Biomedical subjects

I Ivanov

Publications and source records attributed to I Ivanov.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1999

N-terminal methionine in recombinant proteins expressed in two different Escherichia coli strains.

Two genes coding for chloramphenicol acetyltransferase and human interferon gamma, respectively, were overexpressed constitutively in two different strains of Escherichia coli (E. coli LE392 and E. coli XL1). The N-terminal amino acid analysis of the purified proteins showed that: (a) the N-terminal methionine is processed more efficiently in E. coli LE392 rather than in E. coli XL1 cells; (b) the N-terminal methionine is removed better from the heterologous human interferon gamma in comparison with the homologous chloramphenicol acetyltransferase protein: and (c) there is no strong correlation between the efficiency of N-terminal procession and the yield of recombinant protein.

Amino Acid Sequence↗

PubMed1999

A method of one-step enzyme labelling of short oligonucleotide probes for filter hybridisation.

Here we describe a method of labelling short oligonucleotide probes with enzyme without purification or chemical modifications. Biotinylated oligonucleotides as short as 10 nt are coupled with streptavidin-conjugated enzyme, hybridised and detected with enzyme-triggered chemiluminescence. The detection of hybridisation signal is linear for two orders of magnitude of target dilution. It is shown to be comparable in sensitivity with standard procedures and with radioactive detection. The method is quick, simple and has potential for automation of large-scale oligo-nucleotide hybridisation and multiplex sequencing.

Alkaline Phosphatase↗

PubMed1999

Mouse monoclonal antibodies against Phytolacca americana antiviral protein PAP I.

Four hybridoma lines are constructed producing monoclonal antibodies against the pokeweed (Phytolacca americana) antiviral protein PAP I. Two of the antibodies, 4E8 and 5D3, are characterized in more detail. They recognize amino acid sequences rather than conformational changes and their epitopes are 65% distinct. One of these antibodies (5D3) is used to study localization of recombinant PAP I in Escherichia coli cells by immuno-gold electron microscopy.

Animals↗

PubMed1999

Effect on neonatal oestrogen and hCG treatment on the genital organs of adult female rats.

The ovaries and the uterine as well as vaginal mucous membranes of 80-, 180- and 365-day-old intact female rats and females neonatally treated with a single dose of oestrogen and repeated doses of human chorionic gonadotropin (hCG) were studied. Numerous follicles, interstitial cells and corpora lutea (CL) were present on the ovary of intact females from 80 up to 365 days of age. The number of primary and secondary follicles decreased in the intact female rats between Day 180 and Day 365 of life. On the ovaries of 180- and 365-day-old female rats neonatally treated with oestrogen, interstitial glandular cells and cystic follicles predominated. No CL were present on these ovaries. The height of epithelial cells of the uterine and vaginal mucous membranes increased in intact female rats from 80 to 365 days of age, whereas in oestrogen-treated females the height of epithelium decreased. From 80 up to 365 days of age, the height of epithelial cells of uterine and vaginal mucous membranes of rats neonatally treated with repeated doses of hCG was similar to that in the corresponding control animals.

Animals↗

PubMed1999

New method for the histochemical demonstration of dipeptidyl aminopeptidase I activity using a novel anthraquinoyl hydrazide substrate.

A new method for the histochemical localization of dipeptidyl aminopeptidase I (DPP I, cathepsin C), based on a newly synthesized substrate-Gly-L-Phe-5-chloro-1-anthraquinoyl hydrazide.HCl (Gly-Phe-CAH), is proposed. The enzyme activity liberates 5-chloro-1-anthraquinoyl hydrazine (CAH)--a water-insoluble brown-reddish compound, which precipitates on the enzyme locations. The primary reaction product reacts simultaneously or, otherwise, by post-coupling with p-anisaldehyde (p-AA), thus converting to the reddish-violet amorphous hydrazone--the final reaction product. The validity of enzyme localization is thus assured by the insolubility of the primary reaction product and does not depend on the rate of the second reaction step. The enzyme studied is successfully localized in different rat organs using the newly proposed technique.

Animals↗

PubMed1998

Omega-oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site.

During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the omega-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of omega-modified fatty acids by 15-lipoxygenases and found that omega-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, omega-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403-->Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of omega-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the omega-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases.

Animals↗

PubMed1998

Inorganic phase composition of remineralisation in porous CaP ceramics.

The formation of bone tissue in pores, and around, a block made of partially dehydroxylated porous hydroxyapatite ceramic and implanted into a rat's femur using X-ray diffraction (XRD), infrared spectroscopy (IRS) and histological methods were investigated. Structural characteristics and composition of new bone tissue formed three months after implantation, in fact, do not differ from those of the normal femur. A model of osteogenesis based on dissolution-precipitation reactions is given, taking into account peculiarities of structure, deficiency and impurity composition for the material of the implant.

Animals↗