Does dihydromyricetin impact on alcohol metabolism.

Dihydromyricetin (DHM) is a natural flavonoid showing several health promoting effects such as protective activity during severe alcohol intoxication. The mechanism underlying the effects of DHM on alcohol metabolism is virtually unknown. The present paper is focused on clarifying the role of DHM in the liver alcohol elimination at its molecular level. First, impact of DHM on alcohol dehydrogenase (ADH) activity in vitro and the enzyme induction in vivo was examined. Neither the ADH activity nor the enzyme expression were influenced by DHM. Next, the effect of DHM during alcohol intoxication were studied on primary hepatocytes isolated from EtOH-premedicated and untreated rats. The viability of cells exposed to alcohol, estimated based on the released enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), was slightly affected by DHM. Although the expected hepatoprotective effect of DHM was not fully achieved, DHM (in a concentration manner) proved to reduce the level of ROS/RNS in hepatocytes. However, no change in the rate of alcohol metabolism in vivo was found when rats were administered with a single or repeated dose of ethanol supplemented with DHM. In conclusion, the proposed positive effect of DHM during alcohol intoxication has not been proven. Moreover, there is no effect of DHM on the alcohol metabolism. The "hoped-for" DHM hepatoprotective activity can be attributed to the reduction of ROS/RNS levels in cells.

Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues.

The effect of cryopreservation on the protein phosphorylation/dephosphorylation pattern of common carp (Cyprinus carpio) sperm is described. Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol (EG)-based extenders, followed by equilibration, freezing, and thawing. Proteins extracted from fresh and cryopreserved spermatozoa were separated on SDS-PAGE and two-dimensional gel electrophoresis, blotted on polyvinylidene difluoride membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. For the subsequent protein identification we used matrix-associated laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated that cryopreservation with either DMSO or EG extender significantly altered the phosphorylation state of sperm proteins on tyrosine or threonine residues. A dramatic decrease in tyrosine phosphorylation was detected in the cryopreservation procedures with DMSO extender. Endoplasmin, transketolase, and S-adenosylhomocysteine hydrolase were identified as proteins that play a key role in cellular stress responses and oxidation and/or reduction reactions. Results indicate that the phosphorylation and/or dephosphorylation modifications of sperm proteins that occur during cryopreservation could stimulate a series of biochemical effects interfering with spermatozoa function and leading to a loss of motility and fertilization ability. Our findings indicated that use of EG extender provided superior protein preservation during sperm storage.

Comparison of protein fractions in seminal plasma from multiple sperm collections in sterlet (Acipenser ruthenus).

Seminal plasma of sterlet Acipenser ruthenus was evaluated using comparative proteomics to characterize its protein fractions and to determine any influence of multiple sperm collections on these proteins. An experimental group of fish was used, in which sperm was collected three times at 5 h intervals. Protein fractions of seminal plasma were determined by SDS-gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis high-resolution gels (2D). At all stripping times, five protein bands with molecular weights of 93, 53, 48, 33 and 28 kDa were identified using SDS-PAGE. No significant differences (p > 0.05) in relative mass of protein bands among collections were observed. At the third collection, 20 protein spots were detected from the two-dimensional gels, compared to 17 found at the first and second collections. Ten protein spots, from the third stripping, were analysed. Screening of these spots by mass spectrometric analysis showed positive results for spot 10. Direct comparison across public databases revealed sequence similarity with two hypothetical proteins, MCAG_00854 and IscW_ISCW011489. Differences in the seminal plasma protein fractions were found at the third stripping compared to the first two. It is hypothesized that these extra proteins after the third collection could be involved in some step of intracellular mechanism which is responsible for regulating of spermatozoa motility. However, protein identification revealed no significant distinction for any protein spot and protein sequences available in public databases. These results highlighted the need for a complete genome sequences for sturgeons.

Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L.

Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.

Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity.

The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1-1.9 U mg(-1) dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30 degrees C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile >> chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92-100, 70-90 and 30-51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.

Hydroxylated anthraquinones produced by Geosmithia species.

