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T Kulik

Publications and source records attributed to T Kulik.

16 recordsLinked to original sources
PubMed2004

Role of putative membrane receptors in the effect of androgens on human vascular cell growth.

We have reported previously that dihydrotestosterone (DHT) induces a biphasic effect on DNA synthesis in human vascular smooth muscle cells (VSMC), i.e. stimulation at low concentrations and inhibition at high concentrations. In contrast, DHT dose-dependently stimulated [(3)H]thymidine incorporation in a human endothelial cell line (ECV304). Additionally, DHT increased the specific activity of creatine kinase (CK) in both vascular cell types. In the present study, we have determined whether some of these effects are exerted via membrane-binding sites. We measured changes in DNA synthesis and CK after treatment with DHT and the membrane-impermeant testosterone-3-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (T-BSA). High concentrations of either DHT or T-BSA inhibited VSMC proliferation (by 52+22% and 51+25% respectively). DHT as well as T-BSA increased DNA synthesis in ECV304 cells dose-dependently. In contrast, T-BSA did not affect CK in either cell type. In both cell types, DHT as well as T-BSA increased mitogen-activated protein kinase (MAPK) kinase activity as measured by total phosphorylated MAPK. Further, the inhibitory effect of either the free or protein-bound androgens on DNA synthesis was blocked by UO126, an inhibitor of MAPK kinase activity. T-BSA conjugate labeled with Europium showed binding to whole VSMC, which could be displaced by excess T-BSA, but not by estradiol-BSA or the free hormones. Finally, using T-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for androgen in VSMC. Hence, the inhibitory effects of testosterone on DNA synthesis in VSMC are apparently exerted by membrane-binding sites for androgen, do not require intracellular entry of the hormone and its binding to the classical nuclear receptors and are linked to MAPK activation.

Butadienes↗
PubMed2003

Carboxy derivatives of isoflavones as affinity carriers for cytotoxic drug targeting in adrenocortical H295R carcinoma cells.

Carboxy derivatives of isoflavones that exhibit oestrogenic/anti-oestrogenic properties were used as carriers for affinity drug targeting to H295R adrenocortical carcinoma cells that express transcripts of oestrogen receptor (ER) alpha and beta. These derivatives were prepared by introducing a carboxymethyl group at the 6-position of genistein and of biochanin A, yielding 6CG and 6CB respectively. In transactivation assays, 6CG displayed mixed agonist/antagonist activity for ERalpha, whereas 6CB displayed only weak antagonist activity. Low concentrations of oestrogen, 6CG and 6CB were capable of inducing proliferation in H295R cells and of stimulating creatine kinase (CK) specific activity, suggesting that these cells were sensitive to oestrogenic compounds. In in vivo experiments, both 6CG and 6CB were capable of inhibiting oestrogen-induced CK specific activity in rat tIssues. For affinity drug targeting, the cytotoxic drug daunomycin was coupled to 6CB and 6CG, yielding 6CB-Dau and 6CG-Dau respectively. These conjugates were tested for their antiproliferative ability to inhibit DNA synthesis as assessed by incorporation of [(3)H]thymidine in H295R cells. A dose-dependent cytoxicity was observed with both conjugates. At 0.3-3 nM, both conjugates were 10 to 30 times more potent than daunomycin. At 30 nM these conjugates were two to three times more potent than daunomycin. At concentrations ranging between 300 and 3000 nM, no difference in cytotoxicity was observed between the conjugates and daunomycin. When the cells were treated over a wide range of concentrations with a combination of 6CG plus daunomycin, the observed cytotoxicity was less than with daunomycin alone. When non-transformed rat enterocytes, which do not express ER, were treated with 6CG-Dau or daunomycin, the antiproliferative effect of 6CG-Dau was the same as that of daunomycin over the concentration range tested. These pilot studies suggest that the ready availability of oestrogenic binding sites in H295R cells can be exploited for site-directed chemotherapy.

Adrenal Cortex Neoplasms↗
PubMed2002

6-Carboxymethyl genistein: a novel selective oestrogen receptor modulator (SERM) with unique, differential effects on the vasculature, bone and uterus.

