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Thymidylate synthase ThyA (EC 2.1.1.45;-encoded by the Tyms gene), having been for 60 years a molecular target in chemotherapy, catalyses the dUMP pyrimidine ring C(5) methylation reaction, encompassing a transfer of one-carbon group (the methylene one, thus at the formaldehyde oxidation level) from 6R-N5,10-methylenetetrahydrofolate, coupled with a reduction of this group to the methyl one, with concomitant generation of 7,8-dihydrofolate and thymidylate. New facts are presented, concerning (i) molecular mechanism of the catalyzed reaction, including the substrate selectivity mechanism, (ii) mechanism of inhibition by a particular inhibitor, N4-hydroxy-dCMP, (iii) structural properties of the enzyme, (iv) cellular localization, (v) potential posttranslational modifications of the enzyme protein and their influence on the catalytic properties and (vi) non-catalytic activities of the enzyme.

Purine and pyrimidine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage of the glycosidic bond of purine and pyrimidine nucleosides, and are key enzymes of the nucleoside salvage pathway. This metabolic route is the less costly alternative to the de novo synthesis of nucleosides and nucleotides, supplying cells with these important building blocks. Interest in nucleoside phosphorylases is not only due to their important role in metabolism of nucleosides and nucleotides, but also due to the potential medical use of the enzymes (all phosphorylases in activating prodrugs - nucleoside and nucleic base analogs, high-molecular mass purine nucleoside phosphorylases in gene therapy of some solid tumors) and their inhibitors (as selective immunosuppressive, anticancer and antiparasitic agents, and preventing inactivation of other nucleoside drugs). Phosphorylases are also convenient tools for efficient enzymatic synthesis of otherwise inaccessible nucleoside analogues. In this paper the contribution of Professor David Shugar and some of his colleagues and coworkers in studies of these remarkable enzymes carried out over nearly 40 years is discussed on the background of global research in this field.

Poly-ADP-ribose polymerases (PARP) are involved in a number of processes that are vital for every living cell. Once activated by the presence of DNA damage they trigger poly-ADP-ribosylation of various proteins which are crucial for DNA repair, preserving of genom integrity, regulation of transcription, proliferation and apoptosis. PARP1, which is the best known enzyme of PARP protein family, plays a role in single-strand breaks (SSB) repair. Decrease of its activity results in accumulation of single strand DNA breaks (SSB) which leads as a consequence to double-strand breaks (DSBs). This disorder is particularly harmful to cells with deficiency of BRCA1/2 protein which is involved in repair of DNA double-strand breaks. This phenomenon is an example of "synthetic lethality" concept and contributes to research on application of PARP inhibitors in treatment of cancers associated with BRCA1/2 protein defect (breast or ovarian cancer). Noticed synergism between PARP inhibitors and genotoxic chemotherapy or radiotherapy determined another direction of research on application of these medicaments. After promising results of phase I and II trials with most commonly investigated PARP inhibitors--iniparib and olaparib--which recruited patients with triple negative breast cancer and ovarian cancer, further studies started. This paper presents theoretical basis of PARP inhibitors action as well as critical review of most important clinical trials of these medicaments.

Bacterial type II DNA topoisomerases are essential enzymes for correct genome functioning and cell growth. Gyrase is responsible for maintaining negative supercoiling of bacterial chromosome, whereas topoisomerase IV acts in disentangling daughter chromosomes following replication. Type II DNA topoisomerases possess an ATP binding site, which can be treated as a target for antibacterial drugs. Resolving crystal structures of protein fragments consisting of an ATP binding site complexed with ADPNP/antibiotics have proven to be valuable for the understanding of the mode of action of existing antibacterial agents and presented new possibilities for novel drug design. Coumarins, quinolones and cyclothialidines are diverse group of antibiotics that interfere with type II DNA topoisomerases, however their mode of action is different. Recently a new class of antibiotics, simociclinones, was characterized. Their mechanism of action towards gyrase is entirely distinct from already known modes of action, therefore demonstrating the potential for development of novel anti-bacterial agents.

