Biomedical subjects

J Novotná

Publications and source records attributed to J Novotná.

At least 19 recordsLinked to original sources

PubMed2002

Possible role of matrix metalloproteinases in reconstruction of peripheral pulmonary arteries induced by hypoxia.

Exposure to chronic hypoxia results in hypoxic pulmonary hypertension characterized by structural remodeling of peripheral pulmonary vasculature. An important part of this remodeling is an increase of collagen turnover and deposition of newly formed collagen fibrils in the vascular walls. The activity of collagenolytic metalloproteinases in the lung tissue is notably increased in the first days of exposure to hypoxia. The increased collagenolytic activity results in the appearance of collagen cleavages, which may be implied in the triggering of mesenchymal proliferation in peripheral pulmonary arteries. We hypothesize that radical injury to pulmonary vascular walls is involved in collagenolytic metalloproteinase activation.

Animals↗

PubMed2001

Major proteins related to chlortetracycline biosynthesis in a Streptomyces aureofaciens production strain studied by quantitative proteomics.

Changes in synthesis and abundance of proteins associated with chlortetracycline (CTC) production in Streptomyces aureofaciens were investigated by two-dimensional polyacrylamide gel electrophoresis of proteins pulse-labelled in vivo with L-[35S]methionine. Eleven individual protein spots were selected as being related to formation of the antibiotic. Expression of these prominent proteins was not observed in the non-producing mutant; moreover, they were overexpressed in cultures grown in the presence of benzyl thiocyanate, a specific stimulator of CTC biosynthesis used in industrial fermentations. The expression kinetics of the selected proteins was assessed using the technique of computer-assisted image analysis with the EQIAS software and the elongation factor Tu as an internal standard. Interestingly, the kinetic profiles were generally not identical. including those of anhydrotetracycline monooxygenase and the 13-kDa subunit of tetracycline dehydrogenase, two enzymes involved, in the terminal sequential steps of the CTC biosynthetic pathway. The presence of more forms of these enzymes with different charge characteristics was observed. The data presented demonstrated how dramatically the industrial microorganism can change its protein repertoire during the production phase; at least five proteins were nearly comparable in level to the most prominent proteins, exemplified by elongation factor Tu.

Anti-Bacterial Agents↗

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

Hyperoxia and recovery from hypoxia alter collagen in peripheral pulmonary arteries similarly.

Chronic hypoxia causes pulmonary hypertension, the mechanism of which includes altered collagen metabolism in the pulmonary vascular wall. This chronic hypoxic pulmonary hypertension is gradually reversible upon reoxygenation. The return to air after the adjustment to chronic hypoxia resembles in some aspects a hyperoxic stimulus and we hypothesize that the changes of extracellular matrix proteins in peripheral pulmonary arteries may be similar. Therefore, we studied the exposure to moderate chronic hyperoxia (FiO2 = 0.35, 3 weeks) in rats and compared its effects on the rat pulmonary vasculature to the effects of recovery (3 weeks) from chronic hypoxia (FiO2 = 0.1, 3 weeks). Chronically hypoxic rats had pulmonary hypertension (Pap = 26 +/- 3 mm Hg, controls 16 +/- 1 mm Hg) and right ventricular hypertrophy. Pulmonary arterial blood pressure and right ventricle weight normalized after 3 weeks of recovery in air (Pap = 19 +/- 1 mm Hg). The rats exposed to moderate chronic hyperoxia also did not have pulmonary hypertension (Pap = 18 +/- 1 mm Hg, controls 17 +/- 1 mm Hg). Collagenous proteins isolated from the peripheral pulmonary arteries (100-300 microm) were studied using polyacrylamide gel electrophoresis. A dominant low molecular weight peptide (approx. 76 kD) was found in hypoxic rats. The proportion of this peptide decreases significantly in the course of recovery in air. In addition, another larger peptide doublet was found in rats recovering from chronic hypoxia. It was localized in polyacrylamide gels close to the zone of alpha2 chain of collagen type I. It was bound to anticollagen type I antibodies. An identically localized peptide was found in rats exposed to moderate chronic hyperoxia. The apparent molecular weight of this collagen fraction suggests that it is a product of collagen type I cleavage by a rodent-type interstitial collagenase (MMP-13). We conclude that chronic moderate hyperoxia and recovery from chronic hypoxia have a similar effect on collagenous proteins of the peripheral pulmonary arterial wall.

Animals↗

PubMed2000

In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA.

