Biomedical subjects
Publications and source records attributed to G Ambrus.
A 50-year historical survey is presented on the R & D activities of the Institute for Drug Research, Budapest, in the areas of antibiotics, enzyme inhibitors and immunosuppressants of microbial origin as well as in the field of the microbial bioconversion of steroids. Research projects that have led to the elaboration of industrial processes applied by Hungarian pharmaceutical companies have mainly been selected.
The complement system is a major effector arm of the immune defense contributing to the destruction of invading pathogens. There are three possible routes of complement cascade activation: the classical, the alternative and the lectin pathways. The activation of the classical and lectin pathways is initiated by supramolecular complexes, which resemble each other. Each complex has a recognition subunit (C1q in the classical and mannose-binding lectin (MBL) in the lectin pathway), which associates with serine protease zymogens (C1q with C1r and C1s, and MBL with MBL-associated serine proteases: MASP-1, MASP-2) to form the C1 and MBL-MASPs complexes, respectively. As the recognition subunits bind to activator structures, subsequent activation of the serine protease zymogens occurs. The precise structure of the complexes and the exact mechanism of their activation have not been solved, yet. In this review we summarize the recent advances about the structure and function of the individual subcomponents of both complexes achieved by genetic engineering, molecular modeling, physico-chemical and functional studies. Special emphasis will be laid on the serine proteases: the role of the individual domains in the assembly of the C1s-C1r-C1r-C1s tetramer and in the control of the protease activity will be discussed. We will then focus on recent functional models of the supramolecular complexes. The question of how a non-enzymatic signal (the binding of C1q or MBL to activators) can be converted into enzymatic events (activation of serine protease zymogens) will be addressed. The similarities and differences between C1 and MBL-MASPs will also be discussed.
A survey is presented on the last 50 years of biotechnological R & D activities in the Institute for Drug Research, Budapest. In the 1950s and 1960s this Institute played an important role in the industry of antibiotics in Hungary, contributing to the development of manufacturing processes for streptomycin, oxytetracycline, neomycin and nystatine. In the late 1950s a microbial screening program was initiated, which led to the discovery of several new antibiotics and the isolation of microorganisms producing medically important, known antibiotics and other therapeutical agents of microbial origin from natural sources. In the 1970s and 1980s the biotechnological research group elaborated new industrial processes for the production of several antibacterial antibiotics, such as gentamycin C, sisomicin, tobramycin, apramycin, kanamycin B and mupirocin and the antitumor antibiotic daunomycin. In the last 15 years new processes have been developed for manufacturing the immunosuppressants cyclosporin A and mycophenolic acid and the hypocholesterolemic agents mevinolin and pravastatin as well as recombinant hirudin, a thrombin inhibitor. Research on steroid bioconversions has also been continued from the mid 1950s up to now. In the early 1960s manufacturing processes were developed for the anti-inflammatory prednisolone and the anabolic drug methandrostenolone. The results on microbial transformations (stereoselective reduction, hydroxylation) were utilized in the synthesis of contraceptive drugs. Since the mid 1960s several new microbial processes have been discovered for the selective side chain cleavage of natural sterols, resulting in various key intermediates for the synthesis of a wide variety of steroid drugs.
The activation of the C1s-C1r-C1r-C1s tetramer in the C1 complex, which involves the cleavage of an Arg-Ile bond in the catalytic domains of the subcomponents, is a two-step process. First, the autolytic activation of C1r takes place, then activated C1r cleaves zymogen C1s. The Arg463Gln mutant of C1r (C1rQI) is stabilized in the zymogen form. This mutant was used to form a C1q-(C1s-C1rQI-C1r-C1s) heteropentamer to study the relative position of the C1r and C1s subunits in the C1 complex. After triggering the C1 by IgG-Sepharose, both C1s subunits are cleaved by the single proteolytically active C1r subunit in the C1s-C1rQI-C1r-C1s tetramer. This finding indicates that the tetramer is flexible enough to adopt different conformations within the C1 complex during the activation process, enabling the single active C1r to cleave both C1s, the neighboring and the sequentially distant one.
