Organochlorine pesticides (OCPs), dioxin-like polychlorinated biphenyls (dl-PCBs), polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo furans (PCDD/Fs) in edible fish from Lake Volta, Lake Bosumtwi and Weija Lake in Ghana.

Organochlorine pesticides (OCPs), dioxin-like polychlorinated biphenyls (dl-PCBs), polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo furans (PCDD/Fs) were measured by a high resolution gas chromatograph-high resolution mass spectrometer (HRGC/HRMS) in selected edible fish from three freshwater bodies, Lake Volta, Lake Bosumtwi and Weija Lake in Ghana. The levels of organochlorine pesticides measured in this study were generally low. The highest concentration of OCPs was measured for dichlorodiphenyltrichloroethane compounds (DDTs) (p,p'-DDT, o,p'-DDT, p,p'-DDE, o,p'-DDE, p,p'-DDD and o,p'-DDD), followed by chlordane compounds (CHLs) (trans-chlordane, cis-chlordane, trans-nonachlor and cis-nonachlor), hexachlorobenzene (HCB) and gamma-hexachlorocyclohexane (γ-HCH). The relatively high ratio of p,p'-DDT/p,p'-DDE in tilapia and catfish with an extremely high value in catfish purchased from a local market at Madina, a suburb of Accra, however, suggests the fresh contamination of technical DDT in Ghana. Although PCDD/Fs and dl-PCBs showed relatively low levels, the concentrations are, however, comparable with recent data of some developed countries. There is a potential health risk from DDTs, PCDD/Fs and dl-PCBs for the general population of Ghana because fish is one of their important protein sources. It is therefore necessary to estimate the total intake of DDTs, PCDD/Fs and dl-PCBs, and to assess the health risks for the general population of Ghana.

Oncostatin M is produced during pregnancy by decidual cells and stimulates the release of HCG.

Oncostatin M (OSM) is a member of the interleukin-6 superfamily and a multifunctional cytokine that effects the growth and differentiation of many different cell types. OSM concentrations in the sera of pregnant women were found to be significantly higher than those of non-pregnant women. Western blot analysis revealed that the OSM protein was present in the decidua and chorionic tissue in each trimester. Throughout pregnancy, the amount of the OSM protein in the decidua was larger than that in the chorionic tissue. Immunohistochemistry using an anti-OSM monoclonal antibody demonstrated that OSM was mainly localized in the decidual glands and stroma. OSM transcripts in the decidua and the chorionic tissue were detected during each trimester by reverse transcription-polymerase chain reaction (RT-PCR). The regulation of human chorionic gonadotrophin (HCG) release by the placenta in first trimester stimulated with recombinant OSM was also investigated. Stimulation of the placenta by OSM augmented HCG release in a time- and dose-dependent manner. HCG release induced by recombinant human OSM was completely blocked by antibodies against OSM and the signal transducer, gp130, but only partially inhibited by antibodies against the leukaemia inhibiting factor (LIF) receptor. These results suggest that OSM molecules produced by decidual glands and stromal cells during pregnancy have an important role in placental endocrine function.

Suicide gene therapy for human uterine adenocarcinoma cells using herpes simplex virus thymidine kinase.

In gene therapy, the herpes simplex virus thymidine kinase (HSV-tk) gene is widely used as a suicide agent. Tumor cells expressing HSV-tk are sensitive to nucleoside analogs such as ganciclovir (GCV). An advantage of this system is the bystander killing effect whereby HSV-tk-positive cells exposed to GCV are lethal to surrounding HSV-tk-negative cells. We transfected the HSV-tk gene into a human cervical adenocarcinoma cell line, BU25TK-, and a human endometrial adenocarcinoma cell line, HHUA, by the Lipofectine method. The sensitivity of HSV-tk-positive cells to GCV and bystander killing effect on HSV-tk-negative cells were examined in vitro. HSV-tk-positive cells were sensitive to GCV at concentrations of 1 to 100 microg/ml in a dose- and time-dependent manner. The growth of HSV-tk-negative cells was inhibited when the population of cultured cells contained more than about 3% HSV-tk-positive cells. Moreover, for BU25TK- cells, HSV-tk-positive cells were injected into SCID mice subcutaneously and the effects of GCV therapy and bystander killing at a daily concentration of 25 mg/kg for 14 days were examined. HSV-tk-positive tumors transduced into SCID mice almost disappeared upon GCV treatment. Furthermore, tumor reduction was observed when mixtures of HSV-tk-negative cells containing more than 20% HSV-tk-positive cells were injected into SCID mice. In conclusion, the HSV-tk/GCV system might be applied to both cervical and endometrial adenocarcinoma.

