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J Janata

Publications and source records attributed to J Janata.

At least 19 recordsLinked to original sources
PubMed2006

A new evolutionary variant of the streptogramin A resistance protein, Vga(A)LC, from Staphylococcus haemolyticus with shifted substrate specificity towards lincosamides.

We found a new variant of the streptogramin A resistance gene, vga(A)LC, in clinical isolates of Staphylococcus haemolyticus resistant to lincomycin and clindamycin but susceptible to erythromycin and in which no relevant lincosamide resistance gene was detected. The gene vga(A)LC, differing from the gene vga(A) at the protein level by seven amino acid substitutions, was present exclusively in S. haemolyticus strains resistant to both lincosamides and streptogramin A (LS(A) phenotype). Antibiotic resistance profiles of the ATP-binding cassette (ABC) proteins Vga(A)(LC) and Vga(A) in the antibiotic-susceptible host S. aureus RN4220 were compared. It was shown that Vga(A)LC conferred resistance to both lincosamides and streptogramin A, while Vga(A) conferred significant resistance to streptogramin A only. Detailed analysis of the seven amino acid substitutions, distinguishing the two related ABC proteins with different substrate specificities, identified the substrate-recognizing site: four clustered substitutions (L212S, G219V, A220T, and G226S) in the spacer between the two ATP-binding cassettes altered the substrate specificity and constituted the lincosamide-streptogramin A resistance phenotype. A transport experiment with radiolabeled lincomycin demonstrated that the mechanism of lincosamide resistance in S. haemolyticus was identical to that of the reported macrolide-streptogramin B resistance conferred by Msr(A).

Amino Acid Sequence↗
PubMed2006

The prospects of advanced molecular methods in the detection of periodontal pathogens: hybridization in situ.

The paper summarizes the present state in the diagnostics of periodontal pathogens. Both the main advantages and drawbacks of the classic cultivation methods and those of the new DNA techniques are discussed. From the emerging methods of molecular diagnostics, the method of in situ hybridization is presented in more details. Its principle, various modifications of performance and possibilities of use are explained, including examples of its application in the detection of periodontal pathogens.

Bacteria↗
PubMed2003

Spore-specific modification of DNA-dependent RNA polymerase alpha subunit in streptomycetes--a new model of transcription regulation.

At the very beginning of spore germination in streptomycetes the full-length alpha subunit of DNA-dependent RNA polymerase is shortened from its C-terminus. The C-terminal domain of the protein is required for binding of DNA and transcription regulators but its regulatory role in streptomycetes was not extensively studied. Comparison of the sequences of E. coli and S. coelicolor RNA polymerase alpha subunit (RNAP alpha) C-terminal domains reveals that the majority of amino acid residues responsible for the interaction with transcription regulators is conserved in both microorganisms. The spore specific modification of streptomycete RNAP alpha could thus have its regulatory role. The nature of the proteolytic enzyme, responsible for the RNAP alpha cleavage is discussed.

DNA-Directed RNA Polymerases↗
PubMed2003

LmbJ and LmbIH protein levels correlate with lincomycin production in Streptomyces lincolnensis.

AIMS: To demonstrate the expression of two overlapping genes lmbJ and lmbIH in Streptomyces lincolnensis and to document LmbJ and LmbIH protein levels during the lincomycin production phase. To analyse presumable function of the LmbIH protein. METHODS AND RESULTS: Lincomycin production was monitored by thin-layer chromatography, proteins LmbJ and LmbIH were assayed in the cell-free extracts of S. lincolnensis by immunodetection. LmbJ occurred at stable level (2-4 mg x g(-1) of total proteins) for a long time period (36-96 h of cultivation) covering the whole production phase. This fairly corresponds to the catalytic function of the protein in the antibiotic biosynthesis (N-demethyllincomycin methyltransferase). On the contrary, LmbIH reached the detectable level (0.1 and 0.7 mg x g(-1)) just for a short period at 60-72 h. CONCLUSIONS: The absence of LmbIH protein at a detectable level during the major part of the antibiotic production phase casts doubt on its possible catalytic function. Rather a different connection with the final biosynthetic steps, e.g. regulatory, can be envisaged. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of a newly found putative regulatory gene was demonstrated during production of industrial antibiotic, lincomycin.

Bacterial Proteins↗
PubMed2001

Chemical plume tracking. 1. Chemical information encoding.