14-3-3 proteins compose a large family of proteins that exist primarily as homo- and heterodimers within all eukaryotic cells. They are engaged in the regulation of numerous cellular processes, including melatonin biosynthesis. Melatonin, the hormone of darkness, is synthesized in a diurnal or circadian rhythm, with high levels at night. It has been demonstrated that cAMP levels and PKA activity in melatonin-synthesizing cells (pinealocytes and retinal photoreceptors) increase at night. PKA phosphorylates serotonin N-acetyltransferase (AANAT; the penultimate and key regulatory enzyme in melatonin biosynthesis pathway) at its N- (Thr31) and C-(Ser205)terminal region. Phosphorylated of AANAT bind to 14-3-3 proteins. The formation of pAANAT/14-3-3 complex stabilizes the enzyme and protects it against proteolytic destruction. Furthermore, this complex induces allosteric changes of the AANAT molecule resulting in an increase of the enzyme activity; this in turn enhances melatonin production by several fold. Exposure to light at night decreases intracellular cAMP level with concomitant dephosphorylation of pAANAT, its dissociation from 14-3-3 dimers, proteosomal proteolysis of free AANAT molecules, and finally turning off the melatonin production.

Introduction: The aim of this study was to determine the effect of reduced availability of dihydrotestosterone (DHT) on the expression of estrogen receptors alpha and beta (ERalpha and ERbeta) in the epididymis in vivo and in vitro. Expression of estrogen receptors (ERs) is interesting because of the fact that the male reproductive system is controlled not only by androgens but also, in a far-reaching and complex manner, by estrogens. Control by estrogens is exercised through activation of ERs widely distributed in the epididymal epithelium. Epididymal epithelial cells contain a 5alpha-reductase (5alpha-red) which catalyzes the irreversible conversion of testosterone (T) into the most potent and chief androgen of the epididymis, dihydrotestosterone, known to maintain and regulate the structure and functions of the epididymis. Two isoforms of the 5alpha-red were identified: type 1 (5alpha-redl) and type 2 (5alpha-red2). 5alpha-reductase type 2 is more widely expressed in the epididymis than 5alpha-redl. DHT deficit was produced by inhibition of 5alpha-red2 using finasteride (Proscar, MSD Sweden), a steroid inhibitor of this enzyme., Material and Methods: The study was performed in the adult, male Wistar rats randomly divided into control (K) and study (Fin56) groups (5 animals in each). Animals in the study group received 5mg finasteride/kg b.w., orally during 56 days (duration of one spermatogenesis). Immunoexpression of ERs was also studied in epididymal epithelial cells cultured with or without finasteride., Results: It was shown that DHT deficiency, both in vivo and in vitro condition, modulated ERs expression in comparison to the epididymis from control rats and to epididymal cells cultured without finasteride. Distribution of ERalpha and ERbeta in epididymal cells changed (from nucleus to cytoplasm) and the level of ERs expression was markedly decreased., Conclusion: The present findings show that the DHT deficiency caused by finasteride altered the expression of ERalpha and ERbeta in the epididymis and possibly may have destabilized the functioning of this organ.

The aim of the study was to examine the influence of free radicals: nitric oxide (NO), hydrogen peroxide (H202) and superoxide anion (O2-) on Trichinella spiralis infection in mice. The studies were performed on two strains of mice: C57BL/6 and BALB/c, which differ in immunological response to T. spiralis infection. Also the influence of AG--inhibitor of inducible nitric oxide synthase (iNOS) administered in the first days after T. spiralis infection (1-5 dpi) on the cytotoxic immune response and on the number of adult parasites as well as the influence of AG administered at the beginning of muscle phase of the T. spiralis infection (16-29 dpi) on the cytotoxic immune response and the number of muscle larvae was studied. Activation of macrophages can cause pathology. Contact of macrophages with antigens stimulates these cells to produce, among others, highly reactive inorganic compounds. There are free radicals: NO, H2O2 and O2-. NO, O2-, and their metabolites are highly toxic for most pathogens, including parasites. However, little is known about their role in the defense against T. spiralis infection. The performed studies have proved, that free radicals play role in the host immune response during both intestinal and muscle phase of T. spiralis infection in mice. In the intestinal phase of the T. spiralis infection cytotoxic immune response is activated in mice peritoneal cavity and in the muscle phase, the local immune response activated in the neighborhood of larvae in muscles appeared as the higher level of free radicals in blood and urine. Administration of AG between 1-5 dpi causes opposite reactions in two different strains of mice. In BALB/c mice AG causes fast expulsion of adult T. spiralis from the intestine but in C57BL/6 mice the expulsion of parasites is slower after AG. However, there are no differences between two strains of mice after treatment with AG between 16 and 29 dpi. AG causes diminution of larvae in muscles of both BALB/c and C57BL/6 mice. Inflammatory response in peritoneal cavity is observed later during the infection in "low responders" (C57BL/6) mice in comparison with "high responders" (BALB/c) mice. Thl like mice (C57BL/6) react stronger to AG treatment than Th2 like mice (BALB/c). It occurs as changes and fluctuations in free radicals levels and the number of peritoneal cells after AG treatment in C57BL/6 mice. Weak or no reaction on AG injections in BALB/c mice is responsible for more stabile and more sufficient defense response of the host to T. spiralis infection.

