Biomedical subjects

A Baici

Publications and source records attributed to A Baici.

At least 37 records · Page 2Linked to original sources

PubMed1994

Cephem sulfones as inactivators of human leukocyte elastase. 5. 7 alpha-Methoxy- and 7 alpha-chloro-1,1-dioxocephem 4-ketones.

Studies on cephem sulfones as inhibitors of human leukocyte elastase (HLE) have been extended to the new class of cephem 4-ketones. tert-Butyl and phenyl ketones were prepared from 4-carboxycephem derivatives, at either the sulfide or sulfone oxidation level, by chemoselective Grignard reaction. Obtained products were functionalized with heterocyclothio and acyloxy substituents at C-3', C-2, or both positions. tert-Butyl ketones of the 7 alpha-chlorocephem series were in general at least as potent as the corresponding esters at inhibiting the enzyme, but improvements in hydrolytic stability were only marginal. On the other hand, tert-butyl ketones of the 7 alpha-methoxycephem series combined potent biochemical activity with acceptable hydrolytic stability, thus overstepping the esters, thiolesters, and amides reported previously. In particular, the tert-butyl ketones possessing a heterocyclothio group at C-3' or C-2 were at least as active as the corresponding tert-butyl esters but 1 order of magnitude more stable in physiologic buffers (pH 7.4, 37 degrees C). Introduction of acyloxy groups at C-2 delivered the most potent HLE inhibitors of the cephem class ever reported, with inhibition parameters often outside the determination limits of our standard protocol (second-order rate constant kon > 2,000,000 M-1 s-1; Ki at steady state < 2 nM). Keto-enol tautomerism was found to depress activity and boost hydrolytic stability. Thus, double substitution with heterocyclic thiols produced compounds with diverging properties, according to the extent of enolate formation at the investigated pH (7.4): the weakly acidic tert-butyl ketones (pKa > or = 5.8) proved to be potent inhibitors (kon over 10(4) M-1 s-1) with reasonable hydrolytic stability (t1/2 = 30-75 h), while the phenyl ketones (pKa < 4) were fair inhibitors (kon over 10(3) M-1 s-1; Ki at steady state approximately 50 nM) with hydrolytic half-lives exceeding 1000 h. Selected compounds efficiently inhibited the degradation of insoluble bovine neck elastin by HLE in a concentration-dependent manner. Intracellular HLE of polymorphonuclear leukocytes was in general unaffected; however, a lipophilic cephem sulfone apparently able to inactivate the enzyme in living cells was identified.

Cells, Cultured↗

PubMed1994

Kinetics of molecular chaperone action.

Molecular chaperones of the Hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. Target peptides were labeled with an environmentally sensitive fluorophore so that their binding to the molecular chaperone DnaK of Escherichia coli could be followed in real time. The two-step process was characterized by relaxation times of 27 seconds and 200 seconds with 2 microM DnaK and 0.1 microM ligand at 25 degrees C. In the presence of adenosine triphosphate, the formation of the complex was greatly accelerated and appeared to be a single-exponential process with a relaxation time of 0.4 second. The binding-release cycle of DnaK thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the adenosine triphosphatase activity of the chaperone (turnover number, 0.13 per minute at 30 degrees C).

2-Naphthylamine↗

PubMed1993

Inhibition of the human leukocyte endopeptidases elastase and cathepsin G and of porcine pancreatic elastase by N-oleoyl derivatives of heparin.