It is shown experimentally that chemical information can be encoded and preserved in flowing liquid streams. It can be retrieved by chemical sensing arrays using correlation analysis. This finding is important for understanding of the mechanism of chemotaxis as practiced by some aquatic animals and also is a necessary prerequisite for construction of chemical plume tracking robots.

Chemotaxis↗
PubMed2001

Chemical plume tracking. 2. Multiple-frequency modulation.

The purpose of this research is to estimate the effect of turbulent mixing on the information content in a chemically encoded signal, to investigate the effect of the presence of multiple encoded frequencies, and to evaluate the information contained in the higher harmonics of the coherence spectra. The virtual plume instrument is used to mimic the flow conditions and signal patterns in a real chemical plume. Two-frequency modulation experiments are performed using solenoid valves to introduce concentration plugs of a marker into the carrier flows at certain constant frequencies. In our experiments, the length of the delay elements and the dispersion were varied to mimic different characteristics of the turbulent plume. In addition, an artificial but uncorrelated white noise was added to the raw amperometric signals in order to simulate the "noisy" conditions existing in a real plume. Our experiments reveal that the introduced turbulence has only a marginal effect on the coherence spectra. Moreover, it is shown that when the second frequency is present in the plume, both fundamental frequencies can be unambiguously assigned. Higher harmonics in the coherence spectra have been found to depend on the distance from the source. These findings are important for understanding of the mechanism of chemotaxis and may also lead to the design of optimized search algorithms for chemical plume tracking robots.

Journal Article↗
PubMed2001

Headspace analysis of engine oil by gas chromatography/mass spectrometry.

This study establishes the rationale necessary for determining the time to change engine oil. This is based on identifying gaseous components in new and used automobile lubricants. Key compounds, so-called "signature", are separated and identified qualitatively by coupled gas chromatography/mass spectrometry. Volatile antioxidants at zero miles and fuel contaminants at low mileage are observed in the headspace of engine oil. Several oxidative degradation components have been positively identified in the used oil, which include the following: acetaldehyde, acetone, butanal, 2-propanol, acetic acid, 2-hexanol, benzoic acid, benzaldehyde, and 1-pentanol. This study strongly suggests that the status of lubricating oil can be determined by the analysis of the gas phase above the oil. Most importantly, it opens the possibility of performing conditional maintenance of the combustion engine based on information obtained from gas sensors.

Journal Article↗
PubMed2001

Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase.

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.

Amino Acid Sequence↗
PubMed2001

Putative lmbI and lmbH genes form a single lmbIH ORF in Streptomyces lincolnensis type strain ATCC 25466.

The lincomycin-production gene cluster of the industrial overproduction strain Streptomyces lincolnensis 78-11 has been sequenced (Peschke et al. 1995) and twenty-seven putative open reading frames with biosynthetic or regulatory functions (lmb genes) identified. Two distinct hypothetical genes, lmbI and lmbH, were found downstream of the lmbJ gene, coding for LmbJ protein, which is believed to participate in the last lincomycin biosynthetic step, i.e. conversion of N-demethyllincomycin (NDL) to lincomycin. In the present study, we demonstrate the presence of a single larger open reading frame, called lmbIH, in the lincomycin low-production type strain Streptomyces lincolnensis ATCC 25466, instead of two smaller lmbI and lmbH genes. The product, LmbIH, is a protein of an unknown function and is homologous with the T1dD protein family. Escherichia coli T1dD protein was previously shown to be involved in the control of DNA gyrase by LetD protein. Moreover, our experiments indicate co-regulation of lmbJ and lmbIH expression. This translation coupling probably reflects an eight nucleotide overlap between the lmbJ and lmbIH genes, as well as the lack of a Shine-Dalgarno sequence upstream of the lmbIH gene.

Amino Acid Sequence↗
PubMed2001

Plume-tracking robots: a new application of chemical sensors.

Many animals have the ability to search for odor sources by tracking their plumes. Some of the key features of this search behavior have been successfully transferred to robot platforms, although the capabilities of animals are still beyond the current level of sensor technologies. The examples described in this paper are (1) incorporating into a wheeled robot the upwind surges and casting used by moths in tracking pheromone plumes, (2) extracting useful information from the response patterns of a chemical sensor array patterned after the spatially distributed chemoreceptors of some animals, and (3) mimicking the fanning behavior of silkworm moths to enhance the reception of chemical signals by drawing molecules from one direction. The achievements so far and current efforts are reviewed to illustrate the steps to be taken toward future development of this technology.

