Tumor necrosis factor alpha-induced interleukin-8 production via NF-kappaB and phosphatidylinositol 3-kinase/Akt pathways inhibits cell apoptosis in human hepatocytes

Tumor necrosis factor alpha (TNF-alpha) not only induces apoptotic signals but also causes antiapoptotic and regenerative responses in the liver. However, the molecular mechanism(s) of the latter events remains unclear. In the present study, we examined TNF-alpha-induced genes in Hc human normal (unsensitized) hepatocytes by cDNA microarray analysis. Interleukin-8 (IL-8) induction was the most pronounced of the upregulated genes. The IL-8 protein level was also increased. IL-8 belongs to the ELR-CXC chemokine family and appears to exert mitogenic and antiapoptotic functions in other cell systems. IL-8 expression by TNF-alpha was inhibited when two survival signals, nuclear factor kappaB (NF-kappaB) and phosphatidylinositol 3-kinase (PI3K)/Akt, were inhibited by a mutant form of inhibitor of NF-kappaB (IkappaB); by dominant negative (kinase-dead) Akt; or by treatment with LY 294002, an inhibitor of PI3K. TNF-alpha induced apoptosis in Hc cells that were sensitized by inhibition of NF-kappaB and PI3K activation. IL-8 administration protected mice against concanavalin A-induced hepatitis in vivo. IL-8 also rescued the sensitized Hc cells, at least in part, from TNF-alpha-induced apoptosis in vitro. TNF-alpha inhibited DNA synthesis in unsensitized Hc cells in the absence of serum. Exogenous IL-8 reversed, though anti-IL-8 neutralization antibody enhanced, growth inhibition by TNF-alpha. These results indicate that IL-8, the production of which is stimulated by TNF-alpha, inhibits apoptosis of sensitized hepatocytes and releases normal (unsensitized) hepatocytes from growth inhibition induced by TNF-alpha.     

Evidence of the monoclonal composition of human endometrial epithelial glands and mosaic pattern of clonal distribution in luminal epithelium

The endometrium is a highly regenerative tissue that plays a crucial role in implantation. We examined the clonal constitution of glandular cells as well as the luminal epithelium of this unique tissue. Using collagenase-based digestion techniques with microscopic manipulation, we isolated individual human endometrial glands and examined their clonality using a polymerase chain reaction-based assay for nonrandom X chromosome inactivation with an X-linked androgen receptor gene. Most of the glands analyzed were composed of monoclonal populations of epithelial cells and one of the glands exhibited a loss of heterogeneity in the androgen receptor gene. In addition, adjacent glands within a 1-mm(2) area shared clonality, suggesting that clonality of the luminal epithelium is regionally defined. The clonality of endometrium was further confirmed in a study of female mice that harbor the green fluorescent protein gene on either the maternal or paternal X chromosome. Fluorescent microscopy of uterine sections revealed that individual endometrial glands consisted completely of either fluorescent or nonfluorescent cells and that the surface epithelium exhibited a clear boundary between these cell types. These findings suggest that single or multiple stem cells with uniform clonality exist on the bottom of each endometrial gland and genetic alterations occurring in such cells may play a critical role in endometrial carcinogenesis. The possible association between area-specific X inactivation of the endometrial surface and the endometrial receptivity of embryo implantation remains to be clarified.     

Comparative sequence analysis of the M gene among rabies virus strains and its expression by recombinant vaccinia virus

Nucleotide sequences and the deduced amino acid sequences of the gene encoding the matrix (M) protein of the Nishigahara and the CVS strains of rabies virus have been determined. The M gene is 609 nucleotides long and is capable of coding for a peptide composed of 202 amino acids. Sequence comparison of these M genes with those of other stains [Pasteur (PV), ERA, Avol] revealed that there is 89.7–91.5% homology at the nucleotide level, and 90.1–92.1% homology at amino acid level, between almost all combinations of these strains. However, in the combinations of the PV and ERA strains, and the virulent CVS and the avirulent CVS-derived Avol strains, much higher homology was observed both at the nucleotide and amino acid levels. The predicted secondary structure and hydropathy profiles also exhibited similar features. Recombinant vaccinia virus containing the M gene was constructed. Sodium dodecyl sulfate (NaDodSO₄)-polyacrylamide gel electrophoresis of the precipitates obtained by immune reaction of the recombinant virus-infected cell lysate with a monoclonal antibody against the M protein revealed that electrophoretic mobility of the expressed protein is indistinguishable from that of the authentic M protein from rabies virions.The nucleotide sequence data of the M genes of the CVS and Nishigahara (RCEH) strains reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers D90450 and D90451.     

