Biomedical subjects

I Ivanov

Publications and source records attributed to I Ivanov.

At least 19 recordsLinked to original sources

PubMed2002

Characterization of functional domains of human interferon gamma by specific monoclonal antibodies.

For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1E11 and 5D6, were sequence-specific for hIFNgamma. According to the estimated additivity index, they recognized epitopes located at distinct non-overlapping areas of the hIFNgamma molecule. When pre-incubated with hIFNgamma, MAB 1E11 was able to neutralize the antiviral as well as the antiproliferative activity of the cytokine. This indicated that MAB 1E11 was specific either for the domain responsible for binding to the cell receptor or for a domain required for both functions. By contrast, the second MAB 5D6 did not interfere with any of the two hIFNgamma biological activities. The epitope of MAB 5D6 was located between the amino acid residues Leu 135 and Glu 143 by using different forms of C-terminally truncated hIFNgamma. These data allow the conclusion that the last nine C-terminal amino acids are not essential for the receptor binding and biological functioning of this cytokine. The possible role of hIFNgamma C-terminus in the intracellular cascade of events is discussed.

Antibodies, Monoclonal↗

PubMed2002

Potential role of nitric oxide in endothelin-1 provoked inhibition of progesterone secretion by isolated ovarian granulosa cells.

OBJECTIVE: Nitric oxide (NO) is involved in different cell functions including ovarian steroid production. Endothelin-1 (ET-1) was found to regulate the steroidogenesis in ovarian granulosa cells (GC). The present study was designed to receive more information about the mechanism of action of NO in the process of ET-1 induced progesterone (P) inhibition, using nicotine amide dinucleotide phosphate-diaphorase (NADPH-d) histochemistry as a cofactor of oxidoreductase enzymes (e. g. nitric oxide). METHODS: Granulosa cells were isolated from ovaries of: 1. young women with natural cycle or after in vitro fertilization (IVF), 2. premenopausal women. The obtained cells were cultured with endothelin-1 and the concentration of progesterone in conditioned media was determined by RIA. For the estimation of NADPH-d the histochemical reaction was used. RESULTS: The suppressive effect of ET-1 on P production in granulosa cells was more pronounced in young women with natural cycles, slightly weaker after IVF and the most ineffective in premenopausal patients. The number of NADPH-d positive GCs was higher in young non-hormonally stimulated women, slightly lower after IVF and small in premenopausal ones. CONCLUSIONS: The results indicate the possible role of NADPH-d or NOS in the mechanism of ET-1 provoked P suppression.

Adult↗

PubMed2002

Anaerobic sequencing batch reactor as initiating stage in complete pentachlorophenol biodegradation.

Biodegradation of pentachlorophenol (PCP) has been studied in a sequence of two completely mixed reactors. Investigation on the first anaerobic sequencing batch reactor (AnSBR) is discussed in detail in this paper. Key technological and microbiological features were studied: two different types of adaptation process of anaerobic sludge towards PCP detoxication; the influence of the sludge concentration upon the rate of PCP biodegradation; minimum retention time for PCP degradation in dependence on the PCP concentration; modeling of the PCP degradation process; effluent COD and SS concentrations; changes in the micro- and macrostructure of the sludge during acclimatization process.

Bacteria, Anaerobic↗

PubMed2001

Soybean lipoxygenase-1 oxygenates synthetic polyenoic fatty acids with an altered positional specificity: evidence for inverse substrate alignment.

The positional specificity is the decisive enzyme property for classification of lipoxygenases and for the currently used lipoxygenase nomenclature. It has been reported before that soybean lipoxygenase-1, which oxygenates polyenoic fatty acids at alkaline pH to the corresponding n - 6 hydroperoxy derivative, exhibits a different positional specificity when either the reaction conditions or the substrate structure is altered. To investigate the impact of structural substrate modifications on the positional specificity of this enzyme and to force an inverse substrate binding, we synthesized arachidonic acid analogues modified at the omega-terminus. Care was taken that the double bond system remained unchanged so that hydrogen abstraction from all three bisallylic methylenes was theoretically possible. We found that omega-modification of arachidonic acid leads to an impaired substrate affinity and a reduced reaction rate, but we did not detect any 5-lipoxygenation products, suggesting that structural modification of the omega-end may not be sufficient to force an inverse substrate orientation. However, when both ends of the fatty acid chain (omega-terminus and free carboxylate) were modified simultaneously, a considerable share of 5-lipoxygenation products was detected. These results indicate that introduction of polar or bulky groups at the methyl terminus of polyenoic fatty acids was not sufficient to force an inverse substrate orientation. However, simultaneous introduction of an omega-OH group and methylation of the carboxylate led to formation of significant 5-lipoxygenation products, suggesting an inverse head to tail substrate orientation.