Bromouridine-triphosphate is commonly used for in situ immunocytochemical labeling of newly synthesized RNA in living cells. While extranucleolar transcripts do not require special conditions for visualization, special treatment prior to fixation (e.g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in previous studies. We show in the present investigation that bromouridine-triphosphate is efficiently used by both extranucleolar and nucleolar RNA polymerases in living cultured cells. The failure to detect incorporated bromouridine within nucleoli is entirely due to improper treatment of cells after bromouridine incorporation. When methanol/acetone fixation is used, fluorescence signals within nucleoli can be routinely found.

Animals↗

PubMed2000

A possible role of the oxidant tissue injury in the development of hypoxic pulmonary hypertension.

Chronic sojourn in hypoxic environment results in the structural remodeling of peripheral pulmonary arteries and pulmonary hypertension. We hypothesize that the pathogenesis of changes in pulmonary vascular structure is related to the increase of radical production induced by lung tissue hypoxia. Hypoxia primes alveolar macrophages to produce more hydrogen peroxide. Furthermore, the increased release of oxygen radicals by other hypoxic lung cells cannot be excluded. Several recent reports demonstrate the oxidant damage of lungs exposed to chronic hypoxia. The production of nitric oxide is high in animals with hypoxic pulmonary hypertension and the serum concentration of nitrotyrosine (radical product of nitric oxide and superoxide interaction) is also increased in chronically hypoxic rats. Antioxidants were shown to be effective in the prevention of hypoxia induced pulmonary hypertension. We suppose that the mechanism by which the radicals stimulate of the vascular remodeling is due to their effect on the metabolism of vascular wall matrix proteins. Non-enzymatic protein alterations and/or activation of collagenolytic matrix metalloproteinases may also participate. The presence of low-molecular weight cleavage products of matrix proteins stimulates the mesenchymal proliferation in the wall of distal pulmonary arteries. Thickened and less compliant peripheral pulmonary vasculature is then more resistant to the blood flow and the hypoxic pulmonary hypertension is developed.

Animals↗

PubMed1999

Binding of lead to collagen type I and V and alpha2(I) CNBr (3,5) fragment by a modified Hummel-Dreyer method.

Binding of lead (as lead acetate) to collagen type I alpha, and alpha2 chains, collagen type V and a large cyanogen bromide fragment of type I collagen [alpha2(I)CB(3,5)] was investigated by the large-zone Hummel-Dreyer method. It was demonstrated that two categories of binding sites exist in the collagen molecule, the number of which correlates rather well with the available aspartic and glutamic acid residues. Similar results were obtained for all collagen chains (fragments) used. The number of sites thus obtained was compared with the cross-striation pattern (reflecting areas where lead is bound) of the SLS form of collagen type I (alpha1 chain); it is suggested that the number of bands seen in the SLS form reflects primarily the number of available aspartic acid residues in the molecule. The association constants obtained are comparable with the low affinity interactions seen e.g., between Cu and bovine serum albumin.

Collagen↗

PubMed1999

[Mechanisms of remodeling of pulmonary blood vessels in chronic hypoxia].

Chronic lung hypoxia results in the hypoxic pulmonary hypertension, which is caused by the remodeling of peripheral pulmonary blood vessels. Vascular smooth muscle cells proliferate into the prealveolar arteries, the turnover and deposition of connective tissue proteins is increased. We observed an enhanced collagenolytic activity in the extracts from isolated peripheral lung arteries of hypoxic rats. SDS electrophoresis of collagenous proteins extracted from these vessels showed presence of the low molecular weight cleavages of collagen type I. We hypothesize that the activation of collagenolytic metalloproteinases is related to the release of reactive oxygen species, NO and products of their interaction (peroxynitrite). Collagen cleavages may stimulate mesenchymal proliferation in the vascular wall.

Animals↗

PubMed1998

Micropreparation of tissue collagenase fragments of type I collagen in the form of surfactant-peptide complexes and their identification by capillary electrophoresis and partial sequencing.

Combination of standard approaches like pepsin digestion and slab gel electrophoresis with capillary separations allows a relatively easy identification of in vivo occurring collagen fragments. Capillary electrophoresis can be done either in 25 mM phosphate buffer (pH 2.5) or in a 25 mM phosphate buffer (pH 4.5) made 0.1% with respect to sodium dodecyl sulfate (SDS). While in the first case peptides move to the cathode in a molecular mass dependent manner, in the second case they move towards anode (also in a molecular mass dependent manner). The profiles obtained by the two approaches resemble mirror images with low molecular mass peptides moving first in the acid background electrolyte while they move last in the presence of SDS. It is proposed that in the capillary electrophoretic separation at pH 2.5 the separation mechanism involves the interaction of the individual peptides with the capillary wall while in the second case (pH 4.5) the leading mechanism of separation involves the interaction of the analytes with the micellar phase. For micellar phase separation the system must be run at reversed polarity. Capillary electrophoretic separation in the pH 2.5 buffer is considerably affected by the presence of SDS in the previous steps of peptide preparation. If the peptides are obtained from SDS slab gel electrophoresis, their movement in the capillary electrophoresis step is about three times faster that the movement of corresponding peptides which have not been complexed with SDS.

