Several gpt+ transgenic cell lines were derived from hprt V79 cells to study mutagenesis mechanisms in mammalian cells. The G12 cell line was previously shown to be hypermutable by X-rays and UV at the gpt locus compared to the endogenous hprt gene of the parental V79 cells (Klein and Rossman, 1990), and is now shown to be highly mutable by the clastogenic anti-tumor agent bleomycin sulfate. A second transgenic cell line G10, which has a different gpt insertion site, was studied in comparison with G12. Both G12 and G10 cell lines carry the stable gpt locus at a single integration site in the Chinese hamster genome, and neither spontaneously deletes the integrated gpt sequence at a high frequency. Although spontaneous mutation to 6-thioguanine resistance in G10 cells is 3-4 times higher than in G12 cells, the cell lines differ to a much greater extent when mutated by clastogens. In comparison to G12 cells, the gpt locus in G10 cells is up to 13 times more sensitive to bleomycin mutagenesis and 5 times more responsive to X-ray mutagenesis. In contrast, there is much less difference in UV-induced mutagenesis and no differences in mutagenesis induced by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The dose-dependent decrease in survival of the transgenic cells is the same for all mutagens tested, and does not differ from that of V79 cells. Neither transgenic cell line is generally hypermutable, since mutagenesis at an endogenous gene, Na+K+/ATPase, is similar to that of the parental V79 cell line. Although both cell lines can be induced to delete the transgene following clastogen exposure, deletions are not the only recovered mutations, and the cell lines can also be used to study mutations within the PCR recoverable gpt gene. The utility of these transgenic cells to investigate genome position effects related to mammalian mutagenesis mechanisms is discussed.

Transfection of HPRT- L fibroblasts with a plasmid containing two linked selectable markers genes, gpt and neo, regulated by the same eukaryotic control elements, yielded a 6-fold higher transfection frequency on selection for neo than for gpt. Transfection of HPRT- embryonal stem (ES) cells with the same plasmid yielded high levels of transfectants when selected for neo expression with G418, but a level of transfection greater than two orders of magnitude lower was observed when HAT supplemented medium was used to select for gpt expression. Selection for gpt expression in ES cells with medium containing mycophenolic acid and xanthine gave slightly higher frequencies of transfection, but still considerably lower than that for neo selection. In addition, mycophenolic acid exhibited a general cytotoxicity to ES cells with the window between toxicity of this compound to gpt- ES cells and gpt+ ES cells being very narrow. Cells selected with mycophenolic acid and xanthine for expression of gpt remained sensitive to HAT selection. Expression of gpt in a representative ES cell line, selected on mycophenolic acid and xanthine, was verified by Northern analysis and sensitivity to 6-thioguanine. While the level of mRNA expression in this ES cell line was insufficient to support growth via purine salvage when exposed to HAT medium, identical levels of gpt expression in HPRT- L cells, as judged by Northern analysis, allowed for normal growth in HAT medium. This suggests that ES cells place a greater demand on purine nucleotide biosynthesis than L cells. These results are discussed in terms of the use of gpt as a positive and negative selectable marker for gene targeting via homologous recombination in ES cells.

Ultrastructural morphology (transmission electron microscopy) and localisation of cisplatin-induced platinum (Pt)-DNA adducts (immunoelectron microscopy) were analysed in the human small cell lung cancer cell line GLC(4) and its 40-fold in vitro acquired cisplatin-resistant subline GLC(4)-CDDP, which is characterised by, among other things, a decreased DNA platination. Immunolabelling of Pt-DNA adducts was performed with the polyclonal antibody GPt, known to detect the main Pt-containing intrastrand and interstrand DNA adducts. Morphological analysis of GLC(4) and GLC(4)-CDDP at the ultrastructural level showed cells with a high nucleus/cytoplasm ratio with the majority of nuclei containing one or more nucleoli. GLC(4)-CDDP showed, in contrast to GLC(4), an extensive Golgi apparatus and an increased number of mitochondria. DNA platination was detectable in both GLC(4) and GLC(4)-CDDP. Immunoelectron microscopy showed Pt-DNA adducts primarily in the nucleus, preferentially at loci with high-density chromatin (e.g. heterochromatin, pars granulosa around nucleoli, condensed DNA in proliferating and apoptotic cells), and in mitochondria. The level of detectable Pt-DNA adducts was cell cycle status-dependent. In both cell lines, Pt-DNA adduct levels increased from non-dividing interphase cells to dividing cells and were highest in cells undergoing apoptosis. Overall localisation of Pt-DNA adducts was comparable in GLC(4) and GLC(4)-CDDP cells.

Thrombocytopenia during pregnancy is a common diagnostic and management problem. Several differential diagnosis must be considered including manifestations of thrombotic thrombocytopenic purpura (TTP). We report here on a case of a 21-year-old pregnant woman who presented initially severe thrombocytopenia (8 Gpt/l) in the 20(th)+1 week of gestation. The patient had an antibody against ADAMTS13, and enzyme activity was <5%. Immediate plasmapheresis treatment was initiated, followed by plasma infusions, and again plasmapheresis. A male neonate was delivered by caesarean section in the 32(nd )week of gestation. The child had an uncomplicated postnatal development. After delivery, the mother's platelet count and ADAMTS13 activity increased to normal values. This case shows interesting aspects of TTP in pregnancy and a close cooperation between obstetricians, nephrologists and pediatricians is necessary for a successful outcome of the pregnancy.