[Sporadic case of hemolytic uremic syndrome associated with verotoxin producing Escherichia coli O2:K1:H7].

Cases of enterohaemorrhagic Escherichia coli (EHEC) are being increasingly reported in Japan. Bacteriological isolation of EHEC has been a constant problem because the organism is shed for a short duration after onset on illness and generally during the prodromal stages. In order to ease the diagnosis of EHEC infections in this country, we have developed a LPS-based solid phase enzyme linked immunosorbent assay for serological diagnosis of the infection. Adequate knowledge on the prevalence of EHEC serogroups in Japan is mandatory for the success of such a diagnostic test. O157 is currently the prominent serogroup associated with EHEC infections in Japan followed by O111, O26 and O128 in that order of prevalence. In February 1992, a 1 year-old infant developed hemolytic uremic syndrome (HUS) 15 days later the prodrome of watery diarrhoea. Stool sample obtained on day 15 was cultured for detection of EHEC. Ten colonies of E. coli picked from MacConkey agar plate were examined by the polymerase chain reaction (PCR) using primers specific for both verotoxins (VT1 and VT2). Of the 10 colonies, only one was positive for the VT2 gene. The strain was confirmed to be cytotoxic on cultured Vero cells. The strain agglutinated with antisera of E. coli O2, K1 and with H7 serogroups. As of date, none of such strains of O2:K1:H7 EHEC has been reported in Japan. The patient's sera was found to be positive for antibodies against LPS from the homologous isolate as well as against VT2. The patient recovered uneventfully from HUS 40 days after the onset of the disease and was discharged from the hospital.

Effect of glycyrrhizin on lysis of hepatocyte membranes induced by anti-liver cell membrane antibody.

Studies were made on why glycyrrhizin injection decreases the plasma aspartate aminotransferase (AST) and alanine aminotransferase activities in patients with chronic hepatitis. For this, rat hepatocytes were isolated, and incubated with antibody raised against rat liver cell membranes, and the effect of glycyrrhizin on their release of transaminase was investigated. Isolated rat hepatocytes released AST on incubation with anti-liver cell antibody in the presence of complement. At this time, their endogenous phospholipase A2 activity was increased. Cultured hepatocytes also released the transaminase in the presence of venom phospholipase A2. Glycyrrhizin suppressed the release of transaminase in the presence of either anti-liver cell membrane antibody or phospholipase A2. These results suggest that antibody treatment raised the phospholipase A2 activity in liver cell membranes, resulting in release of transaminases, and that glycyrrhizin suppressed this increase in phospholipase A2 activity and so inhibited the release of transaminase.

Simultaneous exposure of sheep to endotoxin and 100% oxygen.

The purpose of this study was to determine the effect of endotoxin on the development of vascular and airway dysfunction during O2 toxicity. Sheep were prepared for chronic measurement of vascular pressures, cardiac output, gas exchange, and collection of lung lymph. Tracheostomies were made for accurate delivery of gas mixtures. Sheep were placed in one of three experimental groups: those receiving endotoxin (n = 9), those breathing 100% O2 and receiving endotoxin (n = 7), and those exposed to 100% O2 alone (n = 6). Sheep had daily measurements of hypoxic vasoconstriction (FIO2 = 0.12), gas exchange, circulating white blood cell counts, lymph flow, and lymph and plasma protein concentrations. Lung neutrophils were counted, and copper-zinc superoxide dismutase and manganous superoxide dismutase were measured in lung samples from some sheep biopsies taken at baseline surgery and postmortem. Endotoxin markedly prolonged survival time and partially protected against the increased lung vascular permeability in sheep breathing 100% oxygen, but impairment of gas exchange, loss of hypoxic pulmonary vasoconstriction, and ultimate progression of respiratory failure were not prevented. Induction of MnSOD occurred in sheep breathing 100% O2, in sheep receiving endotoxin alone, and in those exposed to 100% O2 plus endotoxin. We conclude that endotoxin markedly increases tolerance to O2 toxicity but that some of the pathophysiology of O2 toxicity is unaltered. The role of superoxide dismutase in the observed protection is unclear.

Antioxidant enzymes and lipoperoxide in blood in uremic children and adolescents.

