1. Effect of aqueous-methanolic extract of Artemisia absinthium (Compositae) was investigated against acetaminophen- and CCl4-induced hepatic damage. 2. Acetaminophen produced 100% mortality at the dose of 1 g/kg in mice while pretreatment of animals with plant extract (500 mg/kg) reduced the death rate to 20%. 3. Pretreatment of rats with plant extract (500 mg/kg, orally twice daily for two days) prevented (P < 0.01) the acetaminophen (640 mg/kg) as well as CCl4 (1.5 ml/kg)-induced rise in serum transaminases (GOT and GPT). 4. Post-treatment with three successive doses of extract (500 mg/kg, 6 hr) restricted the hepatic damage induced by acetaminophen (P < 0.01) but CCl4-induced hepatotoxicity was not altered (P>> 0.05). 5. Plant extract (500 mg/kg) caused significant prolongation (P < 0.05) in pentobarbital (75 mg/kg)-induced sleep as well as increased strychnine-induced lethality in mice suggestive of inhibitory effect on microsomal drug metabolizing enzymes (MDME). 6. These results indicate that the crude extract of Artemisia absinthium exhibits hepatoprotective action partly through MDME inhibitory action and validates the traditional use of plant in hepatic damage.

Donor lymphocyte infusions (DLI) were given between June 1990 and March 1996 to 18 patients with chronic myeloid leukemia (CML) for the treatment of cytogenetic (n = 6) or hematologic relapse (n = 12) following an allogeneic bone marrow transplant (BMT). Patients were divided in two groups: patients in group A (n = 8) received a large dose of donor lymphocytes >> or = 1 x 10(8)/kg), whereas patients in group B (n = 10) received escalating numbers of cells (2 x 10(5) up to 2 x 10(8)/kg). The median number of DLI in group A was 2 (range 1-3); the median number of infusions in group B was 7 (range 3-9). Acute GVHD occurred in 12 patients (grades I-III) and was a major cause of death in two. The risk of developing GVHD correlated with the number of cells infused: 37%, 14%, 5% and 0% for DLI with cells>> or = 1 x 10(8), 2 x 10(7)/kg, 2 x 10(6)/kg, and 2 x 10(5)/kg, respectively (P = 0.01). Median transaminase levels were found to be significantly increased in patients with, as compared to patients without, acute GVHD (GPT 412 vs 28 IU/l; P = 0.03). Severe aplasia occurred in four and was a contributing cause of death in two patients. Overall, four patients died as a consequence of DLI and all received>> 1 x 10(8)/kg cells: the actuarial risk was 38% in group A and 14% in group B (P = 0.1). There were 10 complete and three partial cytogenetic responses: the actuarial probability at 5 years of being Ph negative was 69%: it was 46% for group A and 85% for group B (P = 0.1). The longest patient is now 6 years post-DLI, Ph negative, BCR-ABL negative. The actuarial 3 year survival is 38% in group A and 86% in group B (P = 0.06). The study confirms that DLI post-BMT is not innocuous and that there is a definite long-lasting antileukemic effect in patients with CML. It also suggests that: (1) the risk of developing GVHD correlates with the number of infused cells; (2) that significant elevations of serum GPT levels are associated with GVHD; and (3) that the use of escalating doses of cells may allow the identification of side-effects and discontinuation of infusions before life-threatening GVHD has developed.

Liver cirrhosis is responsible for hepatic fibrosis resulting in high mortality and is also a risk factor for developing hepatocellular carcinoma (HCC), which is the fifth most common cancer in men and the seventh in women globally. Several studies have found effective anti-cancer activities of theaflavins, the major black tea polyphenols. The objective of this study was to investigate the protective effects of theaflavin-enriched black tea extracts (TF-BTE) on hepatic fibrosis induced by dimethylnitrosamine (DMN) administration in Sprague-Dawley (SD) rats. Treatment of SD rats with DMN (10 mg per kg bw) for 4 weeks produced inflammation and remarkable liver fibrosis assessed by serum biochemistry and histopathological examination. Fibrotic status and the activation of hepatic stellate cells were improved by oral administration of 40% theaflavins in black tea extracts (40% TF-BTE) as evidenced by histopathological examination. Oral administration of 40% TF-BTE at a low dose of 50 mg per kg bw per day and a high dose of 100 mg per kg bw per day attenuated the DMN-induced elevation of serum GOT (glutamate oxaloacetate transaminase) and GPT (glutamic pyruvic transaminase) levels and reduced necrosis, bile duct proliferation, and inflammation. Western blot analyses revealed that TF-BTE inhibited the expression of liver alpha-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) protein. The histochemical examination showed the inhibitory effect of TF-BTE on the p-Smad3 expression. Overall, these data demonstrated that TF-BTE exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-β1/Smad signaling.

