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A Savchenko

Publications and source records attributed to A Savchenko.

At least 19 recordsLinked to original sources
PubMed2001

A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival.

A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.

Amino Acid Sequence↗
PubMed2001

Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through dephosphorylation of a specific tyrosine residue.

Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca(2+), which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the alpha-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.

Animals↗
PubMed2001

Structure of Thermotoga maritima stationary phase survival protein SurE: a novel acid phosphatase.

BACKGROUND: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase sigma subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. RESULTS: The structure of SurE from Thermotoga maritima was determined at 2.0 A. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. CONCLUSIONS: The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed2001

Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts.

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+i activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1-10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+ exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.

Adenosine Triphosphate↗
PubMed2001

Assay technologies for screening ion channel targets.

The sequence of the human genome will soon provide researchers with hundreds of new ion channel genes. To create a successful ion channel drug discovery program, it is necessary to quickly develop reliable and robust high-throughput screening (HTS) assays for those ion channels implicated in important diseases. Ion channels are dynamic proteins, and therefore require assays that 'sense' their various functional states. Competition-binding assays, although successfully used for other target classes, often fail to identify ligands that modulate specific ion channel functions. Cell-based functional assays, therefore, are preferred for HTS of ion channel targets. In this review, we evaluate the various cell-based assays available for ion channels, and discuss future directions for assay improvements.

Animals↗
PubMed2000

Dissecting the nucleotide binding properties of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase with fluorescent 3'(2)'-o-anthraniloyladenosine 5'-triphosphate.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7, 8-dihydropterin, the first reaction in the folate biosynthetic pathway. Like other enzymes in the folate pathway, HPPK is an ideal target for development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from humans and animals. Using 3'(2')-o-anthraniloyladenosine 5'-triphosphate as a fluorescent probe, a fluorometric competitive binding assay has been developed for measuring the dissociation constants of various compounds that bind to the ATP site of HPPK. The fluorometric assay has been used to determine the nucleotide specificity and dissect the energetics of the binding of MgATP. The order of affinity of various nucleoside triphosphates for HPPK is MgATP>MgGTP>MgITP>MgXTP approximately MgUTP approximately MgCTP. The affinity of MgATP for HPPK (K(d)=2.6+/-0.06 microM) is 260-fold higher than that of MgGTP and more than 1000-fold higher than those of the other nucleoside triphosphates, indicating that HPPK is highly specific with respect to the base moiety of the nucleotide. The affinity of ATP for HPPK in the presence of Mg(2+) is 15 times that in the absence of Mg(2+), indicating that the metal ion is important for the binding of the nucleotide. Removal of the gamma-phosphate from MgATP reduces its affinity for HPPK by a factor of approximately 21. The affinity of AMP for HPPK is about one third that of ADP and almost the same as that of adenosine. The result suggests that among the three phosphoryl groups of MgATP, the gamma-phosphoryl group is most critical for binding to HPPK and the alpha-phosphoryl group contributes little to the binding of the nucleotide. The affinity of MgATP is 18 times that of MgdATP, indicating that the 2'-hydroxyl group of MgATP is also important for binding. van't Hoff analysis suggests that binding of MgATP is mainly driven by enthalpy at 25 degrees C and the entropy of binding is also in favor of the formation of the HPPK.MgATP complex.

Binding, Competitive↗
PubMed2000

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, approximately 20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Cloning, Molecular↗
PubMed2000

Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein.