Geosmithia fungi are little known symbionts of bark beetles. Secondary metabolites of lilac colored species G. lavendula and other nine Geosmithia species were investigated in order to elucidate their possible role in the interactions of the fungi with environment. Hydroxylated anthraquinones (yellow, orange, and red pigments), were found to be the most abundant compounds produced into the medium during the submerged cultivation. Three main compounds were identified as 1,3,6,8-tetrahydroxyanthraquinone (1), rhodolamprometrin (1-acetyl-2,4,5,7-tetrahydroxyanthraquinone; 2), and 1-acetyl-2,4,5,7,8-pentahydroxyanthraquinone (3). Compounds 2 and 3 (representing the majority of produced metabolites) inhibited the growth of G+-bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentration of 64-512 microg/mL. Anti-inflammatory activity detected as inhibition of cyclooxygenase-2 was found only for compound 3 at 1 and 10 microg/mL. Compound 2 interfered with the morphology, compound 3 with cell-cycle dynamics of adherent mammalian cell lines.

Biodegradation of brominated aromatics by cultures and laccase of Trametes versicolor.

2-Bromophenol (1), 4-bromophenol (2), 2,4-dibromophenol (3), 2,6-dibromophenol (4), 2,4,6-tribromophenol (5) and tetrabromobisphenol A (6) (1 mM each) added to growing submerged cultures of Trametes versicolor CCBAS 612 were eliminated by 65-85% from the culture medium within 4d. Extracellular laccase activity in the culture medium was influenced by the type of brominated compound added. Maximum level of laccase (63 U L(-1)) was found in the culture with 2-bromophenol. Tetrabromobisphenol A was degraded by a commercial laccase from Trametes versicolor in absence of any oxidation mediator, hydroxylated dibrominated compounds being detected as soluble reaction products by LC/MS. A significant degradation of brominated phenols by laccase was achieved only in the presence of ABTS structural characterization of major products suggesting reaction between bromophenol and ABTS radicals.

Chemopreventive compounds--view from the other side.

Increasing attention is being paid to the possibility of applying chemopreventive agents for the protection of individuals from cancer risk. The beneficial potential of chemoprotective compounds is usually well documented by extensive experimental data. To assure the desired effect, these compounds are frequently concentrated to produce dietary supplements for human use. The additive and synergistic effects of other food constituents are, however, frequently ignored. Even natural chemopreventive compounds have to be considered as xenobiotics. Thus, as much attention has to be paid to their testing prior to their wide application as is usual in drug development for human treatment. Unfortunately, much of the research in this area is solely based on simplified in vitro systems that cannot take into account the complexity of biotransformation processes, e.g. chemopreventive compound-drug interaction, effect on metabolism of endogenic compounds. Hence, the predicted chemopreventive potential is not attained in respect of cancer prevention; moreover, the administration of high doses of chemopreventive compounds might be even detrimental for the human health.

[Small-cell variant of CD30+ -anaplastic large-cell lymphoma of the skin].

Three cases of the so-called variant of primary cutaneous CD30+ anaplastic large cell lymphoma (ALCL) are presented. All patients were males aged 52, 59, and 78 years old; they had a solitary cutaneous tumor nodule. Their sites included the axilla, thigh, and shoulder. There was no extracutaneous involvement. Microscopically, the tumors were composed of small cells with irregular nuclei that were immunohistochemically positive for CD3, CD5, CD7, and CD30 and negative for B-cell markers; there was focal ALK-1 positivity in 1 case. Two cases had CD4+/CD8-phenotype, while the remaining one exhibited a CD4-/CD8+ immunoprofile. Fhedium to large CD30+ cells were rarely found scattered in the infiltrate. Monoclonal TCR gamma gene rearrangement was detected in 2 cases and rearrangement of IgH (lineage infidelity) was in one case. The tumors were surgically removed in all the patients. Two patients were alive and well 4 and 6 years after surgery, without evidence of cutaneous and extractaneous involvement (including the ALK+ patient). The third patient experienced several relapses of the skin tumor and developed axillary and inguinal lymph node involvement. Chemotherapy was performed and finally the patient underwent allogenic bone marrow transplantation; he died 3 years after the original diagnosis due to acute graft-versus-host disease and sepsis.

