We found that the sequences downstream of the Ig gamma 2b secretory-specific (sec) poly(A) site play an important role in the preferential production of sec Ig mRNA during plasma B cell development. The Ig gamma 2b mRNA production in a deletion mutant (delta-Kpn) lacking the Ig sec poly(A) site and downstream consensus element (dsc) has been previously shown to default to the use of the downstream membrane-specific (mb) poly(A) site. In this study restoration of the Ig sec poly(A) site and dsc to the delta-Kpn gene causes a significant increase in the use of the sec poly(A) site vs mb poly(A) site in stable transfectants of plasma but not memory B cell tumors, indicating plasma cell-specific recognition of the Ig sec dsc. Restoration of the poly(A) cleavage site alone to delta-Kpn did not restore regulation. Substitution of an SV40 downstream poly(A) element for the Ig dsc in the delta-Kpn gene also does not restore regulation. The data further indicate that although the Ig dsc is clearly very important in the plasma cell-regulated expression, the difference in the processing ratios of the restored vs the intact Ig gamma 2b gene in plasma cells suggests that there are other yet to be defined sequences that may also play a role in the intact gene. Insertion of a 130-nucleotide segment of the gene containing the Ig sec poly(A) site and dsc into a heterologous, guanosyl phosphotransferase gene resulted in plasma cell-regulated polyadenylation of the sec poly(A) site. Neither the mb nor the SV40 early poly(A) sites and their respective dscs, in similar gpt chimeras, were regulated. Therefore the region downstream of the Ig sec poly(A) site plays an essential role in regulating polyadenylation at the sec poly(A) site in plasma cells but not memory cells. A model involving a plasma cell-specific recognition factor for the Ig sec dsc is presented.

The Escherichia coli gene coding for the enzyme xanthine-guanine phosphoribosyl transferase (gpt) has been widely used as a dominant selectable marker in a variety of mammalian cells. We have determined the complete nucleotide sequence of the 1057 base pair (bp) segment of DNA containing this gene. The coding sequence for the enzyme is 456 nucleotides long and can code for a 152 amino acid (16.9 Kd) polypeptide. A comparison of the amino acid sequence of the bacterial enzyme with that of the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) reveals no significant homology between the two polypeptides.

BACKGROUND

"Bright" liver at ultrasonography predicts fatty liver.

OBJECTIVE

To assess prevalence and predictors of a bright liver.

METHODS

Prevalence arm--Prospective collection of records of unselected patients undergoing liver ultrasound. Protocol arm--A sample of patients with bright liver underwent routine laboratory investigations, liver tests, HBsAg (EIA) and anti-HCV (ELISA). Equipment--Grey scale real-time scanner. Bright liver diagnosed through the liver-kidney contrast. Statistics--Student's t-test; chi-square test; one-way analysis of variance; stepwise multiple logistic regression analysis, forward and backward.

RESULTS

Prevalence arm--72 out of 363 (19.8%) cases had bright liver. Protocol arm--100 cases underwent laboratory evaluation. No difference was found in mean values of fasting plasma glucose, LDL cholesterol, apo A, Lp(a), uric acid, total bilirubin, GOT, GPT, AP, GGT, and serum bile acids or in the prevalence of serum HBsAg and anti-HCV in bright liver vs control groups. Univariate analysis showed body mass index, age, total cholesterol triglycerides, apo B, albumin, HDL cholesterol to be significantly different between cases and controls. However, the last 2 variables dropped out when logistic regression analysis was applied.

CONCLUSIONS

1. Roughly 20% of Italian patients undergoing US for routine clinical practice will have a bright liver. 2. Body mass index, age, serum cholesterol, triglycerides, and apo B levels are the independent predictors of a bright liver.

OBJECTIVE

To investigate the possible protection provided by oral quercetin pretreatment against hepatic ischemia-reperfusion injury in rats.