Animals↗
PubMed2000

Two forms of DNA-dependent RNA polymerase alpha subunit in streptomycetes.

We demonstrated two different DNA-dependent RNA polymerase (RNAP) alpha subunits in spores of Streptomyces granaticolor with apparent molecular masses of 40 and 43 kDa. The 43-kDa subunit was also found in vegetative cells. These two proteins are highly similar to each other as well as to other bacterial RNAP alpha subunits. The 40-kDa subunit is shortened from its C-terminus, in the portion of the protein, required for binding of DNA and transcription regulators. The gene for RNAP alpha from S. granaticolor was cloned and sequenced and the corresponding protein was overproduced in Escherichia coli. In vitro experiments using purified RNAP alpha showed that the cell free extract from spores of S. granaticolor exhibits proteolytic activity responsible for the alpha subunit shortening, whereas that from vegetative cells does not. This modification of alpha subunit might point to a novel mechanism of transcriptional control in streptomycetes.

Amino Acid Sequence↗
PubMed1999

Mutations in two distinct regions of acetolactate synthase regulatory subunit from Streptomyces cinnamonensis result in the lack of sensitivity to end-product inhibition.

Acetolactate synthase small subunit encoding ilvN genes from the parental Streptomyces cinnamonensis strain and mutants resistant either to valine analogues or to 2-ketobutyrate were cloned and sequenced. The wild-type IlvN from S. cinnamonensis is composed of 175 amino acid residues and shows a high degree of similarity with the small subunits of other valine-sensitive bacterial acetolactate synthases. Changes in the sequence of ilvN conferring the insensitivity to valine in mutant strains were found in two distinct regions. Certain point mutations were located in the conserved domain near the N terminus, while others resulting in the same phenotype shortened the protein at V(104) or V(107). To confirm whether the described mutations were responsible for the changed biochemical properties of the native enzyme, the wild-type large subunit and the wild-type and mutant forms of the small one were expressed separately in E. coli and combined in vitro to reconstitute the active enzyme.

Acetolactate Synthase↗
PubMed1999

Attenuation of microvascular reperfusion injury in rat pancreas transplantation by L-arginine.

BACKGROUND: Despite improving results, exocrine complications remain a major challenge in clinical pancreas transplantation. The etiology of posttransplantation pancreatitis relates almost certainly to cold ischemia/reperfusion-induced microvascular injury with an imbalance of vasoconstricting and vasodilating mediators due to endothelial dysfunction. We therefore studied the effectiveness of a nitric oxide donor on postischemic microvascular reperfusion injury after pancreas transplantation. METHODS: Heterotopic isogeneic pancreaticoduodenal transplantation was performed in spontaneously breathing, chloralhydrate-anesthetized Sprague Dawley rats after 16 hr (n=5) of cold storage of the graft in 4 degrees C histidine-tryptophane-ketoglutarate solution. An additional five animals received L-arginine immediately before (50 mg/kg i.v.) and during the first 30 min of reperfusion (100 mg/kg i.v.). Five animals that did not undergo transplantation served as controls. Intravital fluorescence microscopy was used for analysis of functional capillary density, capillary diameters, and capillary red blood cell velocity in exocrine pancreatic tissue during 120 min of reperfusion. Histology served for quantitative assessment of inflammatory response (leukocytic tissue infiltration) and endothelial disintegration (edema formation). RESULTS: In L-arginine-treated animals, functional capillary density of exocrine tissue of pancreatic grafts was found slightly higher after 30 and 60 min, and significantly higher after 120 min of postischemic reperfusion compared with untreated pancreatic grafts. This was accompanied by a significant increase of capillary diameters. In parallel, pancreatic histology revealed significant attenuation of both leukocytic tissue infiltration and edema formation in the L-arginine-treated animals when compared with the nontreated controls. CONCLUSIONS: Besides reduction of leukocyte-dependent microvascular injury, L-arginine improves postischemic microvascular reperfusion, supposedly by capillary dilatation. Thus, our results suggest that supplement of nitric oxide during reperfusion is effective in attenuating exocrine microvascular reperfusion injury.