This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC>T transitions that induce 40-70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.

A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.

Electrosurgery is a commonly used technology in laparoscopy. Electrosurgical devices are being used in many medical subdisciplines in order to coagulate, cut, and ablate tissue and to produce hemostasis. The latest advance in electrosurgery is the introduction of novel simultaneous hemostatic sealing and cutting bipolar devices that minimize force by delivering energy as high current and low voltage output. One of these systems is the Gyrus Plasma Trissector (GPT), (Gyrus Group PLC, Gyrus International, Ltd.UK), a novel radiofrequency (RF) system based on bipolar technology, which consists of a proprietary PlasmaKinetic (PK) generator and specific instruments, designed as a system. The instrument consists of a 17 mm long gold-coated curved jaw with a ceramic insulated cutting element. The PK system uses high-powered pulsed bipolar energy to produce a plasmakinetic field around the working elements and is designed to operate at temperatures that allow effective tissue dissection but result in minimal collateral damage and adherence to tissue. The GPT was used with the Gyrus G400 WorkStation generator in laparoscopic radical prostatectomies (LRP). Our initial experience with this novel bipolar device demonstrates it to be safe and effective in dissection and hemostasis. The GPT permits us to act quickly when bloody fields are present, improves coagulation, reduces or eliminates sticking, seals large vessels such as dorsal vein complex (DVC) and allows secure grasping and dissecting of tissue. The urologist is familiar with electrosurgery, as we have become comfortable using GPT to dissect, cut, and coagulate tissue. We feel that the GPT can be used in a variety of laparoscopic procedures in urology.

The effect of curcumin on lipopolysaccharide/D: -galactosamine (LPS/GalN)-induced acute shock model of liver injury was examined in mice. The simultaneous administration of LPS (5-20 microg kg(-1), i.p.) and GalN (700 mg kg(-1), i.p.) markedly increased the serum tumor necrosis factor-alpha (TNF-alpha), glutamic oxaloacetic transaminase/glutamic pyruvic transaminase (GOT/GPT), and massive hepatic necrosis and inflammation, leading to 100% lethality. Pre-administration of curcumin (100 mg kg(-1), i.p.) 3 h before induction with LPS/GalN imparted a large extent of protection against acute elevation in serum TNF-alpha and serum GOT/GPT. Hepatic necrosis and lethality caused by LPS/GalN was also greatly reduced by curcumin treatment. The results demonstrated that curcumin could protect mice from LPS/GalN-induced hepatic injury and inflammation through blockading TNF-alpha production, eventually raising the survival rate of septic-shock-induced mice.

The first enzyme in the dolichol pathway of protein N-glycosylation, dolichol-P-dependent N-acetylglucosamine-1-phosphate transferase, GPT, has been implicated in the development of a wide variety of eukaryotes. GPT is encoded by ALG7, an early growth-response gene, whose expression has been shown to affect the extent of N-glycosylation and secretion of proteins. To initiate the molecular characterization of ALG7 involvement in mammalian growth and differentiation, we have used the postnatally developing hamster submandibular gland (SMG) as an experimental paradigm. In this study we report that the ALG7 gene was differentially expressed during postnatal development and in terminally differentiated adult tissues. Throughout development, GPT activity paralleled ALG7 mRNA levels, suggesting that the amount of functional enzyme was determined by modulation of transcript abundance.

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B-D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP-GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.

UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT) is an endoplasmic reticulum (ER) enzyme responsible for synthesis of GlcNAc-P-P-dolichol, the committed step of dolichol-P-P-oligosaccharide synthesis. The sequence of hamster GPT predicted multiple transmembrane segments (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). GPT has also been predicted to act on the cytosolic face of the ER membrane, based on topological studies of its substrates and products. In this report we test these predictions by: (i) immunofluorescence microscopy with antibodies specific for native GPT sequences or epitope tags inserted into GPT, after selective permeabilization of the plasma membrane with digitonin; (ii) insertion of Factor Xa cleavage sites; (iii) in vitro translation of GPT; and (iv) site-directed mutagenesis. The loops between the 1st and 2nd and between the 9th and 10th predicted transmembrane spans of GPT were found to be cytosolic. In contrast, the loop between the 6th and 7th transmembrane spans, as well as the carboxyl terminus, were lumenal. Thus, hamster GPT must cross the ER membrane at least three times, consistent with previous computer-assisted predictions. There was no apparent N-glycosylation or signal sequence cleavage detected by in vitro translation. The cytosolic loop between the 9th and 10th transmembrane spans is the largest hydrophilic segment in GPT and, as judged by site-directed mutagenesis, has a number of conserved residues essential for activity. Hence, these results directly support the hypothesis that dolichol-P-P-oligosaccharide assembly is initiated in the cytosol and that a downstream intermediate must translocate to the lumenal face of the ER membrane.

The red cell GPT phenotypes have been determined in two village populations in Gambia, West Africa. A total of 887 people have been investigated. The results confirm the previous observations that the frequency of the GPT gene is far higher in African populations than Caucasian populations.

BACKGROUND

Prolonged mechanical ventilation and the consequently long stay in the intensive care unit (ICU) appear to be important infection risk factors in patients undergoing liver transplant.

METHODS

We analyzed the data relating to 70 liver transplants performed on 67 patients during the past year's activities. For each patient we have considered the presence of preoperative pulmonary alterations, the first radiological result of the postoperative thorax, the PaO(2)/FiO(2) ratio recorded in the peroperative phase immediately after induction of general anesthesia (T0), and arrival at the ICU at the end of the operation (T1). We also considered the enzyme trend (glutamate-oxalacetic transaminase [GOT] and glutamate-pyruvate transaminase [GPT]) recorded every 6 hours for the first 42 hours of the postoperative period (times T1 to T7).

RESULTS

There was an evident correspondence between the values of PaO(2)/FiO(2) < or = 300 ratio at time T1 and the subsequent duration of mechanical ventilation (P = .001). There was also a correlation between the PaO(2)/FiO(2) < or = 300 ratio at time T1 and the trend of the GPT in the first 24 hours postsurgery (P = .021; P = .026; P = .018; P = .048) or GOT trend over the same span of time (P = .027; P = .035; P = .048).

CONCLUSIONS

Graft malfunction as expressed by the enzyme trend affects both the duration of mechanical ventilation and the postoperative PaO(2)/FiO(2) ratios. This metric may be useful to reinforce infection surveillance and to perform an early percutaneous tracheostomy in these patients.

OBJECTIVE

To investigate the efficacy and safety of oral pantoprazole, 20 mg (0.5 to 1.0 mg/kg/day) once daily for 28 days, in pediatric patients with reflux esophagitis.

METHODS

Patients in this study (n = 15; 6 to 13 years old, 9 boys) had reflux esophagitis grade Ic or II (Vandenplas classification). The efficacy of pantoprazole to reduce esophageal acid exposure time (pH < 4), reduce the number and duration of reflux episodes, and to increase the percentage of time with gastric pH>> 3 was assessed by continuous 24-hour pH monitoring. The intensity of 5 common symptoms of esophagitis was scored before and after treatment on a 4-point scale. Esophagitis was assessed at baseline and after treatment by visual inspection and by the histology of biopsies from the distal third of the esophagus.