METHODS

The quercetin (0.13 mmol/kg) was orally administrated in 50 min prior to hepatic ischemia-reperfusion injury. Ascorbic acid was also similarly administered. The hepatic content of quercetin was assayed by high performance liquid chromatography (HPLC). Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) activities and malondialdehyde (MDA) concentration were measured as markers of hepatic ischemia-reperfusion injury. Meanwhile, hepatic content of glutathione (GSH), activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and xanthine oxidase (XO), total antioxidant capacity (TAOC), contents of reactive oxygen species (ROS) and MDA, DNA fragmentation were also determined.

RESULTS

Hepatic content of quercetin after intragastric administration of quercetin was increased significantly. The increases in plasma GPT, GOT activities and MDA concentration after hepatic ischemia-reperfusion injury were reduced significantly by pretreatment with quercetin. Hepatic content of GSH and activities of SOD, GSH-Px and TAOC were restored remarkably while the ROS and MDA contents were significantly diminished by quercetin pretreatment after ischemia-reperfusion injury. However, quercetin pretreatment did not reduce significantly hepatic XO activity and DNA fragmentation. Ascorbic acid pretreatment had also protective effects against hepatic ischemia-reperfusion injury by restoring hepatic content of GSH, TAOC and diminishing ROS and MDA formation and DNA fragmentation.

CONCLUSIONS

It is indicated that quercetin can protect the liver against ischemia-reperfusion injury after oral pretreatment and the underlying mechanism is associated with improved hepatic antioxidant capacity.

In order to regulate cell volume during hyperosmotic stress, the intertidal copepod Tigriopus californicus, like other aquatic crustaceans, rapidly accumulates high levels of intracellular alanine, proline, and glycine. Glutamate-pyruvate transaminase (GPT; EC 2.6.1.2), which catalyzes the final step of alanine synthesis, is genetically polymorphic in T. californicus populations at Santa Cruz, California. Spectrophotometric studies of homogenates derived from a homozygous isofemale line of each of the two common GPT alleles indicated that the GPTF allozyme has a significantly higher specific activity than the GPTS allozyme. Under conditions of hyperosmotic stress, individual adult copepods of GPTF and GPTF/S genotypes accumulated alanine, but not glycine or proline, more rapidly than GPTS homozygotes. When young larvae were subjected to the same hyperosmotic conditions, GPTS larvae suffered a significantly higher mortality than GPTF or GPTF/S larvae. These results suggest that the biochemical differences among GPT allozymes result in specific physiological variation among GPT genotypes and that this physiological variation is manifested in differential genotypic survivorships under some naturally occurring environmental conditions.

BACKGROUND

Ketamine is widely used as an i.v. anaesthetic agent and as a drug of abuse. Hepatocytes contribute to the metabolism of endogenous and exogenous substances. This study evaluated the toxic effects of S-(+)-ketamine and possible mechanisms using human hepatoma HepG2 cells as the experimental model.

METHODS

HepG2 cells were exposed to S-(+)-ketamine. Cell viability and the release of lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GPT) were measured to determine the toxicity of S-(+)-ketamine to HepG2 cells. Cell morphology, DNA fragmentation, and apoptotic cells were analysed to evaluate the mechanism of S-(+)-ketamine-induced cell death. Amounts of Bax, an apoptotic protein, and cytochrome c in the cytoplasm or mitochondria were quantified by immunoblotting. Cellular adenosine triphosphate levels were analysed using a bioluminescence assay. Caspases-3, -9, and -6 were measured fluorometrically.

RESULTS

Exposure of HepG2 cells to S-(+)-ketamine increased the release of LDH and GPT, but decreased cell viability (all P<0.01). S-(+)-Ketamine time-dependently caused shrinkage of HepG2 cells. Exposure to S-(+)-ketamine led to significant DNA fragmentation and cell apoptosis (P=0.003 and 0.002). S-(+)-Ketamine increased translocation of Bax from the cytoplasm to mitochondria, but decreased the mitochondrial membrane potential and cellular adenosine triphosphate levels (all P<0.01). Sequentially, cytosolic cytochrome c levels and activities of caspases-9, -3, and -6 were augmented after S-(+)-ketamine administration (all P<0.001). Z-VEID-FMK, an inhibitor of caspase-6, alleviated the S-(+)-ketamine-induced augmentation of caspase-6 activity, DNA fragmentation, and cell apoptosis (all P<0.001).