Production of (+)-globulol needle crystals on the surface mycelium of Quambalaria cyanescens.

The structure of unique colorless needle crystals growing from the surface mycelium of the basidiomycete Quambalaria cyanescens and identified as (+)-globulol was followed by mass spectrometry, X-ray diffraction, and polarimetry. The mechanism of (+)-globulol fragmentation is proposed based on collision induced dissociation mass spectrometry. X-Ray analysis revealed that crystal packing is governed by hydrogen bond O-H.....O connecting the molecules into an infinite helix along a 3-fold screw axis propagating along the longest dimension of the needle crystal (c-axis of the unit cell). The X-ray diffraction data correspond well with the proposed structure determined by mass spectrometry.

Monoclonal antibody FsC-47 against carp sperm creatine kinase.

The enzyme creatine kinase (CK) plays a key role in energy homeostasis of cells with high and fluctuating energy requirements. As for spermatozoa, the activity of phosphocreatine shuttle, which directs energy from the mitochondria to sites of ATP consumption, is dependent on individual species. High activities of CK are observed in spermatozoa of nonvertebrate, fish, and birds, contrary to the low-level CK activity in mammalian spermatozoa. A new monoclonal antibody (MAb) to carp sperm creatine kinase was prepared. This antibody is applicable to large-scale immunochemical techniques. In this study, it was applied to the study of carp sperm motility, and the evaluation of the influence of CK on the quality and fertilization ability of carp spermatozoa.

The histological findings in the gastroesophageal junction of fetuses.

The development of the esophageal and gastric mucosa in the gastroesophageal junction was studied in 61 fetuses of 13-41 weeks of the gestational age. During the 13th-15th week, the esophageal multilayered epithelium was covered by a continuous layer of columnar mucous ciliated cells which were present only focally till the 25th week and disappeared later. Before the 15th week, the gastric mucosa was formed by pits only. The glands started as proliferating tubules in the basal parts of the pits in the 15th week. Further, they differentiated into oxyntic glands. The mucosa of the corpus was fully developed in the 27th week. The cardiac mucosa was absent in all the 10 fetuses examined between the 27th and 41st week of gestation. This supports the view that the gastric cardiac mucosa is not a physiological structure but that it results from glandular metaplasia of the distal esophageal mucosa due to gastroesophageal reflux.

Rabbit liver microsomal system: study of interaction with two model N-nitrosamines and their metabolism.

Rabbit liver microsomes of control (non-treated) or animals induced either by ethanol (EtOH) or phenobarbital (PB) were incubated with N-nitrosodimethylamine (NDMA) or N-nitrosomethylaniline (NMA). Difference spectroscopy showed that NMA is bound to the substrate-binding site of cytochrome P-450 (CYP) isoforms as heme ligand in control and EtOH pre-treated microsomes. On the other hand, PB-induced microsomes exhibit with NMA substrate type of spectra. NDMA does not provide any type of binding spectra with used microsomal systems. Oxidative bio-activation of N-nitrosamines by the microsomal CYP isoforms was measured as formaldehyde formation. Analysis of reaction kinetics in control microsomes revealed, for both substrates, two values of Michaelis-Menten constant (K(m)) for, K(m) values of 0.03 and 0.13 mmol/l for NDMA, and 0.30 and 0.82 mmol/l for NMA. Induction of animals with EtOH resulted in a decrease in the K(m) value for both substrates. In contrast, PB treatment caused an elevation of K(m) value for NDMA. Based on these data, we conclude that EtOH-inducible microsomal CYP isoforms (mainly CYP2E1) are responsible for binding and N-demethylation metabolism of both studied N-nitrosamines in rabbit liver microsomal system. The role of the other CYP isoforms involved in the metabolism of mentioned N-nitrosamines is discussed.

Evaluation of comparative cytochrome P450 2B4 model by photoaffinity labeling.