N-oleoyl-heparin derivatives differing in their oleic acid and sulfate contents were synthesized and studied for their abilities to inhibit human leukocyte elastase (HLE), human leukocyte cathepsin G (CatG) and porcine pancreatic elastase (PPE) at pH 8.0, ionic strength 0.05 M and 37 degrees. Heparin (Hep) as well as N-oleoyl-heparins behaved as tight-binding, hyperbolic noncompetitive inhibitors of HLE (KiHep = 75 pM) and CatG (KiHep < 25 pM). The main driving force for the interaction between enzymes and glycosaminoglycans was electrostatic in nature. Under the condition [enzyme] >> Ki, the stoichiometries of the interaction with Hep were 1:2 (Hep:HLE) and 1:4 (Hep:CatG). Coupling one oleic acid residue to three disaccharide units of partially N-desulfated Hep, Ol1:3Hep, lowered HLE inhibition (Ki = 0.3 nM) and the stoichiometry of binding was reduced to 1:1. Re-N-sulfation of a similar derivative, Ol1:5Hep(SO4), containing one fatty acid residue for five disaccharide units, led to a substance with similar HLE inhibitory characteristics as Hep (Ki = 92 pM) and stoichiometry 1:2. Ol1:5Hep(SO4) was also a more efficient inhibitor of CatG (Ki < 33 pM) than Ol1:3Hep (Ki = 9.5 nM). The residual activities of N-oleoyl-Hep complexes with CatG were much lower than the corresponding activities in the presence of Hep. While oleate and Hep could not inhibit PPE, N-oleoyl-Hep, independently of fatty acid substitution and sulfate content, could inhibit this enzyme with Ki congruent to 60 nM and low residual activity. The efficient endopeptidase inhibitory characteristics of N-oleoyl-Hep derivatives, together with their non-anticoagulant properties and their capacity to interact with elastin, may be therapeutically useful in connective tissue degenerative diseases.

Amino Acid Sequence↗

PubMed1992

Analysis of glycosaminoglycans in human serum after oral administration of chondroitin sulfate.

Chondroitin sulfate was administered orally to six healthy volunteers, six patients with rheumatoid arthritis and six patients with osteoarthritis. Blood was collected at intervals before and after treatment and the glycosaminoglycan concentration was analyzed in serum using a sensitive assay based on the metachromatic reaction with 1,9-dimethylmethylene blue. The glycosaminoglycan concentration in serum before and after ingestion of chondroitin sulfate was statistically unchanged in all of the subjects studied. We suggest that chondroprotection by orally administered chondroitin sulfate is a biologically and pharmacologically unfounded theory. Any possible benefit to osteoarthritic patients after ingestion of chondroitin sulfate should be sought at the gastrointestinal rather than at the plasmatic or articular cartilage level.

Administration, Oral↗

PubMed1991

[Arthritis from a biochemical aspect].

The dynamic processes of remodeling and cartilage destruction occurring in osteoarthritis are associated with an altered metabolism of the chondrocytes. Two mechanisms can be made responsible for chondrocyte activation: Stimulation by cytokines (mainly interleukin-1) and phenotypic modulation. Remodeling of osteoarthritic joint structures is associated with cell proliferation and neovascularization, while erosion of the articular cartilage is promoted by a variety of endopeptidases of chondrocytic origin. These aspects are briefly reviewed in the light of recent findings.

Cartilage, Articular↗

PubMed1990

Effect of interleukin-1 beta on the production of cathepsin B by rabbit articular chondrocytes.

Rabbit articular chondrocytes in monolayer culture were stimulated with human recombinant interleukin-1 beta. Under the influence of the cytokine the intracellular pool of the cysteine endopeptidase cathepsin B was increased by a 2-4-fold factor, while enzyme secretion was not stimulated at a significant level. Under the same conditions, the secretion of collagenase, measured as an internal control, was stimulated about 6-fold. The effects of interleukin-1 beta were compared to those caused by phenotypic modulation. Chondrocytes modulated by serial subcultures in monolayer secreted more cathepsin B, but less collagenase than differentiated cells (cultured within collagen gels). Thus, interleukin-1 beta and phenotypic modulation affected differently two endopeptidases which are relevant in the pathogenesis of osteoarthritis.

Animals↗

PubMed1990

Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach.

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.