CONCLUSIONS

This study shows that S-(+)-ketamine can induce apoptotic insults to human HepG2 cells via a Bax-mitochondria-caspase protease pathway. Thus, we suggest that S-(+)-ketamine at a clinically relevant or an abused concentration may induce liver dysfunction possibly due to its toxicity to hepatocytes.

The effect of oral administration of Phyllanthus methanolic extracts (PME) (i.e. P. acidus, P. emblica, P. myrtifolius, P. multiflorus, P. amarus, P. debilis, P. embergeri, P. hookeri, P. tenellus, P. urinaria L.s. nudicarpus, P. urinaria L.s. urinaria) or gallic acid (GA) on the progression of acute liver damage induced by CCl(4) in rats was examined by morphological and biochemical methods. P. acidus, P. urinaria L.s. urinaria, GA at a dose of 0.5 g/kg, and P. emblica, P. urinaria L.s. nudicarpus at a dose of 1.0 g/kg attenuated CCl(4)-induced increase in serum glutamate-oxalate-transaminase (GOT). P. acidus, P. urinaria L.s. nudicarpus, P. urinaria L.s. urinaria, GA at a dose of 0.5 g/kg, and P. emblica, P. amarus, P. hookeri, P. tenellus at a dose of 1.0 g/kg attenuated CCl(4)-induced increase in serum glutamate-pyruvate-transaminase (GPT). Concurrently, P. acidus, P. multiflorus, P. embergeri, P. hookeri, P. tenellus and P. urinaria L.s. urinaria elevated the activity of liver reduced glutathione peroxidase (GSH-Px). Since the protective effects of P. acidus, P. emblica, P. myrtifolius, P. embergeri, P. urinaria L.s. nudicarpus, P. urinaria L.s. urinaria and GA correlate with a reduction in liver infiltration and focal necrosis observed using histological methods, these data demonstrate that P. acidus and P. urinaria L.s. urinaria are hepatoprotective and antioxidant agents.

BACKGROUND

Although radiation-induced bystander effects have been confirmed using a variety of endpoints, the mechanism(s) underlying these effects are not well understood, especially for in vivo study.

METHODS

A 1-cm(2) area (1 cm × 1 cm) in the lower abdominal region of gpt delta transgenic mice was irradiated with 5 Gy of 300 keV X-rays, and changes in out-of-field lung and liver were observed.

RESULTS

Compared with sham-treated controls, the Spi(-) mutation frequency increased 2.4-fold in non-targeted lung tissues at 24 h after partial body irradiation (PBIR). Consistent with dramatic Cyclooxygenase 2 (COX-2) induction in the non-targeted bronchial epithelial cells, increasing levels of prostaglandin, together with 8-hydroxydeoxyguanosine, in the out-of-field lung tissues were observed after PBIR. In addition, DNA double-strand breaks and apoptosis were induced in bystander lung tissues after PBIR.

CONCLUSIONS

The PBIR induces DNA damage and mutagenesis in non-targeted lung tissues, especially in bronchial epithelial cells, and COX-2 has an essential role in bystander mutagenesis.

Elevated expression and activity of N-acetylglucosaminyltransferase V (Mgat5) in hepatocellular carcinoma (HCC) is a common early event involved in tumor invasion during hepatocarcinogenesis. A better understanding of the functional role and the molecular mechanism for Mgat5-targeted protein and downstream signaling pathway behind hepatoma invasion and metastasis is urgently needed. Here, we show that Mgat5 overexpression promoted anchorage-independent growth and inhibited anoikis in hepatoma cells. This effect was reversed by glycosyltransferase inactive mutant Mgat5 L188R transfection, α-mannosidase II inhibitor swainsonine treatment and N-acetyl glucosamine (GlcNAc) phosphotransferase (GPT) inhibitor tunicamycin administration. Mgat5 overexpression increased p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase inactivation with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor P21-activated kinase inhibitor III (IPA3) treatment reversed anoikis resistance in Mgat5-overexpressed hepatoma cells. Furthermore, Mgat5 overexpression upregulated β-1-6-GlcNAc branched N-glycosylation and following phosphorylation of epidermal growth factor receptor (EGFR) in hepatoma cells. EGFR tyrosine kinase inhibitors AG1478 and Iressa treatment declined anchorage-independent growth and anoikis resistance, which could be rescued by constitutive active mutant PAK1 T423E coexpression in Mgat5-overexpressed hepatoma cells. Conversely, knockdown of Mgat5 reduced EGFR/PAK1-dependent anoikis resistance, which could be reversed by PAK1 T423E. These results identified Mgat5-mediated β-1-6-GlcNAc branched N-glycosylation and following activation of EGFR as a potential novel upstream molecular event for PAK1-induced anoikis resistance in hepatoma cells, implicating that molecular targeted therapeutics against Mgat5/EGFR/PAK1 might open a new avenue for personalized medicine in advanced-stage HCC patients.