Three-dimensional distribution patterns of PRL,GH and ACTH cells in the house musk shrew ( Suncus murinus)

Despite a multitude of reports on the classification and distribution of anterior pituitary cells, no previous study has attempted to obtain the three-dimensional (3D) and computer-graphic distribution pattern of each cell type in the whole pituitary. Therefore, we mapped the anterior pituitary cells of the house musk shrew ( Suncus murinus) and found a distinct cellular distribution pattern. Serial horizontal sections of whole shrew pituitaries were stained immunohistochemically for prolactin (PRL), growth hormone (GH), and adrenocorticotropic hormone (ACTH) cells. The contours of positive cells and the anterior, intermediate, and posterior lobes in each section were digitized for 3D visualization by a volume rendering method. The reconstructed images and virtual frontal and sagittal slices were examined in detail. On the 3D reconstructed images, the PRL and GH cells had similar distribution patterns, although the former were concentrated in the dorsolateral and ventrocentral portions, and the latter in the dorsocentral portions of the anterior lobe. On both sides of the pituitary stalk, there lay portions that were conspicuous by scarcity of PRL and GH cells. ACTH cells were widely scattered throughout the whole anterior lobe, but they were very few in the above portions and the dorsocentral portions where GH cells were concentrated. No sex difference in the distribution patterns of each cell type was observed. However, PRL cells in females were more numerous than in males, whereas the opposite was true for GH and ACTH cells. We discuss the relationship between the formation of the spatial distribution patterns and anterior pituitary ontogeny.     

Iron,coronary artery calcification,and mortality in patients undergoing hemodialysis

ObjectiveA high coronary artery calcification score (CACS) may be associated with high mortality in patients undergoing hemodialysis (HD). Recently, effects of iron on vascular smooth muscle cell calcification have been described. We aimed to investigate the relationships between iron, CACS, and mortality in HD patients.MethodsWe studied 173 consecutive patients who were undergoing maintenance HD. Laboratory data and Agatston’s CACS were obtained at baseline for two groups of patients: those with CACS ≥400 (n = 109) and those with CACS <400 (n = 64). Logistic regression analyses for CACS ≥400 and Cox proportional hazard analyses for mortality were conducted.ResultsThe median (interquartile range) age and duration of dialysis of the participants were 67 (60–75) years and 73 (37–138) months, respectively. Serum iron (Fe) and transferrin saturation (TSAT) levels were significantly lower in participants with CACS ≥400 than in those with CACS <400, although the serum ferritin concentration did not differ between the groups. TSAT ≥21% was significantly associated with CACS ≥400 (odds ratio 0.46, p<0.05). TSAT ≥17%, Fe ≥63 µg/dL, and ferritin ≥200 ng/mL appear to protect against 5-year all-cause mortality in HD patients, independent of conventional risk factors of all-cause mortality (p < 0.05).ConclusionWe have identified associations between iron, CACS, and mortality in HD patients. Lower TSAT was found to be an independent predictor of CACS ≥400, and iron deficiency (low TSAT, iron, or ferritin) was a significant predictor of 5-year all-cause mortality in HD patients.     

Purification and Partial Characterization of an Indomethacin Hydrolyzing Enzyme from Pig Liver

Purpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of -chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in -naphthylacetate and -nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.     

Distribution of connectin (titin), nebulin and α-actinin at myotendinous junctions of chicken pectoralis muscles: an immunofluorescence and immunoelectron microscopic study

Summary The distribution of connectin (titin), nebulin and -actinin in the areas of myotendinous junctions of chicken pectoralis muscles was examined by immunocytochemical methods. Staining with antibodies against connectin (4C9, SM1 and P1200) and nebulin formed doublets flanking nonterminal Z-bands; near the end of muscle fibres singlets were seen within the terminal sarcomere on the side adjacent to the terminal Z-bands. The apical regions of muscle processes, where no myosin filaments are present although actin filaments exist, were reactive with anti-nebulin but not with anti-connection. Antibodies against pectoralis (skeletal muscle type) -actinin stained non terminal Z-bands and that against gizzard (smooth muscle type) the sarcolemma. Terminal Z-bands were unreactive with both of these antibodies. These findings indicate that, although terminal and nonterminal Z-bands differ in their molecular composition, connectin and nebulin filaments appear to link myosin and actin filaments, respectively, to both Z-band types.     

