabbits with experimentally induced atherosclerosis. ApoB-containing particles, migrating as LDL, intermediate density lipoproteins and VLDL were the most abundant lipoproteins. Gamma camera in vivo scintigraphy on rabbits with radiolabeled lipoproteins revealed visible signals corresponding to atherosclerotic plaques of the aorta and carotid arteries.

Pentapeptides derived from Abeta 1-42 protect neurons from the modulatory effect of Abeta fibrils--an in vitro and in vivo electrophysiological study.

Short fragments and fragment analogues of beta-amyloid 1-42 peptide (Abeta1-42) display a protective effect against Abeta-mediated neurotoxicity. After consideration of our earlier results with in vitro bioassay of synthetic Abeta-recognition peptides and toxic fibrillar amyloids, five pentapeptides were selected as putative neuroprotective agents: Phe-Arg-His-Asp-Ser amide (Abeta4-8) and Gly-Arg-His-Asp-Ser amide (an analogue of Abeta4-8), Leu-Pro-Tyr-Phe-Asp amide (an analogue of Abeta17-21), Arg-Ile-Ile-Gly-Leu amide (an analogue of Abeta30-34), and Arg-Val-Val-Ile-Ala amide (an analogue of Abeta38-42). In vitro electrophysiological experiments on rat brain slices demonstrated that four of these peptides counteracted with the field excitatory postsynaptic potential-attenuating effect of Abeta1-42; only Arg-Val-Val-Ile-Ala amide proved inactive. In in vivo experiments using extracellular single-unit recordings combined with iontophoresis, all these pentapeptides except Arg-Val-Val-Ile-Ala amide protected neurons from the NMDA response-enhancing effect of Abeta1-42 in the hippocampal CA1 region. These results suggest that Abeta recognition sequences may serve as leads for the design of novel neuroprotective compounds.

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc.

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.

Reopening of L-type calcium channels in human ventricular myocytes during applied epicardial action potentials.

AIMS: Present study was performed to compare the dynamics of human L-type calcium current (ICa,L) flowing during rectangular voltage pulses, voltage ramps, and action potentials (APs) recorded from epicardiac and endocardiac canine ventricular cells. METHODS: ICa,L was recorded in single myocytes isolated from undiseased human hearts using the whole cell voltage clamp technique. RESULTS: The decay of ICa,L was monotonic when using rectangular pulses or endocardial APs as voltage commands, whereas the current became double-peaked (displaying a second rise and fall) during epicardial (EPI) APs or voltage ramps used to mimic EPI APs. These ICa,L profiles were associated with single-hooked and double-hooked phase-plane trajectories, respectively. No sustained current was observed during the AP commands. Kinetics of deactivation and recovery from inactivation of human ICa,L were determined using twin-pulse voltage protocols and voltage ramps, and the results were similar to those obtained previously in canine cells under identical experimental conditions. CONCLUSIONS: ICa,L can inactivate partially before and deactivate during the phase-1 repolarization of the epicardiac AP, and reopening of these channels seems to be associated with formation of the dome.

Preparation and investigation of 99m technetium-labeled low-density lipoproteins in rabbits with experimentally induced hypercholesterolemia.

Low-density lipoproteins (LDL) were radiolabeled in atherosclerosis studies. The aim was to investigate the biodistribution and uptake of 99mTc-labeled LDL by atherosclerotic plaques in experimentally induced hyperlipidemia. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate human LDL aliquots, to prepare radioactive-labeled lipoproteins and in rabbits with induced hyperlipidemia. Preparative density gradient centrifugation was applied for the simultaneous isolation of the major lipoprotein density classes, which form discrete bands of lipoproteins in the preparative tubes. The cholesterol and protein levels in the lipoprotein fractions were determined. LDL was subsequently dialysed against physiological solution and sterilized and apolipoprotein fragments and aggregates were eliminated by passage through a 0.22-micron filter. LDL was radiolabeled with 99mTc by using sodium dithionite as a reducing agent. Radiochemical purity and in vitro stability were controlled by paper chromatography in acetone. The labelling efficiency was 85-90% for human LDL. Two months after the start of cholesterol feeding, the total cholesterol in the blood serum had increased approximately 33-fold in comparison with the basal cholesterol content of hypercholesterolemic rabbits. Investigation of LDL was performed by Schlieren analysis after adjustment of the density of serum and underlayering by salt solution in a spinning ultracentrifugation capillary band-forming cell. Quantitative results were obtained by measuring the Schlieren areas between the sample curves and the reference baseline curve by means of computerized numerical and graphic techniques. In this manner we measured the concentrations of human LDL and analyzed rabbit LDL levels in induced hyperlipidemia. Gamma scintillation camera scanning of the rabbits was performed. Overnight fasted rabbits were injected in the marginal ear vein with 99mTc-labeled human LDL (4-10 mCi, 0.5-1.5 mg protein). The initial scintigram showing a typical blood-pool scan, gradually changing with time to an image of specific organ uptake of radioactivity by the liver, kidneys and brain and in the bladder. Gamma camera in vivo scintigraphy on rabbits revealed visible signals corresponding to atherosclerotic plaques in the aorta and carotid arteries. Our results show that 99mTc-LDL can be used to assess the organ distribution pattern of LDL in the rabbit, and to detect and localize areas of arterial atherosclerotic lesions.