Amino Acid Sequence↗

PubMed1990

The kinetic mechanism of inhibition of human leukocyte elastase by MR889, a new cyclic thiolic compound.

The cyclic thiolic compound 2-[3-thiophencarboxythio]-N-[dihydro-2(3H)-thiophenone-3-il] - propionamide (MR889) was investigated as inhibitor of endopeptidases. The activity of bovine pancreatic alpha-chymotrypsin, human leukocyte cathepsin G and rabbit liver cathepsin B was not affected by MR889, whereas porcine pancreatic elastase and human leukocyte elastase were inhibited. The kinetic mechanism of inhibition of human leukocyte elastase was of the reversible, slow-binding, fully competitive type. The rate constants for complex formation between MR889 and leukocyte elastase, determined by pre-steady-state kinetic analysis in the presence of a tetrapeptide substrate at 37 degrees and pH 7.40, were kon = 2363 +/- 15 M-1 sec-1, koff = 3.01 +/- 0.34 x 10(-3) sec-1. The inhibition equilibrium constant was Ki = koff/kon = 1.27 +/- 0.15 microM. Ki, calculated from steady-state kinetic experiments, was 1.38 microM. MR889 also inhibited the elastolytic activity of leukocyte elastase, as determined with insoluble elastin as the substrate.

Cathepsin B↗

PubMed1990

Cathepsin B secretion by rabbit articular chondrocytes: modulation by cycloheximide and glycosaminoglycans.

Rabbit articular chondrocytes in monolayer culture are modulated away from their differentiated state and undergo morphological and biochemical changes. One of the characteristics of the modulated state is an abnormally high production of the cysteine endopeptidase cathepsin B. Addition to chondrocyte cultures of the protein biosynthesis inhibitor, cycloheximide, resulted in a concentration-dependent reduction of cathepsin B secretion, which was fully restored after removal of cycloheximide. Glycosaminoglycans added to the culture medium of modulated chondrocytes partially reduced the rate of secretion of cathepsin B, this effect being dependent on their structure, the degree of sulfation, and concentration. The age of the chondrocytes and the duration of the treatment also influenced this response. The switching off of cathepsin B release was apparently best favored by a high concentration of negatively charged sulfate groups attached to a polymeric glycosaminoglycan chain; this simulates the natural environment of the chondrocytes in articular cartilage.

Animals↗

PubMed1988

Interaction of site specific hirudin variants with alpha-thrombin.

The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitor's reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of site-directed mutagenesis of a hirudin gene. In comparison to recombinant hirudin (Ki = 19 pM) only those mutant inhibitors which were modified at amino acid position Lys47 showed a higher Ki value for their complexes with thrombin. The observed effects are mainly due to increased koff rate constants.

Algorithms↗

PubMed1988

Cathepsin B as a marker of the dedifferentiated chondrocyte phenotype.

Rabbit articular cartilage does not secrete cathepsin B in organ culture. By established methods for modulating the chondrocyte phenotype in vitro, however, the synthesis, intracellular storage, and secretion of cathepsin B were followed up over a period of two months. With chondrocytes grown in monolayer cultures both the intracellular pool of the enzyme and its secretion were very small initially, but increased progressively to a factor of 110 after eight weeks. The secretion of cathepsin B was strongly depressed after transferring the cells from monolayer to collagen gel cultures. In contrast, collagenase was secreted in almost the same amounts during the whole period in both monolayer and collagen gel cultures. The cells cultured in collagen gels secreted more collagenase than those grown in monolayers. The reversible switch of cathepsin B secretion suggests that this enzyme, unlike collagenase, is a marker of the dedifferentiated chondrocyte phenotype. Cathepsin B was localised within cultured chondrocytes using antibodies raised against rabbit liver cathepsin B and shared with it many catalytic properties. Its Mr, however, was higher (34,000 compared with 27,000) and showed an unusual resistance to denaturation at neutral-alkaline pH, which may confer on this enzyme an important role in the degradation of cartilage matrix.