Serum fluoride as an indicator of occupational hydrofluoric acid exposure

Summary To define the relationship between ionic fluoride concentration in the serum of workers and the amount of hydrofluoric acid (HF) in the work environment, pre-and postshift serum and urine samples of 142 HF workers and 270 unexposed workers were examined. The maximum and minimum concentrations of HF in the air in each workshop varied from the mean by less than 30%. The pre-exposure levels of serum and urinary fluoride in HF workers were higher (P < 0.001) than the control values. This suggests that fluoride excretion from the body continues for at least 12 h. The postshift serum and urinary fluoride concentrations of these workers were significantly higher (P < 0.001) than the preshift concentrations. A good correlation (r = 0.64) was obtained between postshift serum fluoride and postshift urine fluoride. There was a linear relationship between mean serum fluoride concentration and HF concentration in the workshop. A mean fluoride concentration of 82.3 g/l with a lower fiducial limit (95%, P = 0.05) of 57.9 g/l was estimated to correspond to an atmospheric HF concentration of 3 ppm. This is the maximum allowable environmental concentration recommended by the Japanese Association of Industrial Health, and it is also the threshold limit value suggested by the American Conference of Governmental Industrial Hygienists. The results demonstrate that exposure to HF can be monitored by determining the serum fluoride concentration.     

A blood cyanide distribution study in the rabbits intoxicated by oral route and by inhalation

Summary Blood cyanide concentration was determined in rabbits intoxicated orally or by inhalation. Experiments were carried out under urethane anaesthesia. In the inhalation experiments, rabbits inhaled a combustion product containing HCN via the tracheal cannula and in the oral studies animals were administered NaCN solution into the stomach. In addition to the carotid artery and jugular vein blood samples, postmortem samples were obtained from both sides of the heart and the descending vena cava.The arterial cyanide concentration in the inhalation group showed a close relationship with ventilation. After an initial rise, blood levels decreased a little, in some cases with transient apnea. At the last stage it again increased with gasping, reaching its maximal value. After ultimate apnea, the blood cyanide concentration declined. The blood cyanide values were higher in the oral group than in the inhalation group. The difference between the two groups became larger in the inferior order, the left heart blood-the right heart blood-blood in the descending vena cava. The left heart/right heart ratio of the inhalation group was significantly higher than that of the oral group (1.28 ±0.28 vs. 0.95 ±0.09). The coefficient of variation (c.v.) of the inhalation group was larger than that of the other group. Within the inhalation group, the left heart blood showed the largest c.v. values and this was probably due to redistribution of the cyanide by bloodstream after attainment of the maximal concentration.

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Bestimmung der Blutzyanidkonzentration in oral oder inhalatorisch vergifteten Kaninchen

Zusammenfassung Die Blutzyanidkonzentrationen wurden an Zyanid-vergifteten Kaninchen in Urethannarkose bestimmt. Die Kaninchen inhalierten HCN-haltige Verbrennungsgase durch die Trachealkanüle. Eine andere Gruppe von Tieren erhielt NaCN-Lösung per os. Während der Versuche wurden Blutproben aus einer katheterisierten Halsarterie gewonnen. Postmortale Blutproben wurden aus beiden Ventrikeln des Herzens und der kaudalen Hohlvene entnommen.Der arterielle Blutzyanidspiegel der ersten Gruppe zeigte eine enge Beziehung mit der Ventilation auf. Nach einer Konzentrationszunahme im Anfangsstadium nahm der Blutspiegel mit einer vorübergehenden Apnoe ab. Mit der terminalen Atembewegung stiegen die Blutspiegel und erreichten ihre maximalen Werte. Die Blutzyanidwerte verminderten sich nach endgültiger Apnoe. Der Blutspiegel der per-os-Gruppe nahm stetig unabhängig von der Ventilationsgröße zu. Die inhalatorische Gruppe hatte niedrigere Zyanidwerte als die orale Gruppe.

     

In vitro drug-drug interactions with perospirone and concomitantly administered drugs in human liver microsomes.

In vitro metabolism studies were conducted to assess drug-drug interactions between perospirone, an antipsychotic agent, and concomitantly administered drugs--biperiden, flunitrazepam, haloperidol, and diazepam--using human liver microsomes. The metabolism of perospirone in the presence of 100 microg/ml drugs was decreased to 45-73% of that in their absence, whereas no effects were observed with any of the drugs at 1 microg/ml or lower. The effects of perospirone on the metabolism of concomitantly administered drugs were also assessed, and no inhibitory effect was observed. Thus, the metabolism of perospirone and concomitantly administered drugs did not demonstrate any marked mutual inhibition in the human liver microsomes. On the other hand, the perospirone metabolism was markedly reduced by ketoconazole indicating a major role for CYP 3A4. Based on the inhibition constant (Ki) for perospirone metabolism and the plasma unbound concentration of ketoconazole, in vivo perospirone clearance was estimated to be reduced to 64-90% of the control level. Thus careful attention should be paid to the possibility of increase in unchanged perospirone concentration when perospirone is co-administered with drugs that are known as CYP3A4 inhibitors, including macrolide antibiotics and other imidazole antifungals.