The antimalarial activities of crude extracts and 17 fractions from the partition of 80%-methanolic extracts of three plants (the stem bark of Croton mubango, the stem bark of Nauclea pobeguinii and the leaves of Pyrenacantha staudtii) used as antimalarial remedies in the Democratic Republic of Congo were studied both in vitro (against Plasmodium falciparum) and in mice infected with Pl. berghei berghei. The toxic effects of dried aqueous extracts of the plants were also investigated, in uninfected mice. The most active crude extracts in vitro, with median inhibitory concentrations (IC(50)) of <1 microg/ml, were found to be the methanolic and dichloromethane extracts of C. mubango, and the dichloromethane extracts of N. pobeguinii and Py. staudtii. The aqueous extract with the most antimalarial activity in vitro was that of C. mubango (IC(50) = 3.2 microg/ml), followed by that of N. probeguinii (IC(50) = 5.3 microg/ml) and then that of Py. staudii (IC(50) = 15.2 microg/ml). Results from the in-vivo tests of antimalarial activity showed that, at a daily oral dose of 200 mg/kg, all the dichloromethane extracts, the petroleum-ether, chloroformic, ethyl-acetate and residual water-soluble fractions from C. mubango, and the chloroformic, ethyl-acetate and n-butanolic fractions from Py. staudtii produced >80% chemosuppression of the parasitaemias by day 4. The aqueous extracts of C. mubango and N. probeguinii produced a slightly lower but still significant inhibition of parasitaemia (60%-80%) whereas that of Py. staudtii only suppressed the day-4 parasitaemias by 37%. The dried aqueous extract of the stem bark of C. mubango showed some signs of toxicity in mice, with median lethal doses (LD(50)) of 350 mg/kg in the female mice and 900 mg/kg in the male. The extract significantly increased the serum concentrations of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in mice of both sexes, but had no effect on the blood levels of creatinine or urea. No significant toxic effect was observed for the dried aqueous extracts of N. pobeguinii and Py. staudtii (LD(50) >5 g/kg). Neither of these extracts affected the serum concentrations of GPT or the blood concentrations of creatinine and urea, although the N. pobeguinii extract did increase the serum concentration of GOT.

Models of feeding and digestion predict that increased body size should result in longer gut passage time and improved assimilation efficiency. We examined the implications of digestion theory for size-structured interactions in a generalist zooplankton herbivore by studying the relationships between body size, ingestion rate, gut passage time (GPT), assimilation efficiency (AE), and growth rate in a clone of Daphnia pulex feeding on seven taxa of green algae that differed in digestibility. We also tested the effect of varying food concentration on GPT and AE while keeping body size constant. Food quality varied markedly among algal taxa, with mean juvenile growth rates at high food concentrations (1-2 mg/L) ranging from 0.10 to 0.61 d(-1). Juvenile growth rate for high food concentrations was highly correlated with juvenile AE (r2 = 0.96), verifying the importance of digestibility for food quality. AE, measured with 14C-labeled algae, increased with increasing age and body size for each of four digestion-resistant taxa but did not vary with age and body size for three readily digested algae. GPT decreased with decreasing body size, supporting the hypothesis that shorter GPT in juveniles leads to lower AE for digestion-resistant resources. Lower food concentrations led to increased GPT and improved AE for juveniles feeding on two digestion-resistant algae, providing further support for a role of longer gut retention in overcoming digestion defenses. The results suggest that increased abundance of digestion-resistant food will lead to growth and recruitment bottlenecks for juvenile herbivores, but that the effectiveness of digestion defenses will be decreased when large-bodied grazers predominate and when low food concentrations result in longer gut passage times. Gut processing constraints may favor either high concentrations of slow-growing, digestion-resistant resources or low concentrations of fast-growing, undefended resources.