The present study has been designed to evaluate the liver protective and in-vivo antioxidant role of Ethanolic extract (EtAS) and Ethyl acetate extract (EAAS) of roots of Argyreia speciosa, an important 'rasayana' herb in Indian System of medicine, in CCl(4)-induced hepatotoxicity and oxidative stress in rats. Animals were treated with EtAS and EAAS at doses of 200 mg and 400 mg/kg body weight p.o. along with CCl(4) (0.7 ml/kg in olive oil, 1:1 v/v i.p. on every alternate days) for seven days. Serum biochemical parameters such as SGOT, SGPT, ALP, cholesterol, total and direct bilirubin were determined. Antioxidant status in liver was determined by measuring the activities of Super oxide dismutase (SOD), catalase and peroxidase. Histopathological study of isolated liver specimens was also carried out to know the protection offered by the extracts. There was a significant rise in the levels of serum GOT, GPT, and ALP and other biochemical parameters, decrease in the levels of SOD, catalase and peroxidase after administration of CCl(4). Suspensions of EtAS and EAAS (200 and 400 mg/kg) successfully prevented the alterations of these effects in rats (p< 0.001). Histopathological examination demonstrated that CCl(4) treated group induces ballooning degeneration and centrilobular necrosis. Groups treated with EtAS and EAAS showed recovery on ballooning degeneration and centrolobular bridging necrosis was occasionally present. Data also showed that these extracts possessed strong antioxidant activity, and were comparable to Silymarin, a well known liver protecting herbal formulation.

The gonadal steroids estrogen and progesterone have been shown to have neuroprotective properties against various neurodegenerative conditions. Excessive concentrations of glutamate have been found to exert neurotoxic properties. We hypothesize that estrogen and progesterone provide neuroprotection by the autoregulation of blood and brain glutamate levels. Venous blood samples (10 ml) were taken from 31 men and 45 women to determine blood glutamate, estrogen, progesterone, glucose, glutamate-pyruvate transaminase (GPT), and glutamate-oxaloacetate transaminase (GOT) levels, collected on Days 1, 7, 12, and 21 of the female participants' menstrual cycle. Blood glutamate concentrations were higher in men than in women at the start of menstruation (P < 0.05). Blood glutamate levels in women decreased significantly on Days 7 (P < 0.01), 12 (P < 0.001), and 21 (P < 0.001) in comparison with blood glutamate levels on Day 1. There was a significant decrease in blood glutamate levels on Days 12 (P < 0.001) and 21 (P < 0.001) in comparison with blood glutamate levels on Day 7. Furthermore, there was an increase in blood glutamate levels on Day 21 compared with Day 12 (P < 0.05). In women, there were elevated levels of estrogen on Days 7 (P < 0.05), 12, and 21 (P < 0.001), and elevated levels of progesterone on Days 12 and 21 (P < 0.001). There were no differences between men and women with respect to blood glucose concentrations. Concentrations of GOT (P < 0.05) and GPT (P < 0.001) were significantly higher in men than in women during the entire cycle. The results of this study demonstrate that blood glutamate levels are inversely correlated to levels of plasma estrogen and progesterone.