To determine whether oxidant-antioxidant balance is altered in chronic renal failure, antioxidant enzymes and lipid peroxide in peripheral blood cells and lipid peroxide in plasma were measured. Nine children and adolescents maintained on hemodialysis (HD), 9 on continuous ambulatory peritoneal dialysis (CAPD), and 14 controls were studied. Lipid peroxide was assayed fluorimetrically as thiobarbituric acid-reactive substances, superoxide dismutases by radioimmunoassays. Both manganese and copper-zinc superoxide dismutases in lymphocytes and monocytes in the HD and CAPD patients, and manganese superoxide dismutase in polymorphs in the HD patients were higher than in the controls. Copper-zinc superoxide dismutase, glutathione peroxidase, and catalase in erythrocytes were unaltered. The lipid peroxide level in plasma in the dialyzed patients was increased, whereas those in polymorphs and lymphocytes were unaltered. Triglyceride and total cholesterol in plasma in the dialyzed patients were also increased. The plasma lipid peroxide in the patients correlated with the triglyceride and total cholesterol level. This is the first study in which manganese superoxide dismutase is measured in nucleated cells of the patients with chronic renal failure. The present results suggest that increased superoxide dismutases protect against oxidative stress induced by chronic renal failure in nucleated cells but in neither erythrocytes nor plasma.

Structure and organization of Marchantia polymorpha chloroplast genome. II. Gene organization of the large single copy region from rps'12 to atpB.

The nucleotide sequence (56,410 base-pairs) of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha has been determined. The sequence starts from one end (JLA) of the large single-copy region and encompasses genes for 21 tRNAs, six ATPase subunits (atpA, atpB, atpE, atpF, atpH and atpI), two photosystem I polypeptides (psaA and psaB), four photosystem II polypeptides (psbA, psbC, psbD and psbG), five ribosomal proteins (rps2, rps4, rps7, rps'12 and rps14), and three RNA polymerase subunits (rpoB, rpoC1 and rpoC2). In addition, we detected 18 open reading frames ranging from 29 to 2136 amino acid residues long, four of which share significant amino acid sequence homology to those of an Escherichia coli malK protein (designated mbpX), human mitochondrial ND2 (ndh2) and ND3 (ndh3) of a respiratory chain NADH dehydrogenase, or a bacterial antenna protein of a light-harvesting complex (lhcA). Sequence analysis suggests that four tRNA genes and six protein genes might be split by introns; they are trnG(UCC), trnK(UUU), trnL(UAA), trnV(UAC), atpF, ndh2, rpoC1, rps'12, ORF135 and ORF167. In the large single-copy region described here, the gene organization deduced is highly conserved with respect to that of higher plants, but an inversion of some 30,000 base-pairs flanked by trnL(CAA) and trnD(GUC) was seen between the liverwort and tobacco chloroplast genomes.

Structure and organization of Marchantia polymorpha chloroplast genome. I. Cloning and gene identification.

We have determined the complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha, using a clone bank of chloroplast DNA fragments. The circular genome consists of 121,024 base-pairs and includes two large inverted repeats (IRA and IRB, each 10,058 base-pairs), a large single-copy region (LSC, 81,095 base-pairs), and a small single-copy region (SSC, 19,813 base-pairs). The nucleotide sequence was analysed with a computer to deduce the entire gene organization, assuming the universal genetic code and the presence of introns in the coding sequences. We detected 136 possible genes. 103 gene products of which are related to known stable RNA or protein molecules. Stable RNA genes for four species of ribosomal RNA and 32 species of tRNA were located, although one of the tRNA genes may be defective. Twenty genes encoding polypeptides involved in photosynthesis and electron transport were identified by comparison with known chloroplast genes. Twenty-five open reading frames (ORFs) show structural similarities to Escherichia coli RNA polymerase subunits, 19 ribosomal proteins and two related proteins. Seven ORFs are comparable with human mitochondrial NADH dehydrogenase genes. A computer-aided homology search predicted possible chloroplast homologues of bacterial proteins; two ORFs for bacterial 4Fe-4S-type ferredoxin, two for distinct subunits of a protein-dependent transport system, one ORF for a component of nitrogenase, and one for an antenna protein of a light-harvesting complex. The other 33 ORFs, consisting of 29 to 2136 codons, remain to be identified, but some of them seem to be conserved in evolution. Detailed information on gene identification is presented in the accompanying papers. We postulated that there were 22 introns in 20 genes (8 tRNA genes and 12 ORFs), which may be classified into the groups I and II found in fungal mitochondrial genes. The structural gene for ribosomal protein S12 is trans-split on the opposite DNA strand. The universal genetic code was confirmed by the substitution pattern of simultaneous codons, and by possible codon recognition of the chloroplast-encoded tRNA molecules, assuming no importation of tRNA molecules from the cytoplasm. The nucleotide residue A or T is preferred at the third position of the codons (G+C, 11.9%) and in intergenic spacers (G+C, 19.5%), resulting in an overall G+C content that is low (28.8%) throughout the liverwort chloroplast genome. Possible gene expression signals such as promoters and terminators for transcription, predicted locations of gene products, and DNA replicative origins are discussed.