Expression of the purE operon of Salmonella typhimurium was analyzed by using an Escherichia coli F' episome containing a purE-lac fusion. The fusion removes the lacOP and part of the lacZ genes of the lac operon and places the intact lacY and lacA genes under control of the purE operon as shown by inhibition of growth on melibiose (lacY) and repression of thiogalactoside transacetylase (lacA) by various purines. Two classes of regulatory-deficient mutants were found among those resistant to inhibition by purines. One class was trans active (chromosomal) and corresponded to previously described purR mutants involving a deficient cytoplasmic repressor substance. These were also altered in the expression of the purF, purD, purG amd purI genes as evidenced by loss of repressibility of the synthesis of glycinamide ribotide and aminoimidazole ribotide. The other class was cis active (episomal), specific for only purE expression, and thus corresponded to an altered purE operon signal (operator or promoter). The metabolic requirements for the expression of purE were also monitored by measuring repression of the transacetylase in strains with various genetically altered metabolic backgrounds. Repression by guanine required an intact guanine phosphorbosyltransferase (gpt) and repression by adenine and all nucleosides required purine nucleoside phosphorylase (deoD). Synthesis of cyclic AMP (cya) and its receptor protein (crp) were no longer required for the expression of the lac genes under purE control.

Eucaryotic expression vectors containing two selective markers, the herpes simplex 1 thymidine kinase gene (tk) and the Escherichia coli gpt gene (Eco gpt) coding for a xanthine-guanine phosphoribosyl-transferase ( XGPRT ) were constructed. These plasmids were used to transfect mouse Ltk- cells followed by selection of either tk+ or XGPRT + colonies. The transcription and maturation of the Eco gpt mRNA is dependent on the presence of a eucaryotic promoter sequence at its 5' end and on the presence of a viral intron and a poly(A) addition site at its 3' end ( Mulligan and Berg, 1980). Here, we report that both simian virus 40 (SV40) and polyoma early and late promoters permit the transcription of this gene integrated into the cellular genome. Polyoma DNA fragments lacking the TATA box, or both the TATA and CAAT boxes directing early transcription, efficiently promote Eco gpt expression. Furthermore, a fragment terminating approximately 300 nucleotides upstream of the initiation site of early RNA permits Eco gpt synthesis when the early strand is joined to the Eco gpt-coding strand. The SV40 early promoter is 2- to 3-fold more efficient than the controlling sequence of late transcription. These results strongly suggest that the switch from a predominance of early RNA to that of late RNA occurring after the onset of DNA replication is caused by the increase in the abundance of template and by the concomitant repression of early transcription by the T antigen. The presence of the tk gene in all the plasmids constructed permits the analysis of Eco gpt expression as a non-selected marker in tk+ clones selected for growth in HAT medium.(ABSTRACT TRUNCATED AT 250 WORDS)

A general method is described for altering specific genes of vaccinia virus (VV). We demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (PCR). Primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. Linear PCR constructs are transfected into cells infected with wild-type vaccinia virus. Mutant viruses with gpt inserted into the target gene by homologous recombination are then selected by growth in the presence of MPA. This technique was applied to the vaccinia virus thymidine kinase gene and compared to the traditional method of constructing gpt-containing plasmids by cloning. The PCR scheme was found to be highly efficient and could theoretically be used to insert any foreign DNA element into any nonessential target gene for which partial or complete sequence information is available. The procedure can potentially be used for a wide variety of genetic modifications, including the insertion of foreign genes, with poxviruses and other DNA viruses. Genomes of microorganisms, such as bacteria and yeast that can be transformed with linear DNA, are also candidates for manipulation by this methodology.