RESULTS

Before treatment, the median percentage of time with intra-esophageal pH <4 was 9.3%. After 28 days of therapy with pantoprazole, this value decreased to 2.7% (P = 0.0006). The median percentage of time with intragastric pH>> 3 increased from 21% at baseline to 39% on day 28 of therapy (P = 0.005). After 28 days of treatment, all patients experienced at least partial relief from reflux symptoms. Endoscopically confirmed healing of esophagitis was seen in 47% of children (Savary-Miller classification). Histologic evidence of healing was not observed. Median serum gastrin levels were slightly elevated over baseline levels (from 74 pg/ml to 93 pg/ml). In one patient there was a transient elevation of serum GOT and GPT during treatment.

CONCLUSIONS

Oral pantoprazole 20 mg daily provided gastric acid control in 15 pediatric patients with reflux esophagitis with partial clinical improvement of symptoms after 28 days of treatment. Pantoprazole was safe and well tolerated.

The effect of a methanolic extract of Cardiospermum halicacabum L was studied against cyclophosphamide (CTX)-induced toxicity in mice. Administration of CTX (25 mg/kg b.wt, i.p.) for 10 days produced significant myelosuppression as evidenced by a decreased WBC count and bone marrow cellularity. Co-treatment with Cardiospermum significantly increased the total WBC count, bone marrow cellularity and α-esterase positive cells, and the relative organ weights of spleen as well as thymus compared to the CTX alone treated group. Cardiospermum further reduced the enhanced levels of ALP, GPT, LPO, and proinflammatory cytokine TNF-α, and also significantly increased the glutathione (GSH) level in CTX treated animals. The lowered levels of other cytokines like IFN-γ, IL-2, GM-CSF, after CTX treatment were also found to be increased by extract administration. Histopathological analysis of small intestine also suggested reduction of CTX-induced intestinal damage. Moreover the extract down-regulated the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) mRNA expression in LPS stimulated macrophages. These studies indicate that C. halicacabum could reduce cyclophosphamide induced oxidative stress and immunosupression through enhancing the antioxidant status and immunomodulation by stem cell proliferation.

The methanolic extract from the fruit of Piper chaba (Piperaceae) was found to have a hepatoprotective effect on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. From the ethyl acetate-soluble fraction, a new amide constituent named piperchabamide E together with twenty known amide constituents (e.g., piperine, piperchabamides A-D, and piperanine) and two aromatic constituents were isolated as the hepatoprotective constituents. With regard to structure-activity relationships, the amide moiety and the 1,9-decadiene structure between the benzene ring and amide moiety were suggested to be important for strong inhibition of D-GalN/tumor necrosis factor-alpha (TNF-alpha)-induced death of hepatocytes. Furthermore, a principal amide constituent, piperine, dose-dependently inhibited increase in serum GPT and GOT levels at doses of 2.5-10 mg/kg (p.o.) in D-GalN/LPS-treated mice, and this inhibitory effect was suggested to depend on the reduced sensitivity of hepatocytes to TNF-alpha.

Manganese (Mn) is an essential element for normal development and bodily functions in humans. In the present study, we examined whether Mn supplementation can alter the serum lipid parameters and liver function in Ca-deficient ovariectomized (OVX) rats. Sixty female Sprague-Dawley rats (6 weeks) were divided into five groups and bred for 12 weeks: sham-operated control group (Sham), OVX Ca deficiency group (OLCa) with Ca-deficient diet (0.1% Ca modified AIN-93N diet), OVX Ca deficiency and Mn supplementation group (OLCaMn), OVX with adequate Ca group (OACa; 0.5% Ca AIN-93N diet), and OVX with adequate Ca and Mn supplementation group (OACaMn). A low Ca diet increased the liver weight and serum levels of GOT, GPT, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in OVX rats. Mn supplementation decreased these parameters in Ca-deficient OVX rat. The results of our study suggest Mn supplementation results in reductions of the blood cholesterol levels, which show an increase due to Ca deficiency in OVX rats.

BACKGROUND

Gemcitabine is the most promising new agent currently being tested in pancreatic cancer. The present study was conducted to confirm the tolerability of a weekly schedule of gemcitabine at a dose of 1000 mg/m2 in Japanese patients with advanced pancreatic cancer.

METHODS

The primary end-point was to evaluate the frequency of dose-limiting toxicity. Gemcitabine 1000 mg/m2 was administered over 30 min weekly in two schedules: gemcitabine x3 every 4 weeks (Schedule 1) and gemcitabine x7 followed by a week of rest and then gemcitabine x3 every 4 weeks thereafter (Schedule 2). At least three patients entered each schedule and three additional patients were treated in the presence of dose-limiting toxicity.