Numerous laboratory parameters were examined 235 patients with generalized psoriasis treated orally with retinoid and in 35 patients treated topically with anthralin as control. Computer evaluation of the obtained data revealed statistical trends to elevation of the total serum bilirubin level and increasing number of blood monocytes after long-term oral treatment. No other statistically significant changes of the laboratory data were found. Particularly, the liver function tests (transaminases, prothrombin and alkaline phosphatase) showed no significant alterations. Only in a few cases did the retinoid compound have an influence on the GPT and GOT levels. The reasons for this individual sensitivity to the drug remain unknown. No significant alterations were found in the control group treated topically with anthralin.

For the maintenance of good health of very old people such as centenarians, nutrition is very important. However, the information on their nutritional status is very limited. In this study, we evaluated the nutritional status of centenarians by comparison with elderly in their 70 s and young adults. Volunteer subjects were comprised of 71 centenarians (51 females, 20 males) who were not bed ridden, 84 elderly in their 70 s (51 females, 33 males) who had no specific health problems and 27 healthy young adults in their 20 s (17 females, 10 males). We compared 11 items of the activities of daily living (ADL), height, weight, hematological (hemoglobin, hematocrit and red blood cell count) and biochemical [total protein, albumin, total-, HDL-, LDL- and VLDL-cholesterols, triglycerides, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and creatinine] variables among the three age groups. Since the atrophy of muscle is more severe than the atrophies of other tissues in the elderly, we studied the muscle protein breakdown using plasma 3-methylhistidine (3-MH) as a marker. The results of all indices except serum lipids, GOT and GPT, were lower in the centenarians than in the subjects in their 20 s (p < 0.05) and were lower than the lower reference limits. Most of the results of the elderly in their 70s were within the reference intervals. However, as compared to the young subjects, they had: 1) normal ADL; 2) shorter height; 3) heavier weight in females and lighter weight in males; 4) lower total protein and albumin levels and albumin/globulin (A/G) ratios; and 5) higher total- and LDL-cholesterol levels in females and HDL-cholesterol levels in males (p < 0.05). The plasma 3-MH concentration was higher in the centenarian group than in the young and elderly groups (p < 0.05). However, serum creatinine concentrations also tended to be high in the group, suggesting that a high 3-MH concentration did not indicate accelerated muscle protein breakdown but rather poor kidney functions. The results of this study show that the centenarians had shrunken stature, low hematological values and poor protein nutrition but rather normal lipid nutrition, however the elderly subjects in their 70s could still maintain most nutritional parameters in relatively normal condition.

Endotoxin shock can induce the production of several inflammatory mediators such as TNF-alpha, IL-6, and IL-1beta, leading to multiple organ dysfunction and death. Erythropoietin (EPO) has been found to interact with its receptor (EPO-R), expressed in a wide variety of non-hematopoietic tissues, to induce a range of pleiotropic cytoprotective actions. We investigated the effects of low doses of EPO (300U/kg, intravenous administration) on the physiopathology and cytokine levels in endotoxin shock in conscious rats. Endotoxin shock was induced by intravenous injection of Escherichia coli lipopolysaccharide (20mg/kg) in conscious rats. Mean arterial pressure (MAP) and heart rate (HR) were continuously monitored for 48h after LPS administration. Levels of biochemical and cytokine parameters, including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), and creatine phosphokinase (CPK) were measured at 0, 1, 3, 6, 9, 12, 18, 24, and 48h after sepsis. Serum TNF-alpha, IL-6, and IL-1beta level was measured at 1h after sepsis. Endotoxin shock significantly increased blood GOT, GPT, BUN, Cre, LDH, CPK, TNF-alpha, IL-6, IL-1beta levels, and HR, while it decreased MAP. EPO further increased the markers of organ injury (GOT, GPT, BUN, Cre, LDH, and CPK), inflammatory biomarkers (TNF-alpha, IL-6, and IL-1beta) and did not affect MAP and HR after LPS. EPO disserved endotoxin shock-induced liver, kidney, lung, and small intestine damage in conscious rats. In conclusion, pre-treatment with low doses of EPO increased the release of TNF-alpha, IL-6, and IL-1beta, along with aggravating endotoxin shock-induced markers of organ injury in conscious rats.

OBJECTIVE

The present study investigated the effort-reward imbalance (ERI) model-related biological markers of disease risks in Japanese workers of a recently downsized manufacturing corporation.

METHODS

A total of 441 workers was examined to find whether situational effort-reward imbalance or personal overcommitment was associated with hematological and biochemical measurements, serum cortisol, and urine biopyrrins as oxidative metabolites of antioxidant bilirubin.