In previous studies, we have shown that by increasing the brain-to-blood glutamate efflux upon scavenging blood glutamate with either oxaloacetate or pyruvate, one achieves highly significant neuroprotection particularly in the context of traumatic brain injury. The current study examines, for the first time, how the blood glutamate scavenging properties of glutamate-pyruvate transaminase (GPT), alone or in combination with pyruvate, may contribute to the spectrum of its neuroprotective mechanisms and improve the outcome of rats exposed to brain ischemia, as they do after head trauma. Rats that were exposed to permanent middle cerebral artery occlusion (MCAO) and treated with intravenous 250 mg/kg pyruvate had a smaller volume of infarction and reduced brain edema, resulting in an improved neurological outcome and reduced mortality compared to control rats treated with saline. Intravenous pyruvate at the low dose of 31.3 mg/kg did not demonstrate any neuroprotection. However, when combined with 0.6 mg/kg of GPT there was a similar neuroprotection observed as seen with pyruvate at 250 mg/kg. Animals treated with 1.69 g/kg glutamate had a worse neurological outcome and a larger extent of brain edema. The decrease in mortality, infarcted brain volume and edema, as well as the improved neurological outcome following MCAO, was correlated with a decrease in blood glutamate levels. We therefore suggest that the blood glutamate scavenging activity of GPT and pyruvate contributes to the spectrum of their neuroprotective mechanisms and may serve as a new neuroprotective strategy for the treatment of ischemic stroke.

The genes encoding the major putative rice plastidic translocators involved in the carbon flow related to starch metabolism were identified by exhaustive database searches. The genes identified were two for the triose phosphate/phosphate translocator (TPT), five for the glucose 6-phosphate/phosphate translocator (GPT) including putatively non-functional ones, four for the phosphoenolpyruvate/phosphate translocator (PPT), three for the putative ADP-glucose translocator (or Brittle-1 protein, BT1), two for the plastidic nucleotide transport protein (NTT), and one each for the plastidic glucose translocator (pGlcT) and the maltose translocator (MT). The expression patterns of the genes in various photosynthetic and non-photosynthetic organs were examined by quantitative real-time PCR. OsBT1-1 was specifically expressed in the seed and its transcript level tremendously increased at the onset of vigorous starch production in the endosperm, suggesting that the ADP-glucose synthesized in the cytosol is a major precursor for starch biosynthesis in the endosperm amyloplast. In contrast, all of the genes for OsTPT, OsPPT, and OsNTT were mainly expressed in source tissues, suggesting that their proteins play essential roles in the regulation of carbohydrate metabolism in chloroplasts. Substantial expression of the four OsGPT genes and the OspGlcT gene in both source and sink organs suggests that the transport of glucose phosphate and glucose is physiologically important in both photosynthetic and non-photosynthetic tissues. The present study shows that comprehensive analysis of expression patterns of the plastidic translocator genes is a valuable tool for the elucidation of the functions of the translocators in the regulation of starch metabolism in rice.

BACKGROUND

Telephone triage is proposed as a method of managing increasing demand for primary care. Previous studies have involved small samples in limited settings, and focused on nurse roles. Evidence is limited regarding the impact on primary care workload, costs, and patient safety and experience when triage is used to manage patients requesting same-day consultations in general practice.

OBJECTIVE

In comparison with usual care (UC), to assess the impact of GP-led telephone triage (GPT) and nurse-led computer-supported telephone triage (NT) on primary care workload and cost, patient experience of care, and patient safety and health status for patients requesting same-day consultations in general practice.

METHODS

Pragmatic cluster randomised controlled trial, incorporating economic evaluation and qualitative process evaluation.

METHODS

General practices (n = 42) in four regions of England, UK (Devon, Bristol/Somerset, Warwickshire/Coventry, Norfolk/Suffolk).

METHODS

Patients requesting same-day consultations.

METHODS

Practices were randomised to GPT, NT or UC. Data collection was not blinded; however, analysis was conducted by a statistician blinded to practice allocation.

METHODS

Primary - primary care contacts [general practice, out-of-hours primary care, accident and emergency (A&E) and walk-in centre attendances] in the 28 days following the index consultation request. Secondary - resource use and costs, patient safety (deaths and emergency hospital admissions within 7 days of index request, and A&E attendance within 28 days), health status and experience of care.