Animals↗

PubMed1998

Exposure to chronic hypoxia induces qualitative changes of collagen in the walls of peripheral pulmonary arteries.

Qualitative changes of vascular wall matrix collagens in chronic hypoxic pulmonary hypertension were studied by gel electrophoresis. Male adult rats (n = 12) were exposed to hypoxia (FiO2 = 0.1, 3 wks). Control rats (n = 13) were kept in air. Samples of peripheral pulmonary arteries (PPA, diam. 100-400 microm), main branches of pulmonary artery, and aorta were dissected. Arterial samples were treated with 4M guanidine-HCl to remove noncollagenous moieties and the collagenous stroma was dissolved by limited pepsin digestion at low pH. Low molecular mass peptides (M. W. approx. 76 and 66 kD) were detected in the gel electrophoretic profile of collagen peptides of PPA of the chronically hypoxic animals and in aorta of both hypoxic and normoxic groups. These peptides were absent in the PPA of normoxic rats. Since the 76 kD peptide bound anticollagen type I antibodies, it appears to be of collagenous nature and it may be the result of collagenolytic activity in PPA isolated from hypoxic lungs. This was confirmed by zymography. We conclude that exposure of rats to chronic hypoxia results in the presence of low molecular mass peptides in the wall matrix of PPA which resemble those found in aorta of normoxic animals. Collagenolytic activity in the walls of peripheral pulmonary arteries may participate in the mechanism of lung vascular remodelling in chronic hypoxia.

Animals↗

PubMed1998

[Treatment of multiple myeloma with high-dose chemotherapy and transplantation of autologous hematopoietic stem cells and subsequent maintenance therapy with interferon alfa-2b or interferon alfa 2b and dexamethasone. Report of the ongoing study of the "4W" Czech Myeloma Group].

We report our results with high-dose chemotherapy in previously untreated multiple myeloma patients (4 courses of VAD chemotherapy, collection of PBSC after priming with cyclophosphamide, 5 g/m2, high-dose chemotherapy with melphalan, 200 mg/m2). Second transplantation was indicated only for patients who did not achieve remission after the first high-dose therapy (paraprotein lower than 25% of the pretreatment value). For the second transplantation melphalan (200 mg/m2) with methylprednisolone (1.5 g for 5 days) were used as conditioning regimen. After high-dose therapy all patients were randomized into two arms of maintenance therapy: interferon alpha-2b or sequential maintenance therapy (interferon alpha-2b for 3 months followed after 4 week pause by 40 mg of dexamethasone days 1-4, 10-13 and 20-23. The administration of interferon alpha was resumed four weeks after the last dexamethasone for next three months. The maintenance therapy continued for 48 months or until the progression. Fifty-five patients were enrolled in the study from January 1996 to August 1997. Thirty-five patients have undergone the first transplantation and 57% of them reached complete remission. There were 10% of non-responders after the first high-dose regimen. The mean time to reach white blood cell count above 1 x 10(9)/L after the application of high dose melphalan and platelets more than 50 x 10(9)/L were 12.2 (range 6-16 days) and 12.4 (range 0-25 days), respectively. Grade 4 mucositis according to SWOG classification requiring total parenteral nutrition was presented in 40% of the patients. The mean number of 1 unit of platelets and 2 units of packed red blood cells transfusions were given within the posttransplant period. Early transplant related mortality was 3%. This paper describes the response and tolerance of each particular step of therapy. The follow-up has been too short to evaluate event-free and overall survivals.

Adolescent↗

PubMed1998

Accumulation of lead in tissues after its administration in drinking water to laboratory rats.

Lead administered to laboratory rats in drinking water (0.1-0.8%) as lead acetate solution tends to accumulate in collagen-rich tissues such as tendons and the skin. The amount of lead deposited (and also zinc present in the tissue without its supplementation) correlates with the blood supply to the tissue investigated. The highest deposits of lead were observed in placenta and chorionic membranes, though here only about 60% are collagen-bound. No differences in the drinking habits of the animals were observed and also at lower concentrations of lead in the drinking water no dose dependence was revealed. However, at 0.8% of lead in drinking water considerable accumulation of lead was observed in all tissues investigated.

Animals↗

PubMed1997

Quantitation of collagen types I, III and V in tissue slices by capillary electrophoresis after cyanogen bromide solubilization.