Animals↗

PubMed1988

Tumor-host interactions in the rabbit V2 carcinoma: stimulation of cathepsin B in host fibroblasts by a tumor-derived cytokine.

Organ cultures of explanted V2 carcinoma specimens as well as cultured V2 carcinoma cells produced a cytokine which stimulated rabbit skin fibroblasts to synthesize increased amounts of cathepsin B. The cytokine was released by the tumor cells as a heterogeneous family of polypeptides: two inactive forms (Mr = 55,000 and 68,000) which could be activated by limited proteolysis with trypsin and three active forms with Mr values of 12,000, 16,000 and 18,000. The treatment of inactive cytokine-containing tumor-conditioned media with trypsin, followed by chromatographic separation of the products, suggested that the high-Mr inactive components may represent precursors of the active forms. Cathepsin B was immunolocalized in the tumor-host interzone in co-cultures of tumor and host tissues. Some other possible activities of the tumor cytokine which emerged from previous studies, such as the induction of host cells to produce increased levels of collagenase and extracellular matrix, as well as the stimulation of host cell proliferation, are discussed in the light of the new findings and are proposed as an important mechanism in tumor invasion.

Animals↗

PubMed1987

Graphical and statistical analysis of hyperbolic tight-binding inhibition.

Equations are described for the analysis of enzyme-catalysed reactions in the presence of hyperbolic tight-binding inhibitors using both graphical and non-linear-regression methods. The general character of the equations allows the interpretation of several other kinetic mechanisms. As an example, the tight-binding hyperbolic non-competitive inhibition of human leucocyte elastase by a polysulphated glycosaminoglycan is analysed.

Enzyme Inhibitors↗

PubMed1986

Cysteine proteinases produced by cultured rabbit V2 carcinoma cells and rabbit skin fibroblasts.

Rabbit V2 carcinoma cells and normal rabbit skin fibroblasts produced cysteine proteinases with properties similar to those of purified rabbit liver cathepsin B. Both cell types secreted into the culture medium enzymes with an apparent Mr of 43,000, which reacted with synthetic substrates commonly used for cathepsin B. After limited proteolysis with pepsin or treatment at pH 3, the Mr = 43,000 species could be converted into forms with Mr = 34,000 and an increased specific activity. In the intracellular pool of both V2 carcinoma cells and fibroblasts, a cysteine proteinase with the same Mr of cathepsin B (27,000) was found. Despite the similarity in molecular size, substrate specificity and sensitivity to inhibitors, the tumor and fibroblast enzymes were not identical in their stability at pH greater than or equal to 7 and were produced by the 2 cell types in considerably different amounts. In terms of enzyme units and normalized to an equal cell number, the ratios of fibroblast enzyme/tumor enzyme were as follows: secreted 130-150; intracellular, 150-180. The pH stability of the cysteine proteinases was determined quantitatively by measuring the half-life of enzyme activity. At pH 8.0 and 25 degrees C the secreted tumor cysteine proteinase had a half-life of at least 5 hr, whereas the secreted fibroblast enzyme and liver cathepsin B had half-lives of 8.8 min and 4.4 min, respectively.

Animals↗

PubMed1986

Biological studies of ten human squamous carcinoma cell lines: an overview.

Ten cell lines established from surgical specimens of human squamous carcinomas of the tongue and larynx have been investigated with respect to their motility, ultrastructure, karyotypes, certain biochemical features, interaction with normal epithelial and stromal elements and capacity to infiltrate three-dimensional organoid systems. All the cell lines have maintained several morphological and biochemical characteristics indicating a common origin, although the extent to which each line displays this heritage is variable. The phenotypes of each of the individual cell lines are, however, notably stable. Data are provided for epithelial surface markers (including epidermal growth factor, EGF) and for the synthesis and release of prostaglandins and proteases which may be involved in invasive mechanisms. Encounters between the cell lines and organoid substrata (embryonic chick heart spheroids, human amnion, chick chorioallantoic membrane) are described: the results indicate a scale of invasiveness ranging from lack of penetration to full-thickness infiltration by cells showing various distinctive growth patterns. Correlation between in vitro and in vivo findings is discussed, and it is suggested that the biological heterogeneity of the lines may reflect inherent properties of the original carcinoma cell populations which are more distinctly expressed in vitro.