Long-range-acting gene activator elements were randomly isolated from the human genome by functional selection. HeLa cells were transfected with an enhancer trap, a plasmid containing an enhancerless xanthine-guanosine phosphoribosyltransferase (gpt) gene transcribed from the simian virus 40 early promoter, and stably transformed GPT+ cells were selected. From several transformants, human DNA sequences flanking the enhancer trap were cloned. Two gene activators (GA1 and GA2) were found in the cloned human DNAs. GA1 and GA2 showed strong enhancer activity both in a stable transformation assay and in a transient expression assay. They had functional properties similar to those of other known enhancers: GA1 and GA2 activated the expression of a linked gene over distances of at least 5 kilobases both upstream and downstream in an orientation-independent fashion. GA1 may be required for the initial establishment of gene activation but was not essential for the maintenance of active expression. GA1 and GA2 were active not only in HeLa cells but also in other types of human cells, such as neuroblastoma cells. This indicates a limited but relatively broad cell type specificity. The HeLa genome contains multiple copies of GA1, while GA2 exists once in the genome.

A recombinant shuttle vector containing the entire bovine papillomavirus (BPV) genome, sequences from pBR322, and the Escherichia coli gpt gene was used to transform mouse C127 cells. Plasmid extracted from the transformed mouse cells was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. Approximate mutant frequencies of 3-16 X 10(-3) were observed for plasmid molecules which had been passaged through the mammalian cells. Restriction digest analysis indicated that most of the mutant plasmid molecules had gross rearrangements in their DNA structures.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We investigated the localization and role of multidrug-resistance-associated protein (Mrp) 2, a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress. Normal Sprague-Dawley rat (SDR) and Mrp2-deficient Eisai hyperbilirubinemic rat (EHBR) livers were perfused with 500 microM EA. The release of glutamic pyruvic transaminase (GPT) and thiobarbituric-acid-reactive substances (TBARS) from EHBR liver was markedly delayed compared with that from SDR liver. This is mainly due to the higher basal level of glutathione (GSH) in EHBR liver (59.1 +/- 0.3 nmol/mg protein) compared with SDR liver (39.7 +/- 1.5 nmol/mg protein). EA similarly induced a rapid reduction in GSH followed by mitochondrial permeability transition in the isolated mitochondria from both SDR and EHBR. Internalization of Mrp2 was detected before nonspecific disruption of the canalicular membrane and GPT release in SDR liver perfused with 100 microM EA. SDR liver preperfused with hyperosmolar buffer (405 mosmol/L) for 30 min induced internalization of Mrp2 without changing the basal GSH level, while elimination of hepatic GSH by 300 microM EA perfusion was significantly delayed thereafter. Concomitantly, hepatotoxicity assessed by the release of GPT and TBARS was also significantly attenuated under hyperosmolar conditions. In conclusion, preserved cytosolic and intramitochondrial GSH is the key factor involved in the acute hepatotoxicity induced by EA and its susceptibility could be altered by the presence of Mrp2.

Clarifying the participation of oxidative stress among possible contributing factors in potassium bromate (KBrO(3))-induced carcinogenesis is of importance from the perspective of human health protection. In the present study, utilizing the antioxidative effects of alpha-tocopherol (alpha-TP) or sodium ascorbic acid (SAA) to attenuate oxidative stress, alterations in bromodeoxyuridine labeling indices (BrdU-LIs) and reporter gene mutations in kidneys of male and female gpt delta rats given KBrO(3) were examined. Five male and female gpt delta rats in each group were given KBrO(3) at a concentration of 500ppm in the drinking water for 9 weeks, with 1% of alpha-TP or SAA administered in the diet from 1 week prior to the KBrO(3) treatment until the end of the experiment. Increases in 8-hydroxydeoxyguanosine levels in kidney DNA of both sexes of rats given KBrO(3) were significantly inhibited by SAA, but not alpha-TP. While BrdU-LIs in the proximal tubules of female rats were also significantly reduced by SAA, those in the males and gpt mutant frequencies in kidney DNA of both sexes were not affected by SAA or alpha-TP. Immunohistochemical and Western blot analyses for alpha(2u)-globulin strongly suggested that induction of cell proliferation observed in the males might primarily result from accumulation of this protein, independent of oxidative stress. The overall data indicated that while oxidative stress well correlates with induction of cell proliferation in females, its role in males and in generation of in vivo mutagenicity by KBrO(3) in both sexes is limited.