RESULTS

Of 20,990 eligible randomised patients (UC n = 7283; GPT n = 6695; NT n = 7012), primary outcome data were analysed for 16,211 patients (UC n = 5572; GPT n = 5171; NT n = 5468). Compared with UC, GPT and NT increased primary outcome contacts (over 28-day follow-up) by 33% [rate ratio (RR) 1.33, 95% confidence interval (CI) 1.30 to 1.36] and 48% (RR 1.48, 95% CI 1.44 to 1.52), respectively. Compared with GPT, NT was associated with a marginal increase in primary outcome contacts by 4% (RR 1.04, 95% CI 1.01 to 1.08). Triage was associated with a redistribution of primary care contacts. Although GPT, compared with UC, increased the rate of overall GP contacts (face to face and telephone) over the 28 days by 38% (RR 1.38, 95% CI 1.28 to 1.50), GP face-to-face contacts were reduced by 39% (RR 0.61, 95% CI 0.54 to 0.69). NT reduced the rate of overall GP contacts by 16% (RR 0.84, 95% CI 0.78 to 0.91) and GP face-to-face contacts by 20% (RR 0.80, 95% CI 0.71 to 0.90), whereas nurse contacts increased. The increased rate of primary care contacts in triage arms is largely attributable to increased telephone contacts. Estimated overall patient-clinician contact time on the index day increased in triage (GPT = 10.3 minutes; NT = 14.8 minutes; UC = 9.6 minutes), although patterns of clinician use varied between arms. Taking account of both the pattern and duration of primary outcome contacts, overall costs over the 28-day follow-up were similar in all three arms (approximately £75 per patient). Triage appeared safe, and no differences in patient health status were observed. NT was somewhat less acceptable to patients than GPT or UC. The process evaluation identified the complexity associated with introducing triage but found no consistency across practices about what works and what does not work when implementing it.

CONCLUSIONS

Introducing GPT or NT was associated with a redistribution of primary care workload for patients requesting same-day consultations, and at similar cost to UC. Although triage seemed to be safe, investigation of the circumstances of a larger number of deaths or admissions after triage might be warranted, and monitoring of these events is necessary as triage is implemented.

BACKGROUND

Current Controlled Trials ISRCTN20687662.

BACKGROUND

This project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 19, No. 13. See the NIHR Journals Library website for further project information.

The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.

The present study reports the antioxidant activity of Seabuckthorn (Hippophae rhamnoides), family Elaegnaceae, on chromium induced oxidative stress in male albino rats. Oxidative stress was induced in the rats by force-feeding of potassium dichromate equivalent to a dose of 30mg/kg body weight (BW) of chromium(VI) for 30 days. Administration of chromium decreased the body weight and increased organ to body weight ratio significantly. Chromium treatment significantly decreased reduced glutathione (GSH), and increased malondialdehyde (MDA) and creatine phosphokinase (CPK) levels; further it also enhanced glutamate oxaloacetate transferase (GOT) and glutamate pyruvate transferase (GPT) levels in the serum. Different doses of the alcoholic leaf extract of Seabuckthorn were evaluated for the protection against the chromium induced oxidative stress. The results show that the leaf extract at a concentration of 100 and 250mg/kg BW protected the animals from the chromium induced oxidative injury significantly.

Irradiating the plasmid pSV2-gpt with UV (254 nm) doses up to 200 J m-2 caused a dose-dependent increase in the yield of Gpt+ transformants when the plasmid was introduced into human cells by calcium phosphate coprecipitation. UV doses greater than 1 kJ m-2 were required to reduce the efficiency of transformation below that obtained with unirradiated DNA.

