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Biomedical subjects

A Savchenko

Publications and source records attributed to A Savchenko.

34 records · Page 2Linked to original sources
PubMed1997

The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor-operator interactions.

We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis. Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC-lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90 degrees C, whereas B. subtilis AhrC was largely inactivated at 65 degrees C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85 degrees C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus. This pronounced resistance of the repressor-operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.

Amino Acid Sequence↗
PubMed1997

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).

Amino Acid Sequence↗
PubMed1997

TeleInViVo: a collaborative volume visualization application.

Converging technologies in the areas of networks, volume visualization algorithms, and computer performance have made possible the development of a new tool for collaboration, which extends the reach of health professionals, and other consumers of volumetric data around the world. TeleInViVo(tm) is a three-dimensional (3D) collaborative volume visualization tool for medical applications. It extends the capabilities of InViVo(tm), a fast volume visualization tool developed at the Fraunhofer IGD, Darmstadt, Germany [1-3], with efficient and intuitive network collaboration features for remote consultation and new modes of interaction. The software runs on both UNIX and Windows NT platforms. TeleInViVo provides a high degree of interactivity for the medical professional when interacting with the patient data, facilitates explanation and communication between field personnel and medical experts located far from the field, and permits viewing of the data in a multitude of ways designed to support rapid and accurate diagnosis. Current efforts involve architectural enhancements to support multiuser, distributed telemedical scenarios. The application includes the following features: Volume and subvolume data transmission at user specified resolution, Synchronization cues, Integration of Immersion Probe(tm), a 6 degree-of-freedom input device, for ergonomic 3D data exploration, Tools for measuring distances, Tools for planning instrument path, Arbitrary cutting planes in real time, Interactive segmentation tools, Virtual video recorder and playback (cine loops), 3D stereo mode. TeleInViVo is an essential part of the MUSTPAC-1 portable 3D ultrasound system developed by Battelle Pacific Northwest Labs, Richland, WA.

Computer Communication Networks↗
PubMed1996

The arginine operon of Bacillus stearothermophilus: characterization of the control region and its interaction with the heterologous B. subtilis arginine repressor.

Mechanisms of gene regulation have not yet been extensively studied in thermophilic bacteria. In previous studies we showed that the Bacillus stearothermophilus argCJBD gene cluster is subject to specific repression by arginine. Here we report the cloning by colony hybridization, and characterization of the proximal part of the argC gene together with the adjacent control region of the cluster. The promoter was identified by primer extension mapping of the argC transcription startpoint: a sequence overlapping it was found to be similar to the arginine operators of B. subtilis and to a smaller extent of E. coli. Use of an argC-lacZ gene fusion revealed that the argC promoter is strongly repressed by the heterologous B. subtilis arginine repressor/activator AhrC in E. coli cells. Mobility shift and DNase I footprinting experiments revealed tight, specific and arginine-dependent binding of this operator-like sequence to purified AhrC. It is therefore very likely that in B. stearothermophilus the expression of the argCJBD operon is modulated by a repressor that is the thermophilic homologue of AhrC.

Amino Acid Sequence↗
PubMed1995

Chimeric L-type Ca2+ channels expressed in Xenopus laevis oocytes reveal role of repeats III and IV in activation gating.

1. Chimeric alpha 1 subunits consisting of repeat I and II from the rabbit cardiac (alpha 1C-a) and repeat III and IV from the carp skeletal muscle Ca2+ channel (alpha 1S) were constructed and expressed in Xenopus laevis oocytes without co-expressing other channel subunits. Ba2+-current kinetics of five chimeric channel constructs were studied in Xenopus oocytes using the two-microelectrode technique. 2. Exchange of repeats III and IV of alpha 1C-a with sequences of alpha 1S results in a significantly slower and biexponential activation (apparent activation time constants tau 1act = 19.8 +/- 1.8 ms and tau 2act = 214 +/- 28.7 ms, n = 7) of expressed Ca2+ channel currents; no current inactivation was observable during an 800 ms test pulse to 0 mV. 3. Activation of a chimera consisting of repeats I, II and IV from the alpha 1C-a subunit and repeat III from alpha 1S was fast and monoexponential (tau 1act = 6.33 +/- 1.7 ms, n = 5) and the current inactivated during a 350 ms test pulse to 0 mV (tau inact = 175 +/- 22 ms, n = 5). The current kinetics of this construct did not significantly differ from kinetics of a construct consisting of repeats I to IV from alpha 1C-a (tau 1act = 6.6 +/- 2.1 ms; tau inact = 198 +/- 14 ms; n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Improved micro-perfusion chamber for multiple and rapid solution exchange in adherent single cells.