The novel genistein (G) derivative, 6-carboxymethyl genistein (CG) was evaluated for its biological properties in comparison with G. Both compounds showed oestrogenic activity in vitro and in vivo. On the other hand G and CG differed in the following parameters: (i) only CG displayed mixed agonist-antagonist activity for oestrogen receptor (ER) alpha in transactivation assays and (ii) only CG was capable of attenuating oestrogen (E(2))-induced proliferation in vascular smooth muscle cells and of inhibiting oestrogen-induced creatine kinase (CK) specific activity in rat tissues. On the other hand only G enhanced the stimulatory effect on CK specific activity in the uterus. In comparison to the selective oestrogen receptor modulator (SERM) raloxifene (RAL), CG showed the same selectivity profile as RAL in blocking the CK response to E(2) in tissues derived from both immature and ovariectomized female rats. Molecular modelling of CG bound to the ligand binding domain (LBD) of ERbeta predicts that the 6-carboxymethyl group of CG almost fits the binding cavity. On the other hand, molecular modelling of CG bound to the LBD of ERalpha suggests that the carboxyl group of CG may perturb the end of Helix 11, eliciting a severe backbone change for Leu 525, and consequently induces a conformational change which could position Helix 12 in an antagonist conformation. This model supports the experimental findings that CG can act as a mixed agonist-antagonist when E(2) is bound to its receptors. Collectively, our findings suggest that CG can be considered a novel SERM with unique effects on the vasculature, bone and uterus.

Analysis of Variance↗
PubMed2001

Chicken avidin exhibits pseudo-catalytic properties. Biochemical, structural, and electrostatic consequences.

Avidin and its bacterial analogue streptavidin exhibit similarly high affinities toward the vitamin biotin. The extremely high affinity of these two proteins has been utilized as a powerful tool in many biotechnological applications. Although avidin and streptavidin have similar tertiary and quaternary structures, they differ in many of their properties. Here we show that avidin enhances the alkaline hydrolysis of biotinyl p-nitrophenyl ester, whereas streptavidin protects this reaction even under extreme alkaline conditions (pH > 12). Unlike normal enzymatic catalysis, the hydrolysis reaction proceeds as a single cycle with no turnover because of the extremely high affinity of the protein for one of the reaction products (i.e. free biotin). The three-dimensional crystal structures of avidin (2 A) and streptavidin (2.4 A) complexed with the amide analogue, biotinyl p-nitroanilide, as a model for the p-nitrophenyl ester, revealed structural insights into the factors that enhance or protect the hydrolysis reaction. The data demonstrate that several molecular features of avidin are responsible for the enhanced hydrolysis of biotinyl p-nitrophenyl ester. These include the nature of a decisive flexible loop, the presence of an obtrusive arginine 114, and a newly formed critical interaction between lysine 111 and the nitro group of the substrate. The open conformation of the loop serves to expose the substrate to the solvent, and the arginine shifts the p-nitroanilide moiety toward the interacting lysine, which increases the electron withdrawing characteristics and consequent electrophilicity of the carbonyl group of the substrate. Streptavidin lacked such molecular properties, and analogous interactions with the substrate were consequently absent. The information derived from these structures may provide insight into the action of artificial protein catalysts and the evolution of catalytic sites in general.

Animals↗
PubMed2000

Biotin induces tetramerization of a recombinant monomeric avidin. A model for protein-protein interactions.

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine. The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance. To our knowledge, these unexpected results represent the first example of a small monovalent ligand that induces oligomerization of a monomeric protein. This study may suggest a biological role for low molecular weight ligands in inducing oligomerization and in maintaining the stability of multimeric protein assemblies.

Avidin↗
PubMed2000

Recombinant NeutraLite avidin: a non-glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties.

A recombinant non-glycosylated and acidic form of avidin was designed and expressed in soluble form in baculovirus-infected insect cells. The mutations were based on the same principles that guided the design of the chemically and enzymatically modified avidin derivative, known as NeutraLite Avidin. In this novel recombinant avidin derivative, five out of the eight arginine residues were replaced with neutral amino acids, and two of the lysine residues were replaced by glutamic acid. In addition, the carbohydrate-bearing asparagine-17 residue was altered to an isoleucine, according to the known sequences of avidin-related genes. The resultant mutant protein, termed recombinant NeutraLite Avidin, exhibited superior properties compared to those of avidin, streptavidin and the conventional NeutraLite Avidin, prepared by chemo-enzymatic means. In this context, the recombinant mutant is a single molecular species, which possesses strong biotin-binding characteristics. Due to its acidic pI, it is relatively free from non-specific binding to DNA and cells. The recombinant NeutraLite Avidin retains seven lysines per subunit, which are available for further conjugation and derivatization.