In order to evaluate the ability of an exogenous tissue-specific promoter to undergo the same dynamic changes in activity as the endogenous one, a 400-base pair fragment of the rat albumin proximal promoter, upstream of the bacterial gpt gene, has been introduced into rat hepatoma cells. Four clones containing a single integrated copy of the construct and producing substantial amounts of albumin and of xanthine phosphoribosyltransferase were isolated. These clones were subjected to two treatments known to result in silencing of the albumin gene: selection for dedifferentiated variants, and fusion with L-cell fibroblasts. In most cases, the albumin-negative progeny obtained no longer expressed the gpt gene: the exogenous promoter of 400 base pairs must contain the sequences required to respond to the mechanisms that block activity of the endogenous gene. However, exceptions were observed: the albumin-deficient variants of one clone remained xanthine phosphoribosyltransferase positive, and some of the albumin-negative hybrids from a different clone continued to produce xanthine phosphoribosyltransferase. These cases of dissociation in expression of the endogenous and the exogenous genes indicate that the site of integration of the alb-gpt construct in one clone renders the sequences insensitive to the mechanisms responsible for albumin gene silencing in dedifferentiated variants, and in the other clone to the mechanism of extinction. Consequently, the mechanisms causing gene silencing in variants and in intertypic hybrids must be different.

The complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. Isolated peptides were analyzed with a protein sequencer or with a plasma desorption time of flight mass spectrometer and placed in the sequence on the basis of their molecular mass and homology to the sequence of rat GPT. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The Mr of the subunit was calculated to be 54,479, which was in good agreement with a Mr of 55,000 estimated by SDS-PAGE, and also indicated that the active enzyme with a Mr of 114,000 was a homodimer composed of two identical subunits. The amino acid sequence is highly homologous to that of rat GPT (87.9% identity) recently determined [Ishiguro, M., Suzuki, M., Takio, K., Matsuzawa, T., & Titani, K. (1991) Biochemistry 30, 6048-6053]. All of the crucial amino acid residues are conserved in human GPT, which seem to be hydrogen bonding to pyridoxal 5'-phosphate in rat GPT by the sequence homology to other alpha-aminotransferases with known tertiary structures.

OBJECTIVE

Impaired liver function is frequently found in patients who need prolonged total parenteral nutrition. Cyclic total parenteral nutrition can minimize the adverse effects of long-term total parenteral nutrition, such as hepatic complication. The adequate timing to shift to use cyclic total parenteral nutrition for patients with impaired liver function may prevent further hepatic dysfunction.

METHODS

A prospective study of 65 patients who need total parenteral nutrition and have impaired liver functions was performed. Cyclic total parenteral nutrition was used in different groups of patients, when their total bilirubin levels were just over 5 mg%, 10 mg%, or 20 mg% during the course of total parenteral nutrition. The patients of control groups received straight non-cyclic total parenteral nutrition. All the patients had stable vital signs without major stress, such as sepsis or acute bleeding. Ten patients (A2) in Group A were shifted to cyclic total parenteral nutrition when their total bilirubin was just over 5 mg%; the other 10 patients (A1) continued the non-cyclic total parenteral nutrition. Eleven patients (B2) in Group B were shifted to cyclic total parenteral nutrition when their total bilirubin was just over 10 mg%; the other 11 patients (B1) continued the non-cyclic total parenteral nutrition. Ten patients (C2) in Group C were shifted to cyclic total parenteral nutrition when their total bilirubin was just over 20 mg%; the other 13 patients (C1) continued the non-cyclic total parenteral nutrition. The average energy intake among 3 groups had no difference. Their liver functions were examined each week for 2 weeks.