A new method for fast perfusion of single adherent cells during whole-cell and single-channel patch-clamp measurements is described. The main advantages over previous methods are: (1) the solution surrounding a single cell in a Petri dish (or cover slip) can be exchanged without contamination of the entire cell population, and (2) only small quantities of test solution (50 microliters) are required. The method consists of inserting a patch pipette into a micro-perfusion chamber (MPC) where whole-cell and single-channel currents can be studied. A small volume (2-3 microliters) surrounding adherent single cells in a Petri dish is separated by enclosing it in a ring of Silicone rubber which is then pressed to the bottom of the dish. A drug-containing test solution (50 microliters) is supplied to a funnel at the chamber inflow. The MPC volume isolated from the main solution in the Petri dish is rapidly changed when suction is applied to the chamber outflow. The speed of solution exchange in the MPC (16 +/- 5 ms, n = 12) was estimated by observing changes in the tip potential of 1-M omega patch pipettes during rapid chamber perfusion with solutions of different ionic composition. The seal of the MPC with the surface of a Petri dish was approximated by measuring the electrical resistance between the MPC interior and a reference electrode placed in the Petri dish outside the MPC (range 300-500 M omega). Additionally, a radioactive calcium channel ligand [3H]isradipine was added to the MPC and the appearance of radioactivity in the Petri dish (outside the MPC) subsequently measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Amplifiers, Electronic↗
PubMed1994

Cloning and functional expression of a neuronal calcium channel beta subunit from house fly (Musca domestica).

The primary structure of a calcium channel beta subunit (beta M) from housefly (Musca domestica) has been deduced by cDNA cloning and sequence analysis. The open reading frame encodes a 441-amino acid polypeptide with a calculated molecular mass of 48,755 Da. Whole-mount in situ hybridization indicates that beta M mRNA is predominantly expressed in neuronal tissues. Transcription of beta M mRNA is evident from stage 13/14 of embryogenesis up to adulthood. Different expression patterns of splice variants were found in larvae and in adult fly heads. Amino acid identity between beta M and mammalian beta subunits is lower (66-68%) than within mammalian beta subunits (74-80%). Calculation of a phylogenetic tree indicates that beta M is an ancestral form of the four distinct beta subunit gene products identified in mammalian tissues so far. Despite these sequence differences, beta M is able to enhance endogenous calcium channel activity in Xenopus laevis oocytes as well as dihydropyridine binding to membranes from COS 7 cells transfected with rabbit heart alpha 1 cDNA in the same manner as was previously shown for mammalian beta subunits.

Alternative Splicing↗
PubMed1993

A light stabilizer (Tinuvin 770) that elutes from polypropylene plastic tubes is a potent L-type Ca(2+)-channel blocker.

A pharmacologically active agent was easily extracted by aqueous or organic solvents from laboratory plastic tubes (Falcon Blue Max) and has been chemically identified as bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate. This compound (approximately 12 micrograms per tube approximately 25 nmol) blocked 1,4-dihydropyridine-sensitive 45Ca2+ uptake into GH3 cells with an IC50 value of 3.6 microM, inhibited Sr2+ currents through L-type Ca2+ channels in A7r5 smooth-muscle cells in whole-cell patch-clamp experiments after extracellular application, and affected the high-affinity binding of Ca2+ entry-blocker ligands to a variety of preparations. Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate is a highly potent (IC50 values < 10 nM) inhibitor at the phenylalkylamine- and benzothiazepine-selective drug-binding domains of the alpha 1 subunit of L-type Ca2+ channels. This compound behaves as a heterotropic allosteric regulator for the 1,4-dihydropyridine-selective domain in purified Ca(2+)-channel preparations from rabbit skeletal muscle. (+)-Tetrandrine stimulation of 1,4-dihydropyridine binding to the membrane-bound L-type Ca2+ channel is inhibited by the compound in a competitive manner (Ki value = 6.8 nM). Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate is therefore classified as the prototype of another class of L-type Ca(2+)-channel blockers that binds to the alpha 1 subunit at the drug-binding domains selective for (+)-tetrandrine or (+)-cis-diltiazem. This compound is identical to Tinuvin 770, which is used worldwide as a light stabilizer for polyolefins.

Alkaloids↗
PubMed1993

Calcium currents in aged rat dorsal root ganglion neurones.

1. The whole-cell voltage clamp technique was used to record calcium currents in the somatic membrane of rat cultured dorsal root ganglion neurones. 2. Neurones were enzymatically isolated from animals of three age groups (neonatal, 2-7 days; adult, 7 months; and old, 30 months) and maintained in primary culture 3-14 days. 3. The neurones isolated from neonatal and old rats showed two distinct types of Ca2+ currents, a low-threshold transient current and a high-threshold sustained current, whereas neurones from old rats showed only a high-threshold calcium current. 4. The density of the high-threshold calcium current was 28.4 +/- 6.3 pA/pF (mean +/- S.E.M., n = 54) in neonatal, 39.1 +/- 7.2 pA/pF (n = 62) in adult and 11.0 +/- 4.6 pA/pF (n = 64) in old dorsal root ganglion neurones. 5. We found no difference in elementary high-threshold Ca2+ current characteristics in neurones from different age groups. The single-channel conductance was (with 60 mM Ca2+ in the recording pipette) 16.0 +/- 2.7 pS (mean +/- S.E.M., n = 9) in neonatal, 16.2 +/- 1.7 pS (n = 11) in adult and 16.4 +/- 1.2 pS (n = 12) in old neurones. 6. Current-voltage relations and kinetics of high-threshold calcium currents showed no detectable age-dependent difference. 7. The run-down of high-threshold calcium currents in dorsal root ganglion neurones from old rats was practically insensitive to intracellular administration of cyclic AMP and ATP. The same intervention caused a significant deceleration of Ca2+ current run-down in the majority of neonatal and in some adult cells. 8. We suggest that the disappearance of the low-threshold calcium current and reduction of high-threshold calcium current with ageing is due to a depression of calcium channel expression during late ontogenesis. The decrease of sensitivity of high-threshold calcium channels to phosphorylation by cyclic AMP-dependent protein kinase in aged neurones could also be a reason for altered turnover between silent and functional pools of calcium channels, which may underlie the age-dependent decline in the density of high-threshold calcium channels.