Differences in electrophysiological and contractile properties of mammalian cardiac tissues bathed in bicarbonate - and HEPES-buffered solutions.

AIM: The aim of this study was to compare the action potential configuration, contractility, intracellular Ca2+ and H+ concentrations in mammalian cardiac tissues bathed with Krebs and Tyrode solutions at 37 degrees C. RESULTS: In Langendorff-perfused guinea-pig hearts, loaded with the fluorescent Ca2+-indicator Fura-2, or H+-sensitive dye carboxy-SNARF, shifts from Krebs to Tyrode solution caused intra-cellular acidification, increased diastolic pressure and [Ca2+]i, decreased systolic pressure and [Ca2+]i, leading to a reduction in the amplitude of [Ca2+]i transients and pulse pressure. Contractility was also depressed in canine ventricular trabeculae when transferred from Krebs to Tyrode solution. Shifts from Krebs to Tyrode solution increased the duration of action potentials in multicellular cardiac preparations excised from canine and rabbit hearts but not in isolated cardiomyocytes. All these changes in action potential morphology, contractility, [Ca2+]i and [H+]i were readily reversible by addition of 26 mmol L(-1) bicarbonate to Tyrode solution. Effects of dofetilide and CsCl, both blockers of the delayed rectifier K current, on action potential duration were compared in Krebs and Tyrode solutions. Dofetilide lengthened rabbit ventricular action potentials in a significantly greater extent in Tyrode than in Krebs solution. Exposure of canine Purkinje fibres to CsCl evoked early after depolarizations within 40 min in all preparations incubated with Tyrode solution, but not in those bathed with Krebs solution. CONCLUSION: It is concluded that the marked differences in action potential morphology, [Ca2+]i, [H+]i and contractility observed between preparations bathed with Krebs and Tyrode solutions are more likely attributable to differences in the intracellular buffering capacities of the two media.

The role of transmembrane chloride current in afterdepolarisations in canine ventricular cardiomyocytes.

The physiological role of chloride currents (Icl) in cardiac cells is poorly understood. The aim of the present study was to reveal the role of Icl in the genesis of early and delayed afterdepolarisations (EADs and DADs, respectively). First we identified Icl under action potential voltage clamp conditions as the anthracene-9-carboxylic acid (ANTRA) (0.5 mmol/l)-sensitive current. The ANTRA-sensitive current was large and outwardly directed at the beginning, while it was moderate and inwardly directed at the end of the action potential. Application of ANTRA under current clamp conditions decreased the depth of the incisura, shifted the plateau upwards and lengthened the duration of action potentials. The effect of ANTRA was studied in three models of afterdepolarisations: the ouabain-induced DAD model, the caesium-induced EAD model, and in the presence of subthreshold concentration of isoproterenol. Preincubation of the cells with 0.5 mmol/l ANTRA failed to induce afterdepolarisations. Ouabain (200 nmol/l) alone caused DADs in 62.5% of the cells within 15 min. When ouabain was applied in the presence of ANTRA, 60% of the myocytes transiently displayed EADs before the development of DADs, and all cells developed DADs within 7 min. Isoproterenol (5 nmol/l) alone failed to induce afterdepolarisations. However, 75% of the cells produced DADs within 6 min when superfused with isoproterenol in the presence of ANTRA. Incubation of the myocytes with 3.6 mmol/l CsCl caused EADs in 71.4% of the cells within 30 min. Application of CsCl in the presence of ANTRA resulted in immediate depolarisation of the membrane from -79.6 +/- 0.4 to -54.2 +/- 3.5 mV. Summarizing our results we conclude that the ANTRA-sensitive current is an important mechanism of defence against afterdepolarisations. Suppression of Icl may thus increase the incidence and accelerate the rate of development of both EADs and DADs.