A homology model of rabbit CYP 2B4 was constructed on the basis of the crystallographic structure of truncated mammalian CYP 2C5/3 and bacterial soluble CYPs. To validate the CYP 2B4 homology model photoaffinity labeling was employed. Three probes (I-III) containing a photo-labile azido-group and an amino-group on opposite ends of the molecule were designed for photoaffinity labeling of the CYP 2B4 in increasing distance from the heme iron. Spectroscopic data proved probes I (the shortest) and II (a middle sized) to be coordinated with the heme iron via their amino-groups in the enzyme active center while the probe III (the longest) was not bound in this way. This binding orientation of probes I and II is in accordance with the model predicting ion-pairing of the negatively charged side chain of CYP 2B4 Asp 105 and a positively charged nitrogen located in an appropriate position in structures of probes I and II, only. The lack of heme binding of the probe III is clear from its docking into the CYP 2B4 model since no Asp 105 ion-pairing is possible. The target of photoactivated probe II, Arg 197, in a distance of about 16.5 A from the heme iron, exactly matches the position of that amino acid residue, predicted from the CYP 2B4 homology model. Moreover, using this technique, a substrate access channel has been identified. To assess the predicted substrate-binding pocket, an interaction of a specific CYP 2B4 substrate, diamantane, was examined. In "silico" docking revealed strong binding of diamantane in an orientation allowing experimentally observed C4-hydroxylation. Our homology model of CYP 2B4 is thus consistent with experimental metabolic and photoaffinity labeling data.

[Metanephric adenoma. 11 case reports and detection of collagen spherules in one tumor]. / Metanefrický adenom. Popis 11 prípadu s prukazem kolagenních sferul v jednom nádoru.

Eleven cases of metanephric adenoma are reported. The tumors were selected out of 6500 tumorous and pseudotumorous lesions of the kidney in our registry. Female to male ratio was 1:1.2. The average age of the patients was 48.3 years, with a range of 13-79 years. The mean size of the tumors was 7.2 cm. The tumors were spherical in shape, whitish to yellowish in colour. Histologically, they were arranged in a mainly tubular pattern with short pseudopapillae. The tumorous cells were deeply eosinophilic to basophilic with predominantly round nuclei. Psammomatous bodies were numerous. Immunohistochemically, they reacted positively with antibodies against cytokeratins, vimentin, and WT1. Ultrastructurally, the cytoplasm contained mitochondria, RER, and ribosomes. A collagenous spherulosis, identical with those in salivary gland and mammary tumors, was revealed in one case. The spherules were located mainly inside tubular structures. Ultrastructurally, they were composed of basement membrane-like material, which was surrounded by epithelial cells. Follow-up all of our patients was negative (if known) for 10 months to 4 years.

Intestinal necrosis following Calcium Resonium-sorbitol administration in a premature uraemic infant.

Sodium polystyrene sulphonate (Resonium A) in sorbitol given as an enema or orally to treat hyperkalaemia has been described to induce intestinal necrosis in uraemic patients. We report a case of a premature infant with acute renal insufficiency who developed focal transmural necrosis and perforation of the small intestine after 10 days of administration of calcium polystyrene sulphonatum (Calcium Resonium) in sorbitol by enema and by nasogastric tube. On histological examination of the resected part of the small intestine, numerous strongly basophilic angular crystals of resonium were found in the lumen, in the necrotic wall, as well as in the organized exudate on the peritoneal surface. The crystals showed a strong direct Schiff positivity without preoxidation. They were also stained using PAS, Giemsa, Ziehl-Neelsen, Schmorl, and Gram method. In contrast, the crystals were Congo red and Alcian blue (pH 2.5) negative and non-birefringent. The direct Schiff positivity without preoxidation is virtually pathognomonic for resin crystals in routinely processed tissues. The same crystals were observed in the lumen of the small intestine and in peritoneal adhesions at autopsy. Thus our case provides additional evidence that Resonium A/Calcium Resonium in sorbitol administered as an enema or orally can lead to intestinal necrosis in uraemic patients.

alpha-Naphthoflavone acts as activator and reversible or irreversible inhibitor of rabbit microsomal CYP3A6.