RESULTS

Eleven chemo-naive patients with a good Karnofsky performance status of>> or =80 points and distant metastasis were entered into this trial. In Schedule 1, no dose-limiting toxicity was observed in the three patients. In Schedule 2, the evaluation of dose-limiting toxicity was complete in six of the eight enrolled patients and two patients showed dose-limiting toxicity in this Schedule; one patient experienced both grade 4 leukocytopenia and grade 4 neutropenia, and both grade 4 neutropenia and grade 3 GOT/GPT increased in another patient. Two patients (18%) showed a partial response and a clinical benefit response was also achieved in two (29%) of the seven evaluable patients.

CONCLUSIONS

Gemcitabine 1000 mg/m2 weekly x7 followed by a week of rest and weekly x3 every 4 weeks thereafter may be tolerated in Japanese patients with advanced pancreatic cancer.

The translocation of the messenger RNA relative to the ribosome during peptide synthesis represents an example of a mechano-chemical reaction in which the chemical bond energy of GTP is transformed into coordinated motion. Such transformations also occur during the beating of cilia and flagellae, the contraction of muscle and the migration of chromosomes in cell division. In protein synthesis the functional geometric and energetic conditions for this transformation are well defined. For each peptide bond formed, the ribosome moves one codon along the mRNA (towards the 3' end) and one molecule of GTP is hydrolysed. Although the basic requirements of this reaction have been elucidated, the mechanism is still unresolved. We demonstrate here that translocation can be analysed as a series of binding equilibria shifted by one irreversible, GTP-consuming step. The shift in the binding equilibrium is induced by the transfer of the peptidyl moiety to the (A) site-bound aminoacyl (AA)-tRNA. This results in the A site-bound tRNA having an increased affinity for the high-affinity (P) site, and a strengthened association with the mRNA. Elongation factor (EF) G . GPT catalyses removal of the deacylated tRNA, empties the P site and at the same time loosens ribosome-mRNA association. The result of these changes is that peptidyl(PP)-tRNA . mRNA is shifted from the A site to the P site, binding of AA-tRNA . EF-Tu . GPT to the vacant A site ensuring that the process is irreversible.

Elimination of glutamate through enzymatic degradation is an alternative to glutamate receptor blockade in preventing excitotoxic neuronal injury. Glutamate pyruvate transaminase (GPT) is a highly active glutamate degrading enzyme that requires pyruvate as a co-substrate. This study examined the ability of GPT to protect neurons of the hippocampal slice preparation against glutamate toxicity. Two methods were used to elevate the concentration of glutamate in the peri-neuronal space. In an endogenous release paradigm, slices were incubated with 100-500 microM L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate re-uptake. One hour of exposure to PDC in normal, pyruvate-free slice maintenance medium caused a dose dependent increase in neuronal death assessed 24 h later by propidium iodide uptake in dead cell nuclei. GPT (10 U/ml) decreased neuronal death caused by exposure to PDC at all PDC concentrations tested. Neuroprotection in this model was not dependent on added or non-physiologic levels of pyruvate. In a different paradigm, glutamate was added directly to the normal, pyruvate-free slice maintenance medium and not rinsed away, exposing the slices to a range of 1-5 mM glutamate for an extended period. Twenty-four hours later, neuronal death was again assessed by propidium iodide uptake. GPT was again neuroprotective, decreasing neuronal death in the range from 3 to 5 mM glutamate. In the setting of incubation with this large load of glutamate, neuroprotection by GPT was enhanced by adding pyruvate to the medium. GPT is an effective neuroprotectant against glutamate excitotoxicity. When exposure is limited to endogenously released glutamate, neuroprotection by GPT is not dependent on added pyruvate.