Arachidonic Acid↗

PubMed2001

Role of the C-terminal chain in human interferongamma stability: an electrostatic study.

Electrostatic interactions in two structures of human interferon gamma (hIFNgamma), corresponding to interferon molecule alone and bound to its receptor, were analyzed on the basis of a continuum dielectric model. It was found that a number of titratable groups, mainly basic, show large pK shifts and remain in their neutral forms at physiologically relevant pH. The fact that these groups are largely common to both structures and that most of them belong to the set of most conserved sites suggests that this is a property inherent to the hIFNgamma molecule rather than an artifact of the crystal packing. His111 was also found deprotonated at neutral pH. It was concluded that receptor recognition involving His111 is driven by aromatic coupling of His111 and Tyr52 from the receptor rather than by electrostatic interactions. The structure corresponding to hIFNgamma in complex with its receptor shows a reduction in number and in degree of desolvation of the buried titratable sites. This finding suggested that on receptor binding, hIFNgamma adopts energetically more favorable, relaxed, conformation. It was experimentally shown that in contrast to the full-size hIFNgamma, the construct having 21 amino acid residues deleted from the C-terminus is soluble. The hydrophobicity profile analysis suggested that factors other than the exposure of hydrophobic parts of the molecule are responsible for the low stability and propensity for aggregation. On the basis of these results, it was assumed that the electrostatic influence of the C-terminal part contributes particularly to the low solvent exposure of the titratable groups, and hence to the low structural stability and propensity for aggregation of the recombinant hIFNgamma. Proteins 2001;43:125-133.

Escherichia coli↗

PubMed2001

Sintering peculiarities for hydroxyapatite with different degrees of crystallinity.

It has been shown that reduction of sintering temperature of hydroxyapatite is connected to the use of a powder, the particles of which consist of crystalline and amorphous-like constituents. Shrinkage of the pressings made of the powder starts at the middle temperatures (600-700 degrees C) and is realized by a mechanism of a mutual sliding of the particles. If the firing temperature of the pressings increases, the second stage, realized by a diffusion mechanism (over approximately 900 degrees C), begins. A shrinkage curve on the sliding stage has a stepped character. It is caused by a stopping effect on the shrinkage of the pressure of a gas, which is exuded as a result of crystallization of the amorphous-like constituent, and partly accumulates in closed cavities and pores. The contribution of the two mechanisms to the shrinkage process is determined by the degree of crystallinity of the powder (i.e. the ratio of the crystalline and the amorphous-like constituents). If a powder has a higher degree of crystallinity, higher temperatures result, and the stepped character of the shrinkage curve is smoothed. The sliding stage is practically lacking for pressings of crystalline powder, and a noticeable shrinkage starts at high temperatures (over approximately 900 degrees C) as diffusive processes develop.

Ceramics↗

PubMed2001

Alterations of lipoxygenase specificity by targeted substrate modification and site-directed mutagenesis.

BACKGROUND: Mammalian lipoxygenases (LOXs) are categorised with respect to their positional specificity of arachidonic acid oxygenation. However, the mechanistic basis for this classification is not well understood. To gain a deeper insight into the structural basis of LOX specificity we determined the reaction characteristics of wild-type and mutant mammalian LOX isoforms with native and synthetic fatty acids substrates. RESULTS: The rabbit 15-LOX is capable of catalysing major 12-lipoxygenation when the volume of the substrate-binding pocket is enlarged. These alterations in the positional specificity can be reversed when bulky residues are introduced at the omega end of the substrate. Simultaneous derivatisation of both ends of fatty acids forces a 15-LOX-catalysed 5-lipoxygenation and this reaction involves an inverse head-to-tail substrate orientation. In contrast, for arachidonic acid 5-lipoxygenation by the human 5-LOX the substrate fatty acid may not be inversely aligned. The positional specificity of this isoenzyme may be related to its voluminous substrate-binding pocket. Site-directed mutagenesis, which leads to a reduction of active site volume, converts the 5-LOX to a 15-lipoxygenating enzyme species. CONCLUSIONS: The positional specificity of LOXs is not an invariant enzyme property but depends on the substrate structure and the volume of the substrate-binding pocket. 15-LOX-catalysed 5-lipoxygenation involves an inverse substrate alignment but this may not be the case for 5-LOXs. Thus, both theories for the mechanistic basis of 5-lipoxygenation (straight and inverse substrate orientation) appear to be correct for different LOX isoforms.

Animals↗

PubMed2001

Evidence for non-enzymatic glycosylation in Escherichia coli.