Amino Acid Sequence↗
PubMed1999

Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain.

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.

Animals↗
PubMed1998

Engineering of chicken avidin: a progressive series of reduced charge mutants.

Avidin, a positively charged egg-white glycoprotein, is a widely used tool in biotechnological applications because of its ability to bind biotin strongly. The high pI of avidin (approximately 10.5), however, is a hindrance in certain applications due to non-specific (charge-related) binding. Here we report a construction of a series of avidin charge mutants with pIs ranging from 9.4 to 4.7. Rational design of the avidin mutants was based on known crystallographic data together with comparative sequence alignment of avidin, streptavidin and a set of avidin-related genes which occur in the chicken genome. All charge mutants retained the ability to bind biotin tightly according to optical biosensor interaction analysis. In most cases, their thermal stability characteristics were indistinguishable from those of the wild-type avidin. Our results demonstrate that the charge properties of avidin can be modified without disturbing the crucial biotin-binding activity.

Animals↗
PubMed1995

Close similarity among streptavidin-like, biotin-binding proteins from Streptomyces.

Two strains of Streptomyces venezuelae were found to produce high-affinity, biotin-binding proteins, termed streptavidin v1 and v2, respectively. Both proteins were isolated to purity, and their corresponding genes were cloned and sequenced. Compared to streptavidin from S. avidinii, streptavidin v1 had only a single amino acid substitution and streptavidin v2 showed 9 such differences. The substitutions were remarkably conservative, none of which affected the amino acid residues known to be important to the biotin-binding properties or to the structure of the tetrameric protein. The results also indicate that the biosynthesis of such biotin-binding proteins is not simply a curious anomaly in a single species of Streptomyces. It is suggested that the classification of S. avidinii as a unique species should be reconsidered. The occurrence of these proteins appears to be linked to the production of an unusual synergistic antibiotic complex.

Amino Acid Sequence↗
PubMed1995

Preparation of deglycosylated egg white avidin.

A simple procedure for the preparation of deglycosylated avidin is described. Commercially obtained avidin was treated with a mixed microbial culture. The cells were capable of growing on the oligosaccharide residues, but generally ignored the polypeptide portion of the egg white glycoprotein. The resultant deglycosylated avidin retained its biotin-binding characteristics. The major bacterial strain (strain BECH080), responsible for the deglycosylation, was isolated. On the basis of elementary biochemical tests, fatty acid, and phenotypic analyses, the isolate was identified as a strain of Flavobacterium meningosepticum. The primary enzymatic activity that caused the removal of the oligosaccharide residues of avidin appeared to be similar to endoglycosidase F.

Avidin↗
PubMedDate not supplied

Pressure-regulated volume control vs volume control ventilation in infants after surgery for congenital heart disease.

The objective of this investigation was to compare how two modes of positive pressure ventilation affect cardiac output, airway pressures, oxygenation, and carbon dioxide removal in children with congenital heart disease in the immediate postoperative period. The investigation used a one group pretest-post-test study design and was performed in the pediatric cardiac intensive care unit in a university-affiliated children's hospital. Nine infants were enrolled immediately after repair of tetralogy of Fallot (2) or atrioventricular septal defects (7) with mean weight = 5.5 kg (4.2-7.3 kg). Children were admitted to the pediatric cardiothoracic intensive care unit after complete surgical repair of their cardiac defect and stabilized on a Siemen's Servo 300 ventilator in volume control mode (VCV1) (volume-targeted ventilation with a square flow wave pattern). Tidal volume was set at 15 cc/kg (total). Hemodynamic parameters, airway pressures and ventilator settings, and an arterial blood gas were measured. Patients were then changed to pressure-regulated volume control mode (PRVC) (volume-targeted ventilation with decelerating flow wave pattern) with the tidal volume set as before. Measurements were repeated after 30 minutes. Patients were then returned to volume control mode (VCV2) and final measurements made after 30 minutes. The measurements and results are as follows: After correction of congenital heart defects in infants, mechanical ventilation using a decelerating flow wave pattern resulted in a 19% decrease in peak inspiratory pressure without affecting hemodynamics, arterial oxygenation, or carbon dioxide removal.

Analysis of Variance↗