Aging↗
PubMed1993

Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus.

A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes.

Amidohydrolases↗
PubMed1993

Extracellular localization of the benzothiazepine binding domain of L-type Ca2+ channels.

To determine which side of L-type Ca2+ channels forms the benzothiazepine binding domain, we tested the effects of a membrane-impermeable, diltiazem-like, Ca2+ antagonist, SQ32,428 [(cis)-1,3,4,5-tetrahydro-4-(4-methoxyphenyl)-3-methyl-6- (trifluoromethyl)-1-[2-trimethylammonio)ethyl]-2H-1-benzazepin-2-o ne], on Ca2+ channels in smooth muscle-like cells (A7r5 cells) and skeletal muscle-like cells (differentiated BC3H1 cells). This permanently charged, quaternary benzazepine bound to the benzothiazepine-selective domain of skeletal muscle Ca2+ channels with a Ki of 1.2 +/- 0.1 microM. Extracellular application of SQ32,428 reversibly blocked whole-cell barium currents through L-type Ca2+ channels in A7r5 and BC3H1 cells with similar potencies (A7r5, IC50 = 86 microM; BC3H1, IC50 = microM). Block was fully reversible, was independent of stimulation frequency, and did not affect steady state inactivation of the channel in A7r5 cells. Intracellular dialysis of the cells with 100 microM SQ32,428 was without effect, but the same concentration of the quaternary phenylalkylamine D890 blocked channel activity from the cytoplasmic side. Our data demonstrate that the benzothiazepine binding domain of L-type Ca2+ channels binds diltiazem-like benzazepine Ca2+ antagonists and is formed by amino acid residues exposed to the extracellular channel surface.

Animals↗
PubMedDate not supplied

Cyclic-nucleotide-gated channels mediate synaptic feedback by nitric oxide.

Cyclic-nucleotide-gated (CNG) channels in outer segments of vertebrate photoreceptors generate electrical signals in response to changes in cyclic GMP concentration during phototransduction. CNG channels also allow the influx of Ca2+, which is essential for photoreceptor adaptation. In cone photoreceptors, cGMP triggers an increase in membrane capacitance indicative of exocytosis, suggesting that CNG channels are also involved in synaptic function. Here we examine whether CNG channels reside in cone terminals and whether they regulate neurotransmitter release, specifically in response to nitric oxide (NO), a retrograde transmitter that increases cGMP synthesis and potentiates synaptic transmission in the brain. Using intact retina, we show that endogenous NO modulates synapses between cones and horizontal cells. In experiments on isolated cones, we show directly that CNG channels occur in clusters and are indirectly activated by S-nitrosocysteine (SNC), an NO donor. Furthermore, both SNC and pCPT-cGMP, a membrane-permeant analogue of cGMP, trigger the release of transmitter from the cone terminals. The NO-induced transmitter release is suppressed by guanylate cyclase inhibitors and prevented by direct activation of CNG channels, indicating that their activation is required for NO to elicit release. These results expand our view of CNG channel function to include the regulation of synaptic transmission and mediation of the presynaptic effects of NO.

Ambystoma↗
PubMedDate not supplied

[Direct coronary arterial stenting without predilatation in patients with coronary heart disease].

The study was undertaken to evaluate the safety and efficiency of direct stenting versus routine stenting with predilation. It included 133 patients. By the decision of operators, direct stenting was conducted in 66 patients (71 stenoses) (Group 1). The remaining 67 patients (73 stenoses) underwent routine stenting with predilation (Group 2). The initial angiographic success of stenting was 100% in Group 1 and 98 in Group 2. Complications were absent. In the direct stenting group, technical problems occurred during a session in 9 (12%) cases. In this group the mean duration of fluoroscopy and the total duration of a session were much less than in the routine stenting group. The mean number of balloons used at dilation per stenosis and the number of dilation sessions per stenosis were much lower in Group 1 than in Group 2. The results of quantitative angiogram analysis before and after a session were similar in both patient groups. Six months following stenting, angiographic restenosis occurred in 7 (10%) patients in Group 1 and in 9 (12%) in Group 2. Direct stenting is a safe and effective treatment for non-occlusive coronary lesions without marked kinks and calcinosis. Direct stenting reduces the duration of fluoroscopy and the total duration of an operation by 50 and 22%, respectively, as compared to predilation stenting.

Catheterization↗