Non-enzymatic glycosylation (glycation) is a chain of chemical reactions affecting free amino groups in proteins of long-living eukaryotes. It proceeds in several steps leading to the consecutive formation of Schiff bases, Amadori products and advanced glycation end-products (AGEs). To our knowledge, this process has not been observed in prokaryotes so far. However, the present study provides clear-cut evidence that glycation takes place in bacteria despite their short life span. We have detected AGEs in recombinant human interferon gamma (rhIFN-gamma) produced in Escherichia coli as well as in total protein of the same bacterium using three different approaches: (i) Western blotting using two monoclonal antibodies raised against AGEs; (ii) fluorescent spectroscopy; and (iii) investigation of the effect of known AGE inhibitors (such as acetyl salicylic acid and thiamine) on the glycation reaction. Our study shows that non-enzymatic glycosylation is initiated during the normal growth of E. coli and results in AGE formation even after isolation of proteins. This process seems to be tightly associated with some post-translational modifications observed in the cysteineless rhIFN-gamma, such as covalent dimerization and truncation.

Bacterial Proteins↗

PubMed2001

Self-initiation of translation of mRNAs devoid of translational initiators in Escherichia coli.

Recent studies have shown that the canonical SD-anti-SD interaction is dispensable for the initiation of translation of certain mRNAs in Escherichia coli. In this study the cat and tetR genes were modified to either destroy complementarity to E. coli 16S RNA or completely delete their 5' non-translated regions. Thus a series of cat- and tetR-derived genes were constructed, cloned under a strong constitutive promoter and expressed in E. coli cells. The efficiency of expression was evaluated by the yield of CAT (for the cat gene) and cell viability in increasing concentrations of antibiotic (for both cat and tetR genes). The obtained results show that the mRNAs transcribed from both series of reporter genes (cat and tetR) were active in vivo. Their activity was preserved even in the cases when the length of their 5' non-translated leader sequences was reduced to one nucleotide for the cat gene and eight nucleotides for the tetR gene. The yield of protein obtained with the latter constructs was detectable and sufficient for bacteria to survive at 50-100 microg/ml chloramphenicol and 20 microg/ml tetracycline, respectively.

5' Untranslated Regions↗

PubMed2001

Effect of sensory inputs alteration and central sensory disinteraction on postural sway and optokinetic reflex maintaining simultaneously body balance.

We aimed at estimating the effect of central sensory disinteraction on postural sway (PS) and optokinetic reflex (OKR) maintaining concurrently body balance. Healthy humans (N) and patients (P) with multiple sclerosis, and undergone infarct in the middle cerebral artery were examined. They stood on stable platform and foam rubber under three conditions: eyes open; eyes closed; during horizontal central optokinetic stimulation (COKS). On stable platform, a difference between PS of N and P was not found, while on foam rubber, PS of P was significantly greater than PS of N. During COKS on stable platform, PS increased only in P, while on foam rubber it increased in both N and P. OKR gain of N was circa 1 on stable and unstable supports, while that of P was 0.5. We concluded that artificial sensory disinteraction causes strong postural instability, but does not affect OKR.

Adult↗

PubMed2000

Investigation of Thin Aqueous Films on Silica Using a Modified Interferometric Technique.

The thinning behavior of liquid films between free bubbles and silica is investigated using the interferometric technique. Stable films were obtained at higher salt concentrations compared to previous studies because of the improved cleaning procedure. In contrast to the captive bubble technique used in the past, the free bubble method employed more closely approximates such processes as flotation. The results obtained for aqueous films at low and high ionic strengths are in better agreement with theoretical predictions than those in previous studies. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Mass-spectral analysis of human interferon-gamma and chloramphenicol acetyltransferase I produced in two Escherichia coli strains.

Recombinant human interferon-gamma and chloramphenicol acetyltransferase I were isolated from two Escherichia coli strains, E. coli LE329 and E. coli XL1-blue and characterized by electrospray ionization mass spectrometry (ESI-MS). The ESI-MS analysis showed higher masses in comparison with the theoretically calculated for both proteins as well as unexpected molecular heterogeneity. The ESI-MS spectral patterns of the proteins depended on the host strain used and were more heterogenous for the proteins isolated from E. coli LE392. One of the proteins (human interferon-gamma obtained from E. coli XL1-blue) was further subjected to BrCN cleavage. The ESI-MS analysis of the polypeptide mixture revealed shift in the molecular mass for two peptides including the last 26 amino acids of the human interferon-gamma molecule.

Biotechnology↗

PubMed2000

Influence of ochratoxin A and an extract of artichoke on the vaccinal immunity and health in broiler chicks.