This report describes the effect of alpha-naphthoflavone (alpha-NF), a known substrate, inhibitor and activator of several cytochromes P450 (CYP), on rabbit CYP3A6. Hepatic microsomes of rabbit pretreated with rifampicine (RIF), enriched with CYP3A6, as well as purified CYP3A6 reconstituted with isolated NADPH:CYP reductase were used as enzymatic systems in this study. The data from difference spectroscopy experiments showed that alpha-NF does yield a type I binding spectrum. This compound is oxidized by microsomal CYP3A6 into two metabolites (5,6-epoxide and trans-7,8-dihydrodiol). While alpha-NF is a substrate of CYP3A6, it also acts as an enzyme modulator. Under the conditions used, stimulation of 17beta-estradiol 2-hydroxylation by alpha-NF was observed. In contrast, this compound reversibly inhibited N-demethylation of erythromycin and tamoxifen, competitively with respect to these substrates, having the K(i) values of 51.5 and 18.0 microM, respectively. Moreover, alpha-NF was found to be an effective inactivator of progesterone and testosterone 6beta-hydroxylation catalyzed by CYP3A6 in RIF-microsomes. In addition, time- and concentration-dependent inactivation of human CYP3A4-mediated 6beta-hydroxylation of testosterone by alpha-NF, was determined. The inactivation of CYP3A6 followed pseudo-first-order kinetics and was dependent on both NADPH and alpha-NF. The concentrations required for half-maximal inactivation (K(i)) were 80.1 and 108.5 microM and the times required for half of the enzyme to be inactivated were 10.0 and 11.9 min for 6beta-hydroxylation of progesterone and testosterone, respectively. The loss of the enzyme activity was not recovered following dialysis, while 90% of the ability to form a reduced CO complex remained. This indicates the binding of alpha-NF to a CYP apoprotein molecule rather than to a heme moiety. Protection from inactivation was seen in the presence of all tested CYP3A substrates. Progesterone and testosterone protected CYP3A6 against inactivation competitively with respect to inactivator, erythromycin non-competitively and 17beta-estradiol showed a mixed type of protection. Here, we described for the first time that alpha-NF is capable of irreversible inhibition of microsomal rabbit CYP3A6 and human CYP3A4. The obtained results strongly suggest that the CYP3A active center contains at least two and probably three distinct binding sites for substrates.

[Oncocytoma of the kidney--morphologic variation in 102 cases]. / Onkocytom ledviny--morfologická variabilita 102 prípadu.

From the collection of 2500 cases of renal epithelial tumors in our files, 102 renal oncocytomas were analyzed for size, multifocality and a morphologic spectrum of the growth pattern. The size of the tumors ranged from 1.5 to 13 cm in diameter, with a mean of 6.3 cm. Three cases were multifocal, four cases were combined with another primary renal tumor (1x angiomyolipoma, 1x conventional renal carcinoma, 2x papillary renal cell carcinoma). A central fibrosis or a scar was noted in 13 cases, and there was a gross area of hemorrhage in 11 cases. In 4 cases extensive necroses were recognized. Histologically, an alveolar pattern was noted in 70 cases. A tubular pattern was revealed in 31 cases and an unusual tubopapillar ("glomeruloid") pattern was noted in one case. Foci of atypical nuclei were identified in 58 cases. In 4 oncocytomas broad areas of clearance of the oncocytes were found. Psammoma bodies were recognized in 9 tumors and foci of ossification were present in 4 cases. Intracellular and extracellular hyaline globules were noted in two cases. Renal oncocytoma has a variable morphologic spectrum, and its diagnosis should be based on an analysis of structural and cytologic features. Differential diagnosis of renal oncocytomas with various tumors of the kidney which contain granular cytoplasm is discussed. These tumors with granular cytoplasm include conventional renal cell carcinomas, chromophobe cell carcinomas, and rare examples of papillary renal carcinomas.