Effect of a new synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor on cholesterol synthesis and low density lipoprotein uptake by primary cultures of rat hepatocytes.

CS-514 ((+)-sodium (3R,5R)-3,5-dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-2- methyl-8-[(S)-2-methyl-butyryl]-1,2,6,7,8-hexahydro-1-naphthyl] heptanoate) which was recently synthesized as an inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase lowers serum cholesterol levels. This compound inhibited cholesterol synthesis dose-dependently from acetate in primary cultures of rat hepatocytes. At high concentration (0.5 and 5.0 mumol/l), but not at lower concentration, it inhibited bile formation from acetate. Low density lipoprotein (LDL)-[3H]-cholesteryl linoleate incorporation into hepatocytes was increased when the cells were preincubated with 0.5 or 5.0 mumol/l CS-514 for 12 h. Bile formation from LDL-[3H]-cholesterol linoleate was not affected by addition of CS-514. These results suggest that inhibition of de novo cholesterol synthesis by CS-514 enhanced LDL receptor function in primary cultures of rat hepatocytes and lowered LDL-cholesterol level.

Endotoxin increases superoxide dismutase in cultured bovine pulmonary endothelial cells.

Manganous (Mn) and copper zinc (CuZn) superoxide dismutase (SOD) concentrations and glutathione peroxidase (GSH-Px) and catalase (CAT) activities were measured in cultured bovine pulmonary endothelial cells with and without exposure to Escherichia coli endotoxin (10(-1) micrograms/ml) over intervals of 0.5-24 h. The activities of two mitochondrial marker enzymes, fumarase and cytochrome-c oxidase, were also measured. Endotoxin exposure caused a marked increase (9-fold) in endothelial cell Mn SOD content without significant effects on GSH-Px, CAT, fumarase, or cytochrome-c oxidase activities. Endotoxin induced a slight decrease in CuZn SOD content over 24 h. This is the first report of a selective effect of endotoxin on Mn SOD in pulmonary endothelial cells. The response appears to be independent of an increase in mitochondrial activity (no change was observed in cytochrome-c oxidase or fumarase activities). These findings support the notion that endotoxin increases generation of toxic oxygen metabolites within pulmonary endothelial cells. An endotoxin-induced increase in Mn SOD could contribute to the reported protective effect of endotoxin against oxygen toxicity in these cells.

Cytotoxic effects of paraquat and inhibition of them by vitamin E.

Paraquat causes failure of multiple organs including the liver in humans. The kinetics and mechanism of paraquat intoxication were studied using cultured rat hepatocytes. Paraquat induced time- and dose-dependent lactate dehydrogenase release, lipid peroxidation, and cell death, estimated as decrease in protein in cells attached to culture dishes. However, the increase in lipid peroxidation occurred after lactate dehydrogenase release had reached a plateau. Vitamin E inhibited the inductions of all these cytotoxic effects of paraquat. Kinetic studies showed that lipid peroxidation was a better indicator of cell death than lactate dehydrogenase release, because vitamin E inhibited the induction of cell death even when added 6 h after paraquat, when lactate dehydrogenase release had reached a plateau but lipid peroxidation had not. The present results strongly suggest that paraquat exerts its cytotoxicity by a mechanism involving oxidation reactions.

[A study on the changes with age in thyroid function during infancy, childhood and adolescence].