Synthesis and fluorescent labeling of beta-amyloid peptides.

Fluorescent cell analytical techniques require the incorporation of a fluorophore into the target molecule without causing a significant change in the native conformation. Many short peptides have a limited number of reactive groups that can be labeled without affecting the biological activity. In this work we present several methods for labeling beta-amyloid peptides (betaA[25-35], betaA[1-40]) and their derivatives (LPFFD, RIIGL and RVVIA) with different chromophores exclusively at the N-terminus. In the case of liquid-phase labeling, fluorescein isothiocyanate was used. The side-chain amino function of Lys, if present in the sequence, was protected with an Fmoc group, whereby the hydrophobic character of the peptide was further increased. The labeling reaction was carried out in an appropriate deaggregating solvent, DMSO. For solid-phase labeling, 5(6)-carboxyfluorescein and 7-amino-4-methyl-3-coumarinylacetic acid were applied. Several cleavage cocktails were tested for removal of the labeled amyloid peptides from the resin in order to completely suppress the oxidation of Met.

Effects of the antiarrhythmic agent EGIS-7229 (S 21407) on calcium and potassium currents in canine ventricular cardiomyocytes.

Based on earlier pharmacological studies performed using conventional microelectrodes EGIS-7229 (S 21407), the novel antiarrhythmic candidate, was suggested to have a combined mode of action in cardiac tissues isolated from various mammalian species. In order to characterize the electrophysiological effects of the compound, its effects on calcium and potassium currents of isolated canine ventricular cardiomyocytes were studied in the present work using the whole cell configuration of the patch clamp technique. L-type Ca current (ICa) was significantly depressed by EGIS-7229 at concentrations of 3 microM or higher with no concomitant changes in the voltage-dependence of activation and time course of inactivation of ICa. The drug reversibly suppressed the rapid component of the delayed rectifier K current (IKr) in a concentration-dependent manner, having a K0.5 value of 1.1+/-0.1 microM and a slope factor of close to unity (1.23+/-0.16), indicating that probably one single binding site of high affinity may be involved in binding of EGIS-7229 to the IKr channel. In contrast, no changes in the slow component of the delayed rectifier K current (IKs) was observed with the compound up to the concentration of 100 microM, even if the current was fully activated by 8-bromo-cAMP. At a concentration of 10 microM or higher, EGIS-7229 caused also a moderate but significant reduction in the inward rectifier K current (IK1) and the transient outward K current (Ito) with no change in the voltage-dependence of activation and steady-state inactivation of Ito. Present results indicate that EGIS-7229 can be considered as a selective IKr blocker at low (1 microM) concentration; however, its combined (class III + IV) mechanism of action is evident at concentrations of 3 microM or higher. Suppression of ICa may explain the lack of development of early afterdepolarizations in the presence of EGIS-7229, predicting a relatively safe clinical application in contrast to pure class III compounds.

A short-run new analytical ultracentrifugal micromethod for determining low-density lipoprotein sub-fractions using Schlieren refractometry.

We have developed a new analytical ultracentrifugal micromethod for the determination of serum low-density lipoprotein (LDL) subclasses directly from ultracentrifugal Schlieren scans. We have used special software for the analysis of this type of single-spin density-gradient ultracentrifugation. The flotation of LDL patterns was obtained by underlayering a physiological salt solution with serum or isolated lipoprotein fractions raised to a density of 1.3 g/mL in the spinning ultracentrifugation capillary band-forming cell. The repeated analysis of Schlieren curves of the same sample from 10 to 100 microL in the 60-100 min full-speed interval time resulted in quite reproducible results. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by using computerised numerical and graphic techniques. The decomposition of the integrated curve was carried out using a nonlinear regression program followed by deconvolution algorithm analysis in order to determine the parameters of the composing Gaussian subclasses. The LDL particle concentrations were calculated from the area under the integral of the Gaussian curve using a calibration data constant. The flotation range of the LDL Schlieren curves in the cell was identified with serum from which LDL had been removed by means of precipitation reagents and with centrifugation of isolated LDL aliquots. With this technique, we measured the concentration of LDL and analysed its polydispersity without the need for preceding sequential isolation of the LDL. On the basis of the Schlieren curves, the LDL samples were either physically paucidisperse, having a symmetrical peak within a narrow density range, or were polydisperse, showing an asymmetrical pattern distributed over a broader density region. The described method proved to be useful for a clear and immediate visual presentation of the concentration values of the LDL and for the identification of the heterogeneity of LDL variants without the need for the preparative isolation of that density class.