The use of adriamycin, an antitumour agent, is restricted by its cardiotoxicity. The objective of this study was to investigate the role of mitochondrial Ca2+ in adriamycin-induced cardiotoxicity and the effect of either cyclosporin A (CsA) or tacrolimus (FK506) on that cardiotoxicity. A single dose of adriamycin (10 mg/kg body weight) caused myocardial damage that was manifested by elevation of serum enzymes, glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), lactate dehydrogenase isoenzyme (LDH-iso) and creatine phosphokinase isoenzyme (CPK2-MB). The permeability of heart inner mitochondrial membrane of adriamycin-treated rats was examined. Tetraphenyl phosphonium ion (TPP+) uptake, estimated with a TPP+-sensitive electrode was used to monitor changes in heart inner mitochondrial membrane potential. Ca2+ efflux was measured spectrophotometrically with the Ca2+ indicator arsenazo III. The ability of heart mitochondria isolated from adriamycin treated rats to retain accumulated Ca2+ or TPP+ was sharply reduced. The increase of diagnostic serum enzymes and isoenzymes and the reduced ability to retain Ca2+ or TPP+ by heart mitochondria were restored to almost the normal levels when (500 microg/kg body weight) of CsA or FK506 were injected with adriamycin. The data suggested that adriamycin cardiotoxicity might be due to the increase of inner membrane permeability in heart mitochondria as a result of increasing the sensitivity of a Ca2+ dependent-pore of the inner mitochondrial membrane to calcium, leading to dissipation of membrane potential and release of pre-accumulated Ca2+. Suitable antagonists of Ca2+-dependent pore formation such as CsA or FK506 may improve heart tolerance to adriamycin.

Mitochondrial (mt) DNA deletions and low folate status, proposed characteristics of carcinogenesis, in relation to human hepatocellular carcinoma (HCC) susceptibility are not clearly understood. We hypothesised that low folate status may modify frequencies of mtDNA deletions in humans, both of which could predispose individuals to HCC development. Biochemical folate status of serum and lymphocytes, and frequencies of mtDNA deletions in lymphocytes were determined in ninety HCC cases and ninety cancer-free healthy controls, individually matched by age and sex. The data revealed that HCC patients had lower levels of serum folate (P = 0.0002), lymphocytic folate (P = 0.040) and accumulated higher frequency of lymphocytic mtDNA deletions (P < 0.0001) than the controls. In the total studied subjects, frequencies of lymphocytic mtDNA deletions were associated with hepatitic B infection (P = 0.004) and HCC incidents (P = 0.001), and were correlated with serum folate (r - 0.155; P = 0.041), lymphocyte folate (r - 0.314; P = 0.0001), levels of glutamate-oxaloacetate transaminase (GOT) (r 0.206; P = 0.006), glutamate-pyruvate transaminase (GPT) (r 0.163; P = 0.037) and alpha-fetal protein concentrations (r 0.212; P = 0.005). After adjustment for age, sex, lifestyle and one-carbon metabolite factors, individuals with low blood folate ( < 11.5 nmol/l) or high mtDNA deletions (Delta threshold cycle number (Ct)>5.3) had increased risks for HCC (OR 7.7, 95 % CI 1.9, 29.9, P = 0.003; OR 5.4; 95 % CI 1.7, 16.8, P = 0.003, respectively). When combined with folate deficiency (serum folate < 14 nmol/l), the OR of HCC in individuals with high levels of lymphocytic mtDNA deletions was enhanced (OR 13.3; 95 % CI 1.45, 122; P = 0.008). Further controlling for GOT and GPT levels, however, negated those effects on HCC risk. Taken together, the data suggest that biochemical folate status and liver injuries are important modulators to lymphocytic mtDNA deletions. The mt genetic instability that results from a high rate of mtDNA deletions and/or low folate status increased the risk for HCC, which is mediated by clinical hepatic lesions.