RESULTS

The results showed that the patients with non-cyclic total parenteral nutrition had significant increase of direct-total bilirubin and alkaline phosphatase (P < 0.05) in Group A and significant decrease of albumin accompanied with increase of GOT, GPT, direct/total bilirubin (P < 0.05) in Group B. The patients either using cyclic or non-cyclic total parenteral nutrition showed significant decrease of albumin and increase of direct/total bilirubin (P < 0.05) in Group C.

CONCLUSIONS

We conclude that the early use of cyclic total parenteral nutrition may prevent deterioration of liver function for the patients with jaundice and need prolonged total parenteral nutrition.

99mTc-DTPA-galactosyl human serum albumin (99mTc-GSA) liver scintigraphy was performed in 230 patients with chronic active hepatitis type C, and its quantitative indices were compared with histological findings. 99mTc-GSA findings correlated well with four indices of the histology activity index (HAI), especially with the fibrosis score. Ninety patients were given interferon treatments, and 99mTc-GSA findings were compared with the results of the treatments. We classified the effects of interferon treatment into three groups according to clinical outcome: group 1: good effect (HCV-RNA negative, n = 34), group 2: moderate effect (HCV-RNA positive, but the value of GPT was normal for six months after the end of treatment, n = 19) and group 3: no effect (n = 37). Quantitative indices of 99mTc-GSA showed significant differences between groups. Follow-up study with 99mTc-GSA scintigrams was obtained in eight patients. The results of 99mTc-GSA improved in three patients in group 1 and deteriorated in five patients in group 3. There is a possibility that 99mTc-GSA scintigraphy can be used to predict the clinical outcome of chronic active hepatitis type C after interferon treatment.

Traumatic brain injury (TBI) and stroke lead to elevated levels of glutamate in the brain that negatively affect the neurological outcomes in both animals and humans. Intravenous administration of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) enzymes can be used to lower the blood glutamate levels and to improve the neurological outcome following TBI and stroke. The objective of this study was to analyze the pharmacokinetics and to determine the glutamate-lowering effects of GOT and GPT enzymes in naïve rats. We determined the time course of serum GOT, GPT, and glutamate levels following a single intravenous administration of two different doses of each one of the studied enzymes. Forty-six male rats were randomly assigned into one of 5 treatment groups: saline (control), human GOT at dose 0.03 and 0.06 mg/kg and porcine GPT at dose 0.6 and 1.2 mg/kg. Blood samples were collected at baseline, 5 min, and 2, 4, 8, 12, and 24 h after the drug injection and GOT, GPT and glutamate levels were determined. The pharmacokinetics of both GOT and GPT followed one-compartment model, and both enzymes exhibited substantial glutamate-lowering effects following intravenous administration. Analysis of the pharmacokinetic data indicated that both enzymes were distributed predominantly in the blood (central circulation) and did not permeate to the peripheral organs and tissues. Several-hour delay was present between the time course of the enzyme levels and the glutamate-lowering effects (leading to clock-wise hysteresis on concentration-effect curves), apparently due to the time that is required to affect the pool of serum glutamate. We conclude that the interaction between the systemically-administered enzymes (GOT and GPT) and the glutamate takes place in the central circulation. Thus, glutamate-lowering effects of GOT and GPT apparently lead to redistribution of the excess glutamate from the brain's extracellular fluid into the blood and can reduce secondary brain injury due to glutamate neurotoxicity. The outcomes of this study regarding the pharmacokinetic and pharmacodynamic properties of the GOT and GPT enzymes will be subsequently verified in clinical studies that can lead to design of effective neuroprotective treatment strategies in patients with traumatic brain diseases and stroke.