This study was designed to investigate the changes in plasma and tissue endothelin-1/endothelin-2 (ET) after liver ischemia and to assess the protective effect of anti-ET 1/ET 2 monoclonal antibody (ET antibody) against ischemia-reperfusion injury. The ET levels in the liver tissue, hepatic venous blood of the ischemic and non-ischemic sides, and in the portal venous blood were measured before and after partial liver ischemia for 1 hour in the adult dog. The ET levels in the liver tissue and hepatic venous blood on the ischemic side increased slightly during ischemia and markedly after reperfusion, whereas those on the nonischemic side showed no significant increases. The ET levels in the portal venous blood peaked at 1 to 3 hours after ischemia, which was significantly higher than the levels in the hepatic venous blood on the ischemic side and which correlated with the portal venous pressure elevated because of the partial liver clamping. The administration of antibody (2 mg/kg, intravenous) before reperfusion resulted in a significant inhibition of the postreperfusion elevations of serum-glutamic-oxaloacetic transaminase (GOT), S-glutamic pyruvic transaminase (GPT), and the indocyanine green (ICG) dye retention rate. In conclusion, ET was produced both in the liver tissue exposed to ischemia and in the vascular endothelium of the portal bed exposed to portal congestion. The ET released from the vascular endothelium, including the liver and the portal bed, was found to be a possible factor of ischemia-reperfusion injury.

The crossing of tumor cells through basement membranes represents a critical step in the metastatic process. We have used a reconstituted basement membrane (matrigel) coated on filter in a Boyden chamber to assess the invasive potential of tumor and normal cells. No correlation was found between chemoinvasion in vitro and the metastatic potential in vivo. Normal human fibroblasts and murine 3T3 fibroblasts penetrated filters coated with matrigel. On the other hand, the tumoral cells (MCF7, MCF7 gpt, MCF7 ras, BeWo, JAR, NUC-1 cells) were unable to cross the matrix. Our results suggest that in our conditions, this widely used model to assess tumoral invasion does not provide a universal assay to test the invasiveness of tumor cells. Penetration of the matrigel appears to be related to chemotactic or haptotactic responses depending upon cell types. Our data emphasize the variability of molecular events associated with basement membrane invasion.

An extensive series of oligodeoxyribonucleotides was reacted with reductively activated mitomycin C (MC), and the resulting cross-linked drug-oligonucleotide complexes were isolated by reverse-phase HPLC and characterized by nucleoside and MC-nucleoside adduct analysis. HPLC also served for assay of the yield of cross-linked oligonucleotides. AT-rich duplex oligonucleotides, containing a single central CG.CG, gave high yields of cross-links between the two guanines while those having GC.GC, instead, gave none. In another series, the central sequences CGC.GCG and CGC.ICG both yielded 50% cross-link while CGC.GCI was completely resistant. Cross-linking was conducted also in two steps: Oligonucleotides substituted monofunctionally by MC at guanine at either a CG or GC sequence were annealed with their complementary strands followed by reductive reactivation of the bound MC to form a cross-link. The CG oligomers were cross-linked quantitatively while the GC ones were again resistant. These results show unambiguously that the MC cross-link is absolutely specific to the CG.CG duplex sequence, confirming our previous finding [Chawla, A.K., Lipman, R., & Tomasz, M. (1987) in Structure and Expression, Volume 2: DNA and Its Drug Complexes (Sarma, R.H., & Sarma, M.H., Eds.) Adenine Press, Guilderland, NY]. Evidence is presented that this specificity is due to the specific orientation of the monofunctionally attached MC in the minor groove. Superimposed on the CG.CG requirement, a four-base-pair sequence preference was observed at PuCGPyr.PuCGPyr sequences. This suggests that the guanine N2 atom of GpPyr is more reactive toward the drug than that of GpPu, due to the favorable effect of the negative dipole of the O2 of the Pyr on the reaction; in accordance, GpT was more reactive than GpC.(ABSTRACT TRUNCATED AT 250 WORDS)

Chinese hamster ovary cell lines (AA8 and AS52) were stably transfected to overexpress hOgg1 protein, the human DNA repair glycosylase for 7,8-dihydro-8-oxoguanine (8-oxoG). In the transfectants, the repair rate of 8-oxoG residues induced by either potassium bromate or the photosensitizer [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbo nyl ]-2-pyrrolidinemethanolplus light was up to 3-fold more rapid than in the parental cells. However, the improved repair had little effect on the mutagenicity of potassium bromate in the guanine phosphoribosyl transferase (gpt) locus of the OGG1-transfected AS52 cells. The steady-state (background) levels of DNA base modifications sensitive to Fpg protein, which include 8-oxoG, in cells not exposed to a damaging agent were not reduced by the overexpression of Ogg1 protein. Moreover, the spontaneous mutation rates in the gpt locus were similar in OGG1-transformed and vector-only-transformed cells. The results demonstrate the potential of Ogg1 protein to remove its substrate modifications from most of the chromosomal DNA. They indicate, on the other hand, that the Ogg1 protein alone may not be rate limiting for the repair of the residual substrate modifications observed in cells under normal growth conditions.