Animals↗
PubMed1999

Exocrine, but not endocrine, tissue is susceptible to microvascular ischemia/reperfusion injury following pancreas transplantation in the rat.

While post-transplant pancreatitis is still a frequently occurring complication of whole pancreas transplantation, dysfunction of the endocrine tissue is rarely observed. Given that microcirculatory disorders play a major role in the pathogenesis of pancreatitis, we hypothesized a dissociation of endocrine and exocrine microvascular control in pancreas transplantation (cold ischemia-reperfusion) and studied this dissociation quantitatively, analyzing the pancreatic microcirculation after heterotopic isogeneic pancreaticoduodenal transplantation in rats by means of fluorescence microscopy. Functional capillary density (FCD) of both exocrine and endocrine tissue of pancreatic grafts after 1 h of cold storage in HTK solution did not differ when compared to sham-operated, time-matched controls. Intermittent capillary perfusion, which is absent under sham control conditions and which is proposed to be operative as a compensatory mechanism to counteract malperfusion, was observed in 52% of the exocrine, but in only 8% of the endocrine, tissue studied (p < 0.05). In contrast, cold storage of pancreatic grafts for 6 h in HTK resulted in a complete loss of intermittent capillary perfusion in exocrine tissue and, consequently, marked exocrine perfusion failure (decrease in FCD), while FCD of pancreatic endocrine tissue was preserved without any significant change in the incidence of intermittent capillary perfusion. Thus, our results indicate a higher susceptibility of the exocrine pancreas to cold ischemia/reperfusion events that is associated with significant alterations in nutritive perfusion and, thus, with limitations of the oxygen supply to the tissue. This may lead to inflammatory tissue reactions in the clinical setting of pancreas transplantation.

Animals↗
PubMed1997

Expression and kinetic characterization of methylmalonyl-CoA mutase from patients with the mut- phenotype: evidence for naturally occurring interallelic complementation.

L-Methylmalonyl-CoA mutase (MUT) is an adenosylcobalamin (AdoCbl)-requiring mitochondrial matrix enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Inherited defects in the gene encoding this enzyme result in the mut forms of methylmalonic acidemia. Expression of mature human MUT cDNA in Escherichia coli at a post-induction cultivation temperature of 12 degrees C, rather than 37 degrees C, led to the folding of the majority of the synthesized protein to a soluble form, with an activity of 0.2-0.3 U/mg protein in the cell-free extract, 10-15 times higher than that in human liver homogenate. Six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V, were detected in MUT cDNA of patients suffering from the mut- form of methylmalonic acidemia, resulting from defective AdoCbl binding. Two (G623R and G717V) had been reported in other patients. Three (G94V, Y231N and R369H) are the first changes in the NH2-terminal part of the enzyme reported to cause the mut- phenotype. Enzymes with the mutations were individually expressed, and their kinetic parameters were generally in accord with published biochemical data from extracts of fibroblasts from these patients. The mutations increased the K(m) for AdoCbl by 40- to 900-fold, while V(max) values varied from 0.2% to nearly 100% of that of wild-type protein. In one case of a doubly heterozygous cell line, however, neither of the constituent mutant enzymes had a K(m) corresponding to the lower of the two estimated from the extract data. This finding may reflect the natural occurrence of interallelic complementation in vivo in this cell line.

Cell Line↗
PubMed1995

Translation initiation factors of a tetracycline-producing strain of Streptomyces aureofaciens.

Protein synthesis initiation factors from a tetracycline-producing strain of Streptomyces aureofaciens were purified and characterized. Two forms of IF3 (M(r) = 24,000 and 22,500) were found. By Western blot analysis, only one form of protein IF2 cross-reactive with anti-IF2 of Escherichia coli was revealed. The molecular mass of purified IF2 was 69,000 as determined by SDS-polyacrylamide gel electrophoresis. In spite of differences in molecular mass between the IF2 forms of E. coli and the factor from S. aureofaciens, the latter could substitute IF2 of E. coli in the stimulation of codon-specific binding of initiator tRNA. In contrast to the reported absence of IF1 in some Gram-positive microorganisms, we found "protein IF1" (M(r) = 9,000) in S. aureofaciens that increased the IF2-dependent binding of initiator tRNA to ribosomes.

Bacterial Proteins↗
PubMed1994

Chemical sensors.

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Biosensing Techniques↗