Recombination values were used to calculate the gene-centromere map distances for four electrophoretically detected loci, Aat3, Idh1, Idh4, and Mpi, in chum salmon (Oncorhynchus keta). We also report the results from 39 pairwise examinations for joint segregation for 10 loci in nine testcross families. Only two loci assorted nonrandomly--either Aat1 or Aat2 with Gpt. Gene-centromere distances for Aat3 and Mpi differed significantly from those reported previously for rainbow trout (Salmo gairdneri), a closely related species. This difference indicates either the presence of chromosome rearrangements or a different rate of recombination between the species. These results contrast with the conservation of linkage distances previously reported within and between other salmonid genera.

Manganese superoxide dismutase (MnSOD) has been found to be depleted in a variety of tumor cells as well as in in vitro transformed cell lines, suggesting that MnSOD may function as an anticarcinogen by protecting the cell from oxidant-induced carcinogenesis. The relationship between MnSOD expression and tumor promotion was studied by transfection of a human MnSOD cDNA into the promotable mouse epidermal cell line JB6 clone41. The effect of MnSOD overexpression on the promotion-sensitive phenotype of JB6 cells was assessed by measuring growth characteristics such as growth rate and the ability to form colonies in soft agar. Compared with the parental and vector-transfected (gpt) control cells, MnSOD-overexpressing cells had a slower growth rate and their ability to form colonies in soft agar was significantly decreased in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Since the transformation-sensitive phenotype of JB6 clone41 cells is associated with increased expression of the transcription factor AP-1, we compared c-jun and c-fos mRNA expression in MnSOD-transfected and control JB6 cells. Overexpression of MnSOD led to a significant decrease in c-jun and c-fos expression in response to treatment with TPA or the oxidant promoter superoxide. These findings indicate that the promotion-sensitive phenotype of JB6 clone41 cells can be reverted by increasing MnSOD intracellularly. A possible mechanism is that elevated MnSOD expression might change the intracellular redox state by altering the balance of reactive oxygen species. This could lead to a modulation of TPA and oxidant-induced signal transduction pathways controlling cell growth and differentiation.

In roots, nitrate assimilation is dependent upon a supply of reductant that is initially generated by oxidative metabolism including the pentose phosphate pathway (OPPP). The uptake of nitrite into the plastids and its subsequent reduction by nitrite reductase (NiR) and glutamate synthase (GOGAT) are potentially important control points that may affect nitrate assimilation. To support the operation of the OPPP there is a need for glucose 6-phosphate (Glc6P) to be imported into the plastids by the glucose phosphate translocator (GPT). Competitive inhibitors of Glc6P uptake had little impact on the rate of Glc6P-dependent nitrite reduction. Nitrite uptake into plastids, using (13)N labelled nitrite, was shown to be by passive diffusion. Flux through the OPPP during nitrite reduction and glutamate synthesis in purified plastids was followed by monitoring the release of (14)CO(2) from [1-(14)C]-Glc6P. The results suggest that the flux through the OPPP is maximal when NiR operates at maximal capacity and could not respond further to the increased demand for reductant caused by the concurrent operation of NiR and GOGAT. Simultaneous nitrite reduction and glutamate synthesis resulted in decreased rates of both enzymatic reactions. The enzyme activity of glucose 6-phosphate dehydrogenase (G6PDH), the enzyme supporting the first step of the OPPP, was induced by external nitrate supply. The maximum catalytic activity of G6PDH was determined to be more than sufficient to support the reductant requirements of both NiR and GOGAT. These data are discussed in terms of competition between NiR and GOGAT for the provision of reductant generated by the OPPP.