We have synthesized 2-[(5-methylbenz-1-ox-4-azin-6-yl)imidazoline, 3, a potent, peripherally acting alpha 2 adrenoceptor agonist. The agent is conveniently prepared in five steps from 2-amino-m-cresol. The agent has demonstrated good selectivity for alpha 2 adrenoceptors in binding and functional studies. When applied topically to eyes, the agent is efficacious for the reduction of intraocular pressure. The agent does not penetrate the blood-brain barrier and, as a consequence, does not lower blood pressure or induce sedation when administered topically or intravenously. We have determined the pKa and log P in water versus both octanol and dodecane of 3 and a set of related agents. The best physical parameter to explain its lack of central nervous system penetration appears to be log P measured in octanol versus water.
The genetically modified Mycobacterium sp. BCS 396 strain has been used to transform sterols with stigmastane side chain in order to obtain 26-oxidized metabolites. beta-Sitosterol (I) was transformed to 4-stigmasten-3-one (II), 26-hydroxy-4-stigmasten-3-one (III), and 3-oxo-4-stigmasten-26-oic acid (IV), while stigmasterol (V) was converted to 4,22-stigmastadien-3-one (VI), 6 beta-hydroxy-4,22-stigmastadien-3-one (VII), 26-hydroxy-4,22-stigmastadien-3-one (VIII), 3-oxo-4,22-stigmastadien-26-oic acid methyl ester (IX), and 3-oxo-1,4,22-stigmastatrien-26-oic acid methyl ester (X) with that strain. In both beta-sitosterol and stigmasterol, 26-oxidation generates the R-configuration on C-25.
In order to investigate the effect of the different stereochemistry of C-24 on the microbial C-26 oxidation of sterol side-chain the genetically modified Mycobacterium sp. BCS 396 strain was used to transform erogsterol. Ergosterol was converted to 3-oxo-4,22-ergostadien-26-oic acid methyl ester, 3-oxo-1,4,22-ergostatrien-26-oic acid methyl ester, and 3-oxo-1,4,22-ergostatrien-26-oic acid, the structures of which have been determined by IR, 1H NMR, 13C NMR, and mass spectroscopy. The X-ray structure of 3-oxo-4,22-ergostadien-26-oic acid methyl ester revealed that oxidation at C-26 of the ergostane side-chain generates a chiral center with S-configuration at C-25 as a result of chiral induction of the C-24 center.
Human myometrium contains functional hCG/LH receptors. The purpose of this study was to determine whether or not leiomyomas that arise from myometrium also contain these receptors. Northern blotting demonstrated that leiomyomas and normal adjacent myometria contained hCG/LH receptor mRNA transcripts. Western immunoblotting showed that leiomyomas and corresponding normal myometria also contained 60- and 50-kDa receptor proteins. Ligand blotting revealed that only the 50-kDa receptor protein in leiomyomas and corresponding normal myometria could bind 125I-hCG and that this binding inhibited by excess unlabeled hCG. In situ hybridization and immunocytochemistry revealed that smooth muscle cells in leiomyomas and corresponding normal myometria contained the hCG/LH receptor mRNA transcripts and receptor proteins. The receptor levels were lower in leiomyomas than in corresponding normal myometria. However, the receptors were functional, as treatment with hCG resulted in a decrease of connexin-43 protein levels in leiomyomas as in normal myometria. In summary, human uterine leiomyomas express a functional hCG/LH receptor gene at a reduced level compared with that for normal corresponding myometria. This finding could be relevant to an understanding of the growth control mechanisms in leiomyomas and the manner in which medical therapy for leiomyomas might work.
Human myometrium contains hCG/LH receptors. There are fewer of these receptors during labor compared to no labor at preterm or term deliveries. Exogenous hCG can directly inhibit oxytocin-stimulated human myometrial contractions. These findings suggest that hCG may directly maintain myometrial quiescence during pregnancy. As maintenance of uterine quiescence may involve down-regulation of myometrial gap junctions, we investigated the effect of hCG on connexin-43 (CX-43) gene expression from RNA to protein and morphological gap junctions. The addition of 5 or 10 nM highly purified hCG to subconfluent cultures of pregnant myometrial smooth muscle cells resulted in a significant decrease in CX-43 protein levels. Higher hCG concentrations (100 and 1000 nM), however, had no effect. The maximal effect of hCG was seen at 4-8 h of culture, followed by recovery after a longer duration of culture. hCG treatment also concomitantly decreased CX-43 messenger RNA and morphological gap junctions. The hCG effect on CX-43 protein levels is hormone specific and mediated by protein kinase-A signaling. Estradiol and oxytocin increased, whereas progesterone decreased, CX-43 protein levels and morphological gap junctions. The oxytocin-induced increase was reversed by cotreatment with hCG. Although RU 486 alone had no effect on CX-43 protein levels, it prevented the down-regulating action of hCG and progesterone. In summary, our results demonstrate that hCG can directly decrease CX-43 messenger RNA, protein, and morphological gap junctions in cultured pregnant human myometrial smooth muscle cells. The hCG action is concentration and time dependent, hormone specific, and mediated by protein kinase-A signaling and appears to involve progesterone receptors. These data lend support to the concept that hCG could be one of the hormones responsible for maintaining uterine quiescence by down-regulating myometrial gap junctions during pregnancy.