Aged↗

PubMed1986

Hysteretic enzyme response induced by inhibitory antibodies against human leukocyte elastase.

Polyclonal antibodies raised in rabbits against human leukocyte elastase contained two distinct populations of enzyme-inhibiting immunoglobulins. The enzyme-catalyzed reaction in the presence of antibodies (both IgG or monovalent Fab fragments) showed a transient state lasting up to several minutes depending on the inhibitor and substrate concentrations, which was followed by a linear steady-state. The transient was a concave upward or concave downward lag phase depending on whether the enzyme had been preincubated with the antibodies or not, respectively. The kinetic analysis of reaction progress curves showed that both antibody populations were slow inhibitors, which completely and reversibly excluded the substrate from binding to the enzyme. For both antibody populations, the formation of the enzyme-inhibitor complex was characterized by an initial rapid interaction followed by a slow isomerization to a catalytically inactive complex. The apparent pseudo first-order rate constant of the transient slow phase was a hyperbolic function of the inhibitor concentration for both antibodies, from which relevant kinetic constants and the half times for enzyme inactivation could be calculated. For instance, with a total antibody concentration of 1 mg/ml (as IgG), leukocyte elastase was inactivated with t1/2 = 0.31s and 24.8s by the faster and the slower of the two antibodies, respectively. It is suggested that the hysteretic response of the enzyme to the inhibitory action of its antibodies may be due to a kind of memory of the antibody molecule for a special inactive enzyme conformation resulting from inhibition by proteinase inhibitors during the immunization procedure. In turn, the purified antibodies would be able to reversibly induce a slow transition of the enzyme molecule from an active to a substrate-excluding conformation ("induced misfit").

Antibodies↗

PubMed1985

Release of proteinases by cultures of human cell lines derived from squamous carcinomas of the tongue and larynx.

Seven human cell lines derived from squamous carcinomas of the tongue and larynx were examined for their ability to produce and secrete proteinases. All cell lines were able to release into the culture medium cysteine proteinase and plasminogen activator-like activities. All lines differed from each other in the amount of enzymes secreted, in the kinetics of the secretion, in the quality of the enzymes produced an in the intracellular pool of these activities. These features constitute potential criteria for classifying the biochemical behavior of the tumor cell lines and for explaining their different ability to invade soft tissues and bone.

Carcinoma, Squamous Cell↗

PubMed1984

Interaction between human leukocyte elastase and chondroitin sulfate.

Chondroitin sulfate (Structum) interacts with human leukocyte elastase, a potent mediator of articular cartilage degradation, producing a partial inhibition of the enzyme activity (60% at saturation). Kinetically, the inhibition mechanism can be classified as simple intersecting, hyperbolic noncompetitive and is almost identical to that found earlier for similar compounds. The best inhibitory activity of chondroitin sulfate was found in fractions having at the same time a high proportion of chondroitin-6-sulfate relative to the corresponding 4-isomer and a high molecular mass. Thus, a fraction with high Mr and containing 92% of isomer 6 inhibited leukocyte elastase with Ki = 1.8 micrograms/ml, whereas a fraction with low Mr and almost equal composition of the 4- and 6-isomer had Ki = 140 micrograms/ml. Ki for unfractionated chondroitin sulfate was 3.4 micrograms/ml. It is suggested, that the modulation of the extracellular activity of cartilage-degrading enzymes by cartilage-derived factors may explain, at least in part, the beneficial effects of some therapeutically used chondroprotective agents.

Chondroitin↗