Investigation on the toxic effects of pharmaceutical drugs namely clofibric acid (CA) and diclofenac (DCF) were studied in a common carp Cyprinus carpio at different concentrations such as 1, 10 and 100 μg L(-1) for a short-term period of 96 h under static bioassay method. At all concentrations, red blood cell (RBC), plasma sodium (Na(+)), potassium (K(+)), and glutamate oxaloacetate transaminase (GOT) levels were decreased in fish treated with CA and DCF. Contrastingly, white blood cell (WBC), plasma glucose, protein, lactate dehydrogenase (LDH) and gill Na(+)/K(+)-ATPase level were increased. However, a mixed trend was observed in hemoglobin (Hb), hematocrit (Hct), plasma chloride (Cl(-)), mean cellular volume (MCV), mean cellular hemoglobin (MCH), mean cellular hemoglobin concentration (MCHC) and glutamate pyruvate transaminase (GPT) levels. There was a significant (P<0.01 and P<0.05) change in all parameters measured in fish exposed to different concentrations of CA and DCF. In summary, the alterations in hematological, biochemical, ionoregulatory and enzymological parameters can be used as biomarkers in monitoring the toxicity of CA and DCF in aquatic environment. However, more detailed studies on using of specific biomarkers to monitor the human pharmaceuticals are needed.

OBJECTIVE

To investigate the similarities and dissimilarities in patients with hepatitis B and hepatitis C, clinically and metabolically.

METHODS

Fifty patients with hepatitis B virus and hepatitis C virus infection were included in this study, along with fifty healthy controls for comparison purposes. Intravenous blood (10 mL) samples from patients and healthy subjects were collected and made to clot before serum was separated and immediately levels of the enzymes, alkaline phosphatase (ALK), creatinine phosphokinase (CPK), lactate dehydrogenase (LDH), serum glutamate oxaloacetate transaminase (s-GOT) and serum glutamate pyruvate transaminase (s-GPT) were determined by a kit method. For total content of each metal the serum samples were analyzed using atomic absorption spectrophotometry. Levels of cholesterol, triglycerides, urea, creatinine and uric acid were determined using a kit method on Microlab 300.

RESULTS

Serum magnesium and copper levels remained unchanged, whereas the concentration of zinc decreased and iron increased significantly in both groups of patients. Total antioxidant activity was significantly decreased in both hepatitis B and C. Among the enzymes analyzed, ALK, s-GPT, LDH and s-GOT were all significantly increased in both patients with hepatitis B and C whereas CPK was significantly decreased in patients with hepatitis B and remained unchanged in patients with hepatitis C.

CONCLUSIONS

The information accumulated by this study will help provide a better understanding of involved metabolic processes in order to design appropriate therapeutic approaches for treating these patients, so they can recover and lead normal lives.

BACKGROUND

Rhabdomyolysis is one of the causes of acute renal failure. Erythropoietin (EPO) has been found to interact with its receptor (EPO-R) expressed in a large variety of non-haematopoietic tissues to induce a range of pleiotropic cytoprotective actions. In this study, we used recombinant human erythropoietin (rhEPO) to study the effects on the glycerol-induced rhabdomyolysis with acute renal failure in rats.

METHODS

Twenty-four rats were divided into three groups as glycerol group, glycerol+EPO group and normal saline+EPO group. Rhabdomyolysis was induced by intramuscular injection of 10 mlkg(-1) 50% glycerol in rats. Ten minutes later, the rats received an intravenous injection of rhEPO (300 Ukg(-1)). Biochemical substances, including haemoglobin, blood urea nitrogen (BUN), creatinine (Cre), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and creatine phosphokinase (CPK), were measured at 0, 1, 3, 6, 9, 12, 18, 24 and 48 h. Rats were sacrificed 48 h later after glycerol administration and the kidneys were removed immediately for pathology and immunohistochemistry (IHC).

RESULTS

Intramuscular injection of glycerol significantly increased blood BUN, Cre, GOT, GPT and CPK levels and induced severe histopathologic damage in the kidneys. Nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) were increased and E-cadherin was decreased after glycerol administration, as detected by IHC in the kidneys. Post-treatment with rhEPO decreased blood BUN, Cre, GOT, GPT and CPK levels, decreased markers of kidney injury and suppressed the release of NF-κB and iNOS after rhabdomyolysis.