A method for the determination of the proportions of major fiber-forming collagens (types I, III and V) in soft connective tissue was elaborated. The method is based on the release of insoluble collagen by CNBr with subsequent separation of the arising peptides. For routine application the peptides are separated by capillary electrophoresis (50 mM phosphate pH 2.5, 15 kV, 50 degrees C, 70/60 cm x 70 microns I.D. capillary with UV detection at 200 nm). Quantitation of collagen type I can be done either on the basis of spiking the sample with a peptide mixture obtained from a known amount of collagen type I, or by spiking the sample with an equimolar mixture of the two peptides [alpha 1(I)CB2 and alpha 1(I)CB4] (constituting a fused peak) along with alpha 1(III)CB2 and alpha 1(V)CB1. Compared to the previously published methods the procedure is faster and does not require isolation of marker peptides by tedious chromatographic procedures in a preceding preparatory step. Good results are obtained within a wide range of run buffer concentrations and applied voltages; conversely, intensive cleaning of the capillary after every three runs is recommended with a new capillary after 20-30 runs.

Animals↗

PubMed1997

Oxidized collagen stimulates proliferation of vascular smooth muscle cells.

We hypothesize that the vascular smooth muscle proliferation after lung injury results from oxidative damage to the matrix proteins in the walls of pulmonary blood vessels. The smooth muscle cells (SMC) isolated from rat aorta were cultured on the surface coated with oxidized and nonoxidized (control) collagen of type I. Oxidation of collagen was induced by UV irradiation and characterized by fluorescence tridimensional spectral arrays and by gel electrophoresis. From day 1 to 6 of the experiment, SMC proliferated more rapidly on the oxidized collagen than on the control surface. At high SMC population densities (day 9 of experiment) the difference disappeared. After 10 min of trypsinization the cells growing on oxidized collagen rounded and detached completely from the growth surface. The control cells on nonoxidized collagen detached only after 30 min of trypsinization. We conclude that oxidation of collagen of vascular wall matrix may participate in stimulation of SMC proliferation after oxidant tissue injury.

Animals↗

PubMed1996

Capillary zone electrophoresis of collagen type I CNBr peptides in acid buffers.

Collagen type-I CNBr peptides were separated under acidic conditions by capillary electrophoresis. Separation conditions were: 100 mM phosphate buffer pH 2.5, 50 cm x 50 microns capillary (placed in a cartridge), 8 kV, running time 30-45 min, detection by UV at 200 nm. The peptides were separated strictly by their molecular mass and the overall pattern was well comparable to RP-HPLC separations of these analytes. It is proposed that the separation mechanism may involve hydrophobic sorptions to the capillary wall.

Acids↗

PubMed1996

[EEG correlates of therapeutic effects of electroconvulsive therapy].

In a group of 52 patients electroconvulsive treatment by means of a Thymatron apparatus was provided under simultaneous EEG control. In addition to parameters of the EEG curve (type of curve according to intensity of the paroxysmal process, duration of electroconvulsion on EEG, type of termination of paroxysm of EEG) also other variables were investigated in relation to the therapeutic effect. The following were identified as significant predictors for marked clinical improvement: type of EEG curve with more marked paroxysmal activity, shorter cumulative duration of electroconvulsions on EEG before first clinical improvement of the patient and a higher CGI entering score. Other variables proved unimportant, e.g. the patient's age, total number of electroconvulsions, total cumulative duration of electroconvulsions on EEG and type of termination of electroconvulsions on EEG.

Adult↗

PubMed1995

Purification and characterization of a novel valine dehydrogenase from Streptomyces aureofaciens.

The first valine dehydrogenase of S. aureofaciens had been described (Vancurová, I., Vancura, A., Volc, J., Neuzil, J., Flieger, M., Basarová, G. and Bĕhal, V. (1988) J. Bacteriol. 170, 5192-5196). In the present work, a second valine dehydrogenase was detected and purified by hydrophobic and fast protein liquid chromatographies. The enzyme has a relative molecular mass (M(r)) of 240,000 and is composed of 6 identical subunits, each of M(r) 41,000. In the presence of NAD, the enzyme catalyzes the reversible deamination of several branched- and straight-chain amino acids. The enzyme activities with L-2-aminobutyrate and deamino-NAD+ are markedly higher than those with L-valine and NAD+, respectively. The enzyme synthesis is significantly induced by L-valine but severely repressed by ammonia. Molecular and catalytic properties of the enzyme distinguish it from the other described valine dehydrogenases. The results directly demonstrate the presence of two valine dehydrogenases in a single Streptomyces species.

Amino Acid Oxidoreductases↗

PubMed1995

Protein profiles of Streptomyces aureofaciens producing tetracyclines: reappraisal of the effect of benzyl thiocyanate.

Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 microM benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.

Bacterial Proteins↗