The expression of the rat prostatic steroid binding protein gene C3 is androgen-responsive in vivo in the rat prostate. Recombinant transfection vectors were constructed containing a cloned C3 rat gene ligated directly into the dominant acting selection vector pSV2-gpt, and were used to transfect androgen-responsive mouse mammary tumor cells (S115 cells). Transformants containing intact copies of the C3 gene were isolated and shown by primer extension analysis to produce C3 transcripts that had authentic 5' termini. The steady state level of the transcripts was under androgen control in three cell lines tested which had low copy numbers of the intact C3 gene, but was not regulated in a cell line which had a very high C3 gene copy number. Despite this difference in the androgenic regulation of integrated C3 rat genes, both type of cell lines still demonstrated androgen-regulated growth characteristics.

Fasting serum bile acid (SBA) was measured by the enzymic fluorimetric method coupled with the NAD-resazurin system in 23 controls, 35 asymptomatic carriers of HIs antigen including 4 e antigen carriers and 91 patients with various liver diseases. All GHBs and e antigen carriers showed SBA within the normal range. SBA was most significantly correlated with serum bilirubin (gamma=0.74) and was a more sensitive index for impaired liver function than bilirubin or alkaline phosphatase in 164 radomly chosen samples from the liver disease group. In serial determinations of SBA with reference to GOT, GPT, changing patterns of these two parameters were classified into the parallel type and the discrepant type. Thirty two out of 40 cases with chronic liver disease belonged to the parallel type. SBA remained abnormal even after the normalization of transaminase in 12 out of 20 resolving episodes in cases of the parallel type, regardless of diagnosis. Since SBA changes according to the stage of the disease activity, serial and simultaneous estimation of SBA and GOT, GPT was found to be helpful in the observation of liver diseases. Maximum values of SBA elevation in an endogenous bile acid tolerance test after eating two egg yolks were higher than controls in 4 out of 7 cases with liver disease, who were associated with normal fasting SBA.

Triple helix formation requires a polypurine- polypyrimidine sequence in the target DNA. Recent works have shown that this constraint can be circumvented by using alternate strand triplex-forming oligonucleotides. We have previously demonstrated that (T,G)-containing triplex- forming oligonucleotides may adopt a parallel or an antiparallel orientation with respect to an oligopurine target, depending upon the sequence and, in particular, upon the number of 5'-GpT-3' and 5'-TpG-3' steps [Sun et al. (1991) C.R. Acad. Sci. Paris Ser III, 313, 585-590]. A single (T,G)-containing oligonucleotide can therefore interact with two oligopurine stretches which alternate on the two strands of the target DNA. The (T,G) switch oligonucleotide contains a 5'-part targeted to one of the oligopurine sequences in a parallel orientation followed by a 3'-part that adopts an antiparallel orientation with respect to the second oligopurine sequence. We show that a limitation to the stability of such a triplex may arise from the instability of the antiparallel part, composed of reverse-Hoogsteen C.GxG and T.AxT base triplets. Using DNase I footprinting and ultraviolet absorption experiments, we report that a benzo[e]pyridoindole derivative [(3-methoxy- 7H-8-methyl-11-[(3'-amino-propyl) amino] benzo[e]pyrido [4,3-b]indole (BePI)], a drug interacting more tightly with a triplex than with a duplex DNA, strongly stabilizes triplexes with reverse-Hoogsteen C.GxG and T.AxT triplets thus allowing a stabilization of the triplex-forming switch (T,G) oligonucleotide on alternating oligopurine- oligopyrimidine 5'-(Pu)14(Py)14-3' duplex sequences. These results lead to an extension of the range of oligonucleotide sequences for alternate strand recognition of duplex DNA.