CONCLUSIONS

Treatment with rhEPO suppressed the activities of NF-κB and iNOS, decreased BUN, Cre, GOT, GPT and CPK levels, and decreased the markers of kidney injury after rhabdomyolysis. These actions ameliorated rhabdomyolysis-induced acute renal failure in rats.

Ethanol at initial concentrations between 0.75 and 6 g/l produced a dose-dependent release of the enzymes glutamic-pyruvic-transaminase and sorbitol dehydrogenase (GPT, SDH) from the isolated perfused rat liver. At the concentration of 6 g/l, it also decreased the oxygen consumption and elevated the calcium content of the isolated livers. These toxic effects of ethanol were significantly enhanced in livers, the glutathione content of which had been depleted by pretreatment with phorone. Ethanol-induced toxicity in glutathione-depleted isolated livers could be prevented both by inhibition of alcohol dehydrogenase with 4-methylpyrazole and of xanthine oxidase with allopurinol. In rats, in vivo, 1.6 g/kg ethanol injected intravenously produced a small increase in serum GPT and SDH concentrations 4 h after its administration. This increase in enzyme activities was several-fold higher and longer lasting in rats pretreated with phorone. Glutathione depletion per se did not induce hepatotoxicity in vitro or in vivo. Since glutathione is involved in several lines of defense against oxidative damage, our results of an enhanced susceptibility of glutathione-depleted livers to ethanol toxicity favour the hypothesis that ethanol exerts its hepatotoxic action via an activation of molecular oxygen.

An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil. Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E. coli gpt gene was compared to that in the genome overall. A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E. coli gpt genes. The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease. Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA. The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene.

A novel calcium-binding protein regucalcin has been shown to be specifically expressed in the liver of various specifies including human. Regucalcin concentration in the serum of patients with chronic liver injury was estimated by enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG. Serum samples were obtained from 42 persons who were diagnosed as liver disorder. Serum regucalcin concentration in all patients was in the range of 3.7-69.6 ng/ml, although regucalcin was not entirely seen in the serum of normal subjects (10 persons) without hepatitis. Meanwhile, in 18 patients with liver injury, serum glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities were normal value (less than 40 I.U./I). Serum GOT and GPT activities from 24 patients showed a comparatively higher level (50-234 I.U./I). The present results demonstrate the potential sensitivity of regucalcin as a marker of chronic liver injury.

Fifty-one cases of resected hepatocellular carcinoma (HCC) were retrospectively analyzed to evaluate the clinicopathologic features of HCC in patients with negative virus markers. The data were compared between three groups: hepatitis B surface antigen positive (HB, n = 11), hepatitis C virus antibody positive (HC, n = 21), and non-BC (both HbsAg and HCVAb negative, n = 12). Seven patients were excluded from the study because of operative death (n = 3), a history of alcohol abuse (n = 3), or the presence of dual positive HB and HC virus markers (n = 1). The data were analyzed by either an analysis of variance (ANOVA) or a contingency table. The age of the non-BC patients was higher (63.0 +/- 4.1, +/- SE) than that of HB patients (54.0 +/- 3.2, p < 0.05) but was identical to that of the HC group (62.0 +/- 1.8). Among the preoperative laboratory data, the serum glutamic oxaloacetate and glutamate pyruvate transaminoses (GOT, GPT) levels were statistically lower in the non-BC patients (32.8 +/- 4.8 and 28.0 +/- 4.4 IU/L, respectively) than in the HB and HC patients. The pathologic features of the resected specimens in the non-BC patients showed more invasive growth than in specimens from the HB or HC patients. The clinical stages (defined based on the criteria of the Japanese Association of Hepatocellular Carcinoma) were also more advanced in the non-BC patients than in the other groups. Postoperative survival time showed no significant difference among the groups. In conclusion, the non-BC patients had comparatively greater invasive growth and more advanced clinical stages than the HB and HC patients, despite the absence of liver cirrhosis, and so demonstrated the same poor survival data as observed in the HB and HC patients.