In light of the existing knowledge platelets are not able to de novo synthesis of nucleotides. However single report exists about the possession by thrombocytes of the full enzymatic complex, which is essential to such synthesis. An aim of our study was the estimation of the platelets nucleotide pool of patients subjected to immunossuppresion therapy using azathioprine and mycophenolate mofetil, drugs known to block enzymes of de novo synthesis pathways. Fifty-nine patients enrolled to a study were divided to three groups: hemodialysed patients (n = 28), patients after kidney transplantation receiving azathioprine (n = 16) and kidney transplanted patients receiving mycophenolate mofetil (n = 16). Platelets nucleotides concentration was measured using HPLC. In the group of patients after the kidney transplantation receiving AZA concentration of adenine and guanine nucleotides in thrombocytes were statistically lower both with relation to hemodialysed as and with relation to the healthy group. Similarly platelets of patients after the kidney transplant receiving MMF characterized lower adenine and guanine nucleotides concentration comparatively to hemodialyzed patients. One showed also lower ADP, GDP and AMP concentration with relation to of the healthy volunteers. One from possible reasons of the obtainment of above results can be the possession by platelets enzymes, which are vital to de novo nucleotides synthesis.

This article concerns cyclosporine A and cyclosporine--sparing agents. The drugs are evaluated due to their clinical usefulness in elevating cyclosporine blood level and their safety in long-term administration. We analyzed imidazole antifungal agents, calcium channel blockers and, additionally, pharmacological properties of cyclosporine A.

The aim of the study was to evaluate the effect of alcohol dehydrogenase (ADH; E.C. 1.1.1.1) inhibitors and substrates: cimetidine, 4-methylpyrazole (4MP), EDTA, ethanol and methanol on lactate dehydrogenase (LDH; E.C. 1.1.1.27) activity. The activity of LDH was spectrophotometrically determined in in-vitro prepared diluted hemolysates obtained from human erythrocytes with mentioned compounds at the concentrations 0.01, 0.1, 1.0 mM of cimetidine, EDTA, 4MP and 12.5, 25.0, 50.0 mM of ethanol and methanol. The reaction was conducted at 37 degrees C in pH 7.5 and changes of optical density was measured at lambda = 340nm. LDH activity was significantly inhibited by 0.10 mM (p < 0.05) and 1.0 mM (p < 0.01) of cimetidine and EDTA. There were no observed any significant changes vs. control in LDH activity when 4MP, ethanol or methanol was added to environment of reaction.

The contraction of arteries is influenced by many factors. The aim of this research was to analyze how an artery's reactivity, regulated by arginine-vasopressin changes in time, from the moment of death onwards. The research was conducted on rats' perfundated caudal arteries in four different time groups, with reference to two independent empirical models: I--with the sole use of intracellular Ca ions; and model II--with the sole use of extracellular Ca ions. The influence of NO on the arteries was analyzed in the two models, with the use of the NOS inhibitor. The research had shown that with the passing of time, beginning from the moment of death onwards, the process of emission of intracellular Ca ions and the infusion/transport of Ca ions from extracellular areas/spaces is inhibited. The use of the NOS inhibitor increases the artery's reactivity provided that the infusion of Ca ions from extracellular areas is also prevalent.

We present a 45-year-old woman with atypical celiac sprue who was unresponsive to gluten-free diet. Cyclosporine therapy was applied (5 mg/kg body weight daily for 2 months). The patient demonstrated a remarkable clinical and histological response to this drug.

Formestane is an inhibitor of the aromatase enzyme which is uniquely responsible for the generation of estrone and estradiol from androgenic precursors. Formestane is used in the treatment of breast cancer in postmenopausal women. Formestane causes reduction of the estrogen biosynthesis in all tissues where it occurs. Deficiency of estrogens disrypts remodeling of bone tissue. The effect of formestane on bones in not known. The aim of the present study was to investigate the effects of formestane (20 mg/kg s.c. once a week) administered for 4 weeks on the osseous system in bilaterally ovariectomized and non-ovariectomized rats. The experiments were carried out on 4 groups of 3-month-old female Wistar rats:I--Control (non-ovariectomized rats), II--Ovariectomized rats, III--Non-ovariectomized rats + Formestane, IV--Ovariectomized rats + Formestane. In all the groups examined were body weight gain, bone mass, length and diameter, mineral and calcium contents in the tibia and femur, endosteal and periosteal transverse growth, endosteal and periosteal osteoid width transverse cross-section area of the cortical diaphysis and that of the marrow cavity in the tibia, epiphyseal cartilage width, trabeculae width in the epiphysis and metaphysis of the femur. Mechanical properties of the femur were also studied. Bilateral ovariectomy induced osteopenic skeletal changes in female rats. Formestane did not significantly change bone remodeling in non-ovariectomized rats (group III). Formestane administered to the bilaterally ovariectomized rats (group IV) slightly reduced the changes by ovariectomy in the osseus system.