The cGMP sensitivity of cyclic nucleotide-gated (CNG) channels can be modulated by changes in phosphorylation catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. Previously, we used genistein, a PTK inhibitor, to probe the interaction between PTKs and homomeric channels comprised of alpha subunits (RETalpha) of rod photoreceptor CNG channels expressed in Xenopus oocytes. We showed that in addition to inhibiting phosphorylation, genistein triggers a noncatalytic interaction between PTKs and homomeric RETalpha channels that allosterically inhibits channel gating. Here, we show that native CNG channels from rods, cones, and olfactory receptor neurons also exhibit noncatalytic inhibition induced by genistein, suggesting that in each of these sensory cells, CNG channels are part of a regulatory complex that contains PTKs. Native CNG channels are heteromers, containing beta as well as alpha subunits. To determine the contributions of alpha and beta subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RETalpha and OLFalpha), and heteromeric (RETalpha+beta, OLFalpha+beta, and OLFalpha+RETbeta) CNG channels. We found that genistein only inhibits channels that contain either the RETalpha or the OLFbeta subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RETalpha and OLFbeta subunits contain binding sites for the PTK, whereas RETbeta and OLFalpha subunits do not.

Ambystoma↗
PubMed1999

Noncatalytic inhibition of cyclic nucleotide-gated channels by tyrosine kinase induced by genistein.

Rod photoreceptor cyclic nucleotide-gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.

Adenosine Triphosphate↗
PubMed1998

The Bacillus stearothermophilus argCJBD operon harbours a strong promoter as evaluated in Escherichia coli cells.

We have shown that the B. stearothermophilus argCJBD genes form a single operon. In B. stearothermophilus, a specific repressor governs operon expression by binding to the argCo operator site overlapping the Parg promoter sequence (Dion et al., 1997). Therefore, the enzymatic and transcriptional analyses performed in this work did not reflect the potential strength of Parg in the native host. For evaluation of the Parg promoter strength, E. coli was used as a host since its own ArgR repressor does not interact with the B. stearothermophilus heterologous operator. Parg-promoted argC gene expression dramatically increased, reaching up to 38% of the total protein in E. coli cells. An AT-rich sequence upstream of a -35 site of Parg was found to be indispensable for the promoter strength. Plasmids carrying the B. stearothermophilus argCJBD operon linked with its Parg/argCo region were unstable in E. coli. Stabilization of plasmids was achieved by repression of B. stearothermophilus arg genes through the action of the B. subtilis AhrC repressor.

Acetyltransferases↗
PubMed1998

Thermozymes: biotechnology and structure-function relationships.

Recent findings on the biochemical and molecular features of the following thermozymes are presented, based on their biotechnological use: alpha-amylase and amylopullulanase, used in starch processing; glucose isomerase, used in sweetener production; alcohol dehydrogenase, used in chemical synthesis; and alkaline phosphatase, used in diagnostics. The corresponding genes and recombinant proteins have been characterized in terms of sequence similarities, specific activities, thermophilicity, and unfolding kinetics. Site-directed and nested deletion mutagenesis were used to understand structure-function relationships. All these thermozymes display higher stability and activity than their counterparts currently used in the biotechnology industry.

Alcohol Dehydrogenase↗
PubMed1997

Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation.

Cyclic nucleotide-gated (CNG) channels in vertebrate photoreceptors are crucial for transducing light-induced changes in cGMP concentration into electrical signals. In this study, we show that both native and exogenously expressed CNG channels from rods are modulated by tyrosine phosphorylation. The cGMP sensitivity of CNG channels, composed of rod alpha-subunits expressed in Xenopus oocytes, gradually increases after excision of inside-out patches from the oocyte membrane. This increase in sensitivity is inhibited by a protein tyrosine phosphatase (PTP) inhibitor and is unaffected by three different Ser/Thr phosphatase inhibitors. Moreover, it is suppressed or reversed by application of ATP but not by a nonhydrolyzable ATP analog. Application of protein tyrosine kinase (PTK) inhibitors causes an increase in cGMP sensitivity, but only in the presence of ATP. Taken together, these results suggest that CNG channels expressed in oocytes are associated with active PTK(s) and PTP(s) that regulate their cGMP sensitivity by changing phosphorylation state. The cGMP sensitivity of native CNG channels from salamander rod outer segments also increases and decreases after incubation with inhibitors of PTP(s) and PTK(s), respectively. These results suggest that rod CNG channels are modulated by tyrosine phosphorylation, which may function as a novel mechanism for regulating the sensitivity of rods to light.

Adenosine Triphosphate↗
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↗