Expression of cmg1, an exo-beta-1,3-glucanase gene from Coniothyrium minitans, increases during sclerotial parasitism.

During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains beta-1,3-glucan as its major component. A PCR-based strategy was used to clone a beta-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-beta-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiae INVSc1 expressing the cmg1 gene secreted a approximately 100-kDa beta-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 microg x ml(-1), respectively. A single copy of the cmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.

Isolation of a beta-galactosidase-encoding gene from Bacillus licheniformis: purification and characterization of the recombinant enzyme expressed in Escherichia coli.

The Bacillus licheniformis beta-galactosidase gene, lacBl, was cloned on a 5.8-kb HindIII fragment into pBR322 and expressed by its own promoter in Escherichia coli. Deletion and complementation analysis showed that the enzyme-encoding region was located on a 4. 1-kb HindIII-ClaI fragment. The transcription region for the lacBl was identified on this fragment with promoter- and terminator-probe plasmids. The deduced sequence of 149 aa of the N-terminal part of lacBl showed aa sequence homology with beta-Gal from B. stearothermophilus, B. circulans, Haloferax alicantei, Clostridium perfringens, Arthrobacter sp.. No significant homology was shared with those found in the lacZ and lacS families. The recombinant beta-galactosidase (LacB1) was purified by FPLC. The molecular mass of the enzyme (80 kDa) and its optimal pH (5.7) and temperature (45 degrees C) were determined.

Cloning and expression of a beta-1,4-endoglucanase gene from Cellulomonas sp. CelB7 in Escherichia coli; purification and characterization of the recombinant enzyme.

A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA, was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 degrees C) optima were determined.

Nucleotide sequence of the NS5 gene of Banzi virus: comparison with other flaviviruses.

Banzi is a mosquito borne flavivirus which belongs to the Uganda S serocomplex. No nucleotide sequence data have previously been reported from any virus of this serocomplex. We have determined the nucleotide sequence of the NS5 gene from Banzi virus and the predicted amino acid sequence was elucidated. Previously identified conserved RNA polymerase, methyltransferase and flavivirus NS5 amino acid motifs were present in the Banzi virus NS5 protein. These data add to the evidence for the functional importance of the regions. The encoded amino acid sequence was compared with the predicted amino acid sequence of other flavivirus NS5 proteins. Analysis of these sequences suggested that Banzi virus is most closely related to the mosquito-borne flaviviruses and, in particular, yellow fever virus. This pattern of similarity is in accordance with the previously suggested serological classification of flaviviruses.

Transposon Tn917PF1 mutagenesis in Bacillus licheniformis.

The plasmid pTnPFl containing the transposon Tn917PF1 was introduced into the protoplasts of two Bacillus licheniformis strains in the presence of polyethylene glycol. Transpositions were produced at high temperature which inhibited plasmid replication and kanamycin was used for selection.Transposon Tn917PF1 was inserted randomly into the bacterial chromosome,producing different auxotrophic, prophage BLF and bacitracin-non-producing mutants. The auxotrophic mutant phenotypes were characterized by the Holliday-test and some mutations by hybridization with a transposon DNA probe. Insertions for the entire chromosome or for the prophage genophore were found at random, but preferred target sites were detected within limited regions, like the bacitracin synthetase or sulphate reductase genes. The partial physical map of the chromosomal region of bacitracin synthetase was constructed based on the hybridization patterns of insertion mutants.

Rapid identification of flaviviruses based on conserved NS5 gene sequences.

Two conserved regions in the sequence of the NS5 gene of Flaviviruses were identified. Primers were designed from the consensus sequence of these regions and were used in a reverse transcription/polymerase chain reaction (RT/PCR) to amplify a region of the central european tick-borne encephalitis virus Kumlinge NS5 gene. The authenticity of the amplified fragment was confirmed by nucleotide sequencing. A band of the expected size was also obtained when this RT/PCR was applied to 13 other flaviviral RNAs. This method may be useful for characterisation of the NS5 genes of flaviviruses and as a potential pan-flavivirus diagnostic tool.