The combined effect of ochratoxin A (at diet levels of 130, 305 and 790 ppb) and penicillic acid was studied in 100 broiler chicks. Serological investigations revealed significantly lower haemagglutination inhibiting antibody titers in the experimental chicks immunized with vaccine against Newcastle disease. A statistically significant decrease of the body weight and the relative weight of lymphoid organs as well as a significant increase of the relative weight of kidneys and liver were seen. The main degenerative changes were observed in the proximal convoluted tubules in kidneys and slight degenerative changes were found in the hepatocytes. Degenerative changes and depletion of lymphoid cells were observed in the bursa Fabricii, thymus, spleen and Peyer's patches of intestinal mucosa. Serum analyses revealed significant decreases of the total protein and cholesterol, and significant increases of the uric acid and glucose. Haematological analyses showed a slight anaemia, leucocytosis and slightly decompensated metabolic acidosis. A statistically significant protective effect of 5% total water extract of artichoke on humoral immune response (increase of haemaglutination inhibiting antibody titer), relative organ weight as well as on pathomorphological, haematological and biochemical changes induced by ochratoxin A, was established.

Administration, Oral↗

PubMed2000

Tissue gene expression analysis using arrayed normalized cDNA libraries.

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference.

Algorithms↗

PubMed2000

Crystallization of type I chloramphenicol acetyltransferase: an approach based on the concept of ionic strength reducers.

Chloramphenicol acetyltransferase (CAT) is responsible for bacterial resistance to chloramphenicol. It catalyzes inactivation of the antibiotic by acetyl-group transfer from acetyl CoA to one or both hydroxyl groups of chloramphenicol. Type I CAT possesses some unique properties which are not observed in other CAT variants. Type I CAT overexpressed in Escherichia coli was purified and crystals with a resolution limit of 2.22 A have been obtained using a novel procedure which is based on the concept of 'ionic strength reducers'. The crystals have the symmetry of space group P1 and unit-cell parameters a = 96.46, b = 113.86, c = 114.21 A, alpha = 119.9, beta = 94.1, gamma = 98.6 degrees. These dimensions are consistent with four to six trimers per unit cell, corresponding to a solvent fraction ranging from 65 to 47%.

Chloramphenicol O-Acetyltransferase↗

PubMed2000

DNA microarray technology and antimicrobial drug discovery.

The genomics era is providing us with vast amounts of information derived from whole-genome sequencing. This will doubtlessly revolutionise biology and the way novel medicines will be discovered. To leverage this information efficiently, however, technologies in addition to high-throughput sequencing are required. DNA microarray technology is one technology that has already shown great potential for both basic research and drug discovery. With particular emphasis on antibacterial research we will summarise in this review the key technological aspects and most important applications of DNA microarrays demonstrated so far.

Anti-Bacterial Agents↗

PubMed2000

Modulation of steroidogenesis in human ovarian granulosa cells during aging.

OBJECTIVE: To investigate the in vitro effect of endothelin-1 (ET-1) on the steroid production (progesterone [P] and estradiol [E2]) by cultured human granulosa cells (GCs) during aging. MATERIAL AND METHODS: Human ovarian GCs and granulosa-luteal cells (GLCs) were isolated from ovaries of female patients (young and premenopausal) undergoing surgery for non-ovarian benign gynecological conditions. Cells were cultured with ET-1 in the presence or in the absence of FSH. The concentrations of P and E2 in conditioned media were determined by means of RIA. RESULTS: In human GCs and GLCs obtained from young and premenopausal women, ET-1 in vitro can significantly reduce the FSH stimulated biosynthesis of P, whereas the basal P biosynthesis is only insignificantly diminished. The in vitro application of ET-1 have only a sparse inhibitory effect on both the basal and FSH stimulated biosynthesis of E2 in GCs from the two patient groups. CONCLUSIONS: Our findings support the opinion that ET-1 is a local regulator of ovarian steroidogenesis which might modulate the steroid production and the stimulatory effect of FSH in cultured GCs and GLCs obtained from women at various ages.

Adult↗

PubMed2000

Original method for the histochemical demonstration of tripeptidyl aminopeptidase I.

The original histochemical method for the visualization of tripeptidyl aminopeptidase I (TPP I, EC 3.4.14.9) is developed. The method is based on the new synthetic substrates Gly-L-Pro-L-Met(or L-Ala)-5-chloro-1-anthraquinonyl hydrazide (Gly-Pro-Met[Ala]-CAH). The final reaction product is represented as tripeptidyl-5-chloro-1 anthraquinonyl hydrazides (TPP-CAH). Upon the enzyme action the practically in aqueous media insoluble brown-reddish dye 5-chloro-1-anthraquinonyl hydrazine (CAH) is released, which reacts simultaneously with aromatic aldehydes as e.g. 4-anisaldehyde (p-AA) or 4-nitrobenzaldehyde (p-NBA), resulting in microcrystalline or amorphous deeply colored hydrazones. The last compounds mark accurately the locations of enzymatic activity. The biochemically suggested lysosomal localization of this collagen-degrading exopeptidase is thus confirmed on tissue sections. More information about the distribution of TPP I in different rat organs is presented as well.

Aminopeptidases↗