1, The biological effects of metabolites of leukotriene E4 (LTE4) i.e. N-acetyl LTE4 (N-AcLTE4), 20-COOH-LTE4, 20-COOH-N-AcLTE4, as well as 18-COOH-19,20-dinor-LTE4 (dinor-LTE4) and 16-COOH-17,18,19,20-tetranor-14,15-dihydro-LTE4 (tetranor-LTE4) were investigated on superfused strips of guinea-pig trachea (GPT) and lung parenchyma (GPP) in vitro. 2. The actions of LTE4 were studied in isolated, superfused strips of human lung parenchyma (HP) and bronchus (HBr), in comparison with LTD4 and histamine. Effects of N-AcLTE4, the 20-carboxy metabolites, dinor-LTE4 and tetranor-LTE4 were also investigated in HBr. 3. N-AcLTE4 (0.1-10 nmol) induced dose-related contractions of GPT and was approximately 100 times less active than LTD4 (3-100 pmol). 4. In GPP, N-AcLTE4 (0.01-3 nmol) was equiactive with LTE4 (0.01-1 nmol) and approximately one order of magnitude less active than LTD4 (1-300 pmol). Contractions caused by N-AcLTE4 and LTE4 were very similar and approximately twice as sustained as those due to LTD4. 5. LTE4 (0.1-30 nmol) contracted strips of HP and HBr and was about 2-3 orders of magnitude less active than LTD4. As in GPP, the effect of LTE4 was more protracted than that of LTD4. Actions of N-AcLTE4 were similar to those of LTE4 in HBr. 6. 20-carboxy-LTE4, 20-carboxy-N-AcLTE4, dinor-LTE4 and tetranor-LTE4, all at 0.3-30 nmol, were inactive in GPT, GPP and HBr. 7. Indomethacin (2.8 microM) potentiated the effect of N-AcLTE4 in GPT, inhibited its contraction in GPP but did not affect that due to LTE4 in either HP or HBr. FPL 55712 (1.9 microM) antagonised leukotriene-induced contractions in GPT, GPP and HBr. 8. In conclusion, the metabolism of LTD4 to LTE4 or N-AcLTE4 may represent a detoxification but not an inactivation of cysteinyl-containing leukotrienes, since both metabolites still retain considerable biological activity in guinea-pig and human airways in vitro. However, further metabolism of LTE4 and N-AcLTE4 appears to result in inactivation of leukotrienes.

Cadmium is a potent hepatotoxicant for which neither effective preventive methods nor the mechanism of toxicity has been established. We investigated the preventive effect of dexamethasone against cadmium toxicity on cadmium-induced liver injury in rabbits. Pretreatment with dexamethasone at 1 mg/kg increased the rate of survival in rabbits administered 2.5 mg/kg iv cadmium. Cadmium induced acute severe liver injury characterized by hepatocellular necrosis, infiltration by inflammatory cells, and increases of plasma GOT, GPT, LDH, and LDH5. Dexamethasone mitigated the acute hepatotoxic effect of cadmium, but exacerbated cadmium-induced kidney dysfunction, with destruction of renal tubular cells and increases in excretion of protein, glucose, and amino acids into urine. The cadmium concentration in liver and kidney of rabbits administered cadmium was not changed by dexamethasone pretreatment. Although metallothionein mRNA expression induced by cadmium was not affected by dexamethasone in liver or kidney, cadmium-induced metallothionein protein production was augmented at the early phase in liver and decreased at the later phase in kidney. Neutrophilia observed after cadmium administration was enhanced initially by dexamethasone pretreatment. These results indicate that dexamethasone pretreatment potently prevented cadmium-induced liver injury, but exacerbated renal tubular dysfunction.

The initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes tRNALys,3 as a primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNAIle or tRNAHis, to initiate reverse transcription only if the primer binding site (PBS) was made complementary to the 3' terminal 18 nucleotides of the cognate tRNA. However, upon in vitro culture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNALys,3. To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to tRNAIle or tRNAHis. The genomes contain the gene for xanthine-guanosine phosphoribosyl transferase (gpt) in place of env. Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G) results in viruses that undergo a single round of HIV-1 infection; successful infections are scored as cells resistant to the drug mycophenolic acid. Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNAIle or tRNAHis is three- to fivefold less efficient in replication as measured by production of drug-resistant cell colonies compared to the wild-type virus. These viruses predominantly used the cognate tRNA as primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNALys,3. Using an HIV-1 provirus with a PBS complementary to yeast tRNAPhe, we established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNAPhe. The results of our studies suggest that the mechanism for selection of the tRNA primer for initiation of reverse transcription relies primarily on the complementarity between the tRNA primerthe PBS.