Our laboratory previously demonstrated that the human myometrium contains functional hCG/LH receptors. The present study investigated whether hCG can directly regulate oxytocin-stimulated human myometrial contractions. Uterine specimens were obtained from 30- to 40-yr-old women undergoing hysterectomy for leiomyomata, metrorrhagia, or prolapse. Myometrial strips from the lower uterine segment were primed for 24 h with 2.2 nmol/L estradiol. Then, the slices were incubated for 4 h at 37 C with or without 10 nmol/L hCG and stimulated with 1 mumol/L oxytocin, and the contractions were measured. The results showed that hCG inhibited the amplitude while paradoxically increasing the frequency of contractions. The effect of hCG was seen in proliferative, but not secretory, phase myometrial specimens. hCG had no effect on rat hepatic portal vein smooth muscle contractions, suggesting that the hCG action was tissue specific. Oxytocin treatment of human myometrial smooth muscle cells resulted in a dose-dependent increase in intracellular free Ca2+ levels. Pretreatment with hCG resulted in an attenuation of the oxytocin response, suggesting that the action of hCG was mediated by decreasing intracellular free Ca2+ levels. In summary, our results demonstrate that hCG can directly inhibit the amplitude of oxytocin-stimulated contractions of human myometria from the proliferative phase of the cycle. The hCG action is tissue specific and appears to be mediated by decreasing intracellular free Ca2+ levels in myometrial smooth muscle cells.
A derivative of Rhizobium meliloti 41 was constructed (strain GY654) which was resistant to apramycin and kanamycin but remained sensitive to tobramycin. Strain GY654 selectively measures tobramycin and 6"-O-carbamoyltobramycin in the presence of apramycin, kanamycin and 6"-O-carbamoylkanamycin B, therefore it is suitable for the rapid quantitation of 6"-O-carbamoyltobramycin and tobramycin in fermentation broths of Streptomyces tenebrarius and solvents containing antibiotic mixtures.
High-performance liquid chromatography has been used to quantitate benzalkonium chloride (alkylbenzyldimethylammonium chloride) in complex ophthalmic formulations at or below concentration levels of 50 ppm. The method involves a one-step dilution for sample preparation and direct injection; therefore, recovery and/or conversion problems are nonexistent. The assay is quick, specific, reproducible, and simple. This new approach makes routine determinations far simpler than previous methods and is especially useful for product stability studies and quality control procedures.
A synthesis of 16-amino-derivatives of PGF2 alpha is reported. Introduction of an amino group into position 16 of PGF2 alpha has decreased the sensitivity of the compound to metabolic degradation. 16(S)-amino-PGF2 alpha methyl ester shows high abortifacient activity with reduced diarrhoeic side effect.
In order to check the fragmentation mechanisms proposed in the preceding paper of this series the mass spectral behaviour of some deuterated analogues of prostaglandin F2alpha have been studied. In general, the investigation of these compounds confirmed the previous assumptions and shed light on some further details of the fragmentation pathways.
The fragmentation processes occurring in various 9alpha-hydroxy steroids upon electron ionization have been studied. The mechanisms proposed for the formation of the prominent ions in these spectra have been confirmed with the aid of deuterium labelling, measurements on metastable ion decompositions, low eV spectra and high resolution mass measurements. The fragmentation of the corresponding 9-oxo-9,10-seco steroids, which shows analogy to that of the 9alpha-hydroxy compounds in many respects, has also been discussed.
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In studying the transformation of diazepam (7-chloro-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one) by fungi isolated from soil. N1-demethylation and cleavage of the diazepine ring were observed. Three metabolites: 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, 2-acetamido-2"-benzoyl-4"-chloroacetanilide and 2-acetamido-2"-benzoyl-4"-chloro-N-methylacetanilide were isolated and identified.