We studied the influence of the nitric synthase inhibitor (L-NAME) on the induction of uveitis in Wistar rats. The inflammation was induced by a footpad injection of endotoxin in rat. The clinical signs were evaluated in a slit-lamp, the intraocular pressure was measured using Tono Pen tonometer. We showed, that in endotoxin-induced uveitis the intraocular pressure elevation was observed and that L-NAME application reduced the inflammation and the level of the intraocular pressure.

The literature referring to proteolytic enzymes of neutrophilic granulocytes was surveyed. Biosynthesis, subcellular distribution, division according to the catalytic site structure, inhibitors and methods used to determine the activity of these enzymes were discussed. The survey included metaloproteases (granulocytic collagenase, gelatinase B), serine proteases (granulocytic elastase, cathepsin G, protease 3), membraneous proteases (aminopeptidase N, aminopeptidase P, neprilisine), cysteine and aspartic cathepsins. The role of these proteases in the pathology and diagnostics of certain diseases was considered.

The paper present modern views on turnover, mechanism of action and application of methotrexate. Contraindications and the most frequent side effects observed in certain number of patients treated with this drug are discussed.

The treatment of methanol intoxication usually focuses on prevention of methanol conversion to its toxic metabolites due to administration of ethanol or 4-methylpyrasole (4-MP). Nevertheless there is a need for new measures treatment of methanol intoxication. For this reason the influence of some alcohol dehydrogenase inhibitors on the activity of microsomal alcohol oxidising system (MAOS) with methanol as a substrate was assayed. In the present study MAOS activity was measured spectrophotometrically in vitro at physiological pH 7.4 and 37 degrees C, assaying the degree of methanol oxidation. The quantity of arising formaldehyde was measured according with the method of Nash. The source of enzyme was hepatic slices. Our results have shown that MAOS activity was inhibited to different extents by: 4-MP, isovaleric amide, cimetidine, DMSO, EDTA, o-phenantroline, pyrasole and theophylline at concentrations of 0.01 mmol/l, 0.10 mmol/l 1.00 mmol/l. And methylene blue, acetyl-L-carnitine and penicillamine increase MAOS activity in the process of methanol oxidation. In summary, 4-MP which plays an important role as an antidote in methanol intoxication was not an effective MAOS inhibitor. EDTA was found to be a highly effective inhibitor at all investigated concentrations.

In modern anaesthesiology drug interactions are inevitable. The interactions between anaesthetic drugs are well known for anaesthesiologists and even purposely used to obtain optimal anaesthetic effects. More complicated are interactions between anaesthetic drugs and chronic medications of the patients. This kind of interactions may be dangerous for the patients during anaesthesia procedure. In this survey the most common interactions between anaesthetics and popular groups of drugs were discussed. The knowledge of drug interactions is very important and useful for the practice of anaesthesia.

This study was undertaken to check the situation concerning the occurrence of Gram-negative rods producing extended-spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) in clinical specimens from patients hospitalized in National Clinical Hospital No. 1 in Warsaw. Such determinations were not performed in this hospital so far. During three months (April-June, 1997) 200 strains of Gram-negative rods were cultured. The strains were identified in automatic ATB system using strips with biochemical tests: ID 32 E for enteric rods and ID 32 GN for non-fermenting rods. ESBL-producing strains were detected with double disc diffusion test according to Jarlier et al. (1988). Clavulanate was applied as the inhibitor of beta-lactamases (AMO/CLAV disc). Inducible beta-lactamases were determined using double disc method according to Sanders and Sanders (1979). Cefoxitin was the inductor of these beta-lactamases. 82 strains (41% of all strains) belonging to Enterobacteriaceae family, 92 strains (46%) of Pseudomonadaceae rods and 26 strains (13%) of other Gram-negative rods were isolated. 30 ESBL-producing strains (15% of all strains) and 45 strains (22.5%) with IBL activity were detected. The obtained results confirm the necessity of continuous and reliable monitoring of ESBL--and IBL--producing strains among Gram-negative rods isolated from clinical materials. The aims of such procedure are the control and prevention of their dissemination within a hospital as well as the avoidance of therapeutic failures.