Serum total thyroxine (t-T4), free thyroxine (f-T4), total triiodothyronine (t-T3), free triiodothyronine (f-T3), reverse triiodothyronine (r-T3) and thyroxine-binding globulin (TBG) levels were measured by radioimmunoassay in pregnants, infants, children and adolescents. The mean t-T4 and TBG concentrations in cord blood (9.0 +/- 0.3 micrograms/dl, 24.4 +/- 1.1 micrograms/ml, mean +/- SE) were significantly lower than the levels in maternal blood (12.1 +/- 0.6 micrograms/dl, 40.3 +/- 2.3 micrograms/ml), whereas mean f-T4 concentrations in cord blood (1.13 +/- 0.04 micrograms/dl) were significantly higher than the values in maternal blood (0.86 +/- 0.03 micrograms/ml). The mean t-T3 and f-T3 concentrations in cord blood (48 +/- 2 ng/dl, 1.1 +/- 0.1 pg/ml) were much lower than the levels in maternal blood (136 +/- 5 ng/dl, 2.6 +/- 0.1 pg/ml). The r-T3 concentrations in cord blood (192 +/- 4 ng/dl) were markedly higher than the maternal levels (46.6 +/- 3.7 ng/dl). These results suggest that in the fetus the conversion of T4 to r-T3 was greater than that of T4 to T3 due to a greater ratio of activity of 5-monodeiodinase to that of 5'-monodeiodinase. In relation to the changes in thyroid function after birth, t-T4 and f-T4 values showed parallel changes while t-T3 and f-T3 levels increased rapidly during the first month and t-T3 levels remained unchanged between 1 and 11 months, whereas f-T3 levels and f-T3/t-T3 ratio increased gradually during the same time. T-T3 levels in children 1 to 15 years of age were slightly lower than the levels in infants 1 to 11 months. R-T3 values decreased progressively from 5 days to 1 approximately 3 months and continued to decline until 4 years of age. Values between 4 and 15 years were not changed. T-T3/t-T4 ratio increased markedly 1 approximately 3 months after birth. This indicates that the activity of 5'-monodeiodinase was elevated rapidly after birth and suggests that the rapid increase of T3 levels after birth was due to enhanced enzymatic activity as well as higher TSH levels. R-T3/t-T4 ratio decreased rapidly after birth and continued to decrease during years 1 approximately 3. This indicates that the change of activity of 5-monodeiodinase was different from that of 5'-monodeiodinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous quantitative measurement of fourteen adrenal steroids by capillary column gas chromatography-mass spectrometry, and its clinical application.

Until now, there has been little work covering all of the main native adrenal-cortical steroids in blood. We therefore established a method for the simultaneous quantitative measurement of 14 native adrenal-cortical steroids, which involves capillary column gas chromatography-mass spectrometry (GC--MS). Serum steroids were purified from serum with the Extrelut mini-column and then converted into stable derivatives for GC-MS by a combination of boronic cyclization and trimethylsilyl and methyloxime derivatization. The sensitivities (with a signal-to-noise ratio greater than or equal to 7) of our GC-MS method ranged from 0.1 to 1.0 ng/ml of serum, and the coefficients of variation of intra- and inter-assays were less than 19% for each steroid. Our newly devised method involving a capillary column GC-MS system has been proven to be a simple and suitable method for a diagnosis requiring simultaneous detection of many native adrenal steroids in clinical practice. The analysis time is only 4 h.

Effect of glycyrrhizin on stability of lysosomes in the rat arterial wall.

Studies were made on the effect of phospholipase A2 on the stability of arterial wall lysosomes, and the influence of glycyrrhizin on this effect. Lysosomal phospholipase A2 activity was found to be increased in the aorta of hypercholesterolemic rats. Treatment of the lysosomal fraction of the arterial wall with venom phospholipase A2 resulted in release of acid phosphatase, and addition of glycyrrhizin (0.1 mM) inhibited this release. Lysosomal phospholipase A2 in the arterial wall was also inhibited dose-dependently by glycyrrhizin. These results suggest that lysis of lysosomes was due to increase in phospholipase A2 activity, and that glycyrrhizin stabilized the lysosomes by inhibiting phospholipase A2 activity.

Effect of glycyrrhizin on lysosomes labilization by phospholipase A2.

The influence of glycyrrhizin on the effect of phospholipase A2 on lysosomes was studied. Treatment of rat liver lysosomes with venom phospholipase A2 caused release of acid phosphatase. This release of acid phosphatase was inhibited by 0.1 mM glycyrrhizin. Glycyrrhizin also inhibited acid phospholipase A2 with pH optimum of 4.5, which is thought to be present in the lysosomal membrane. These results suggest that glycyrrhizin stabilizes lysosomes by inhibiting phospholipase A2 activity in the lysosomal membrane.