RESULTS

The effort-reward imbalance was positively associated with the values of red blood cells, hemoglobin, hematocrit, triglycerides and glutamic pyruvic transaminase (GPT), and negatively correlated with the high-density lipoprotein cholesterol level. Overcommitment was positively related to the values of hematocrit and glucose levels, but negatively associated with the total protein level. The relationships between effort-reward imbalance and GPT level, and that between overcommitment and glucose level, persisted when potential confounders were adjusted for. The ERI model was not significantly related to either cortisol or biopyrrins levels.

CONCLUSIONS

The ERI model seems to have an impact on the physical health of the downsized Japanese workers, although the results are mixed and are different from those in workers of Western countries.

The effect of ten phytotherapeutic products on CCl(4) intoxicated liver in albino male Wistar rats was investigated. Biochemical parameters, including serum transaminase activity (GPT and GOT), histoenzymological measurements (lactate dehydrogenase, LDH; succinate dehydrogenase, SDH, cytochromoxidase, CyOx; Mg(2+)-dependent adenosine triphosphatase, ATP-ase) and histochemical (Sudan black) and histological examinations (haematoxylin-eosin staining) of the liver were investigated. Some positive effects such as the reduction of hepatocytolysis and steatosis, and a return to normal values of the activity of some enzymes in the following plants: Chrysanthemum balsamita, Echinacea pallida, Calendula officinalis and Corylus avelana were obtained.

The modulating effects of Korean ginseng saponins on ovarian functions were investigated in immature rats superovulated with pregnant mare serum gonadotropin (PMSG). A single dose of 1 mg (0.1 ml/head) of Korean ginseng total saponin (GTS), Korean ginseng protopanaxatriol saponin (GPT), Korean ginseng protopanaxadiol saponin (GPD), or ginsenoside-Rb1 (Gin-Rb1) was intravenously injected via jugular vein catheter three times at 1 h (early follicular phase), 25 h (middle follicular phase), and 50 h (late follicular phase) after 30 IU PMSG administration. GPD and Gin-Rb1 significantly suppressed excessive ovulatory response caused by PMSG (p<0.05). All Korean ginseng saponins significantly improved oocyte quality by decreasing the proportion of abnormal oocytes (p<0.05). Gin-Rb1 significantly decreased preovulatory serum levels of androgens and 17beta-estradiol, while GPD increased preovulatory serum progesterone level (p<0.05). GPD significantly the increased postovulatory serum progesterone level (p<0.05). These results provide strong evidence that Korean ginseng saponins have a curative effect on ovarian dysfunction caused by excessive stimulation with PMSG.

The effect of iron-overload on both hepatic lipid peroxidation and chemiluminescence was studied in early stages after iron-dextran injection. Total hepatic iron content was markedly elevated over control values 2-6 h after iron dose. A 4-fold increase in light emission was detected after 4-6 h after iron injection. Plasma GOT, GPT and LDH activities were not affected by the treatment suggesting that cell permeability was not affected by necrosis. Increases in the generation of thiobarbituric acid reactive substances (TBARS) and chemiluminescence in liver homogenates, were determined as a function of time after iron administration, in the presence of NADPH as cofactor. Under the same experimental conditions, microsomal cytochrome P-450 content was decreased by 40%, 2 h after iron treatment. To evaluate liver antioxidant defenses, catalase, superoxide dismutase and glutathione peroxidase activities were determined. Glutathione peroxidase activity in the homogenate was not affected by the treatment. Catalase and superoxide dismutase activities declined by 25 and 36%, respectively, compared with control values 4 h after the iron dose. Our data suggest that lipid peroxidation occurs after mild iron overload even though the liver remains functional.

1. We have investigated the effects of (i) several guanidines on the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) in murine cultured macrophages and rat aortic vascular smooth muscle cells (RASM); and (ii) 1-amino-2-hydroxy-guanidine, the most potent inhibitor of iNOS activity discovered, on haemodynamics, multiple organ (liver, renal, and pancreas) dysfunction and iNOS activity in rats with endotoxic shock. 2. The synthesized guanidine analogues caused concentration-dependent inhibitions of the increase in nitrite formation caused by lipopolysaccaride (LPS, 1 microgram ml-1) in J774.2 macrophages and RASM cells with the following rank order of potency: 1-amino-2-hydroxy-guanidine>> 1-amino-2-methyl-guanidine>> 1-amino-1-methyl-guanidine>> 1-amino-1,2-dimethyl-guanidine. Interestingly, 1-amino-2-hydroxy-guanidine (IC50: J774.2, 68 microM; RASM, 114 microM) was more potent in inhibiting nitrite formation caused by LPS than NG-methyl-L-arginine, but less potent than aminoethyl-isothiourea. 3. In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 115 +/- 4 mmHg (time 0) to 98 +/- 5 mmHg at 2 h (P < 0.05, n = 10) and 69 +/- 5 mmHg at 6 h (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 mg kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine (10 mg kg-1, i.v. plus 10 mg kg-1 h-1 starting at 2 h after LPS) prevented the delayed hypotension and vascular hyporeactivity seen in LPS-rats. However, 1-amino-2-hydroxy-guanidine had no effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 4. Endotoxaemia for 6 h caused a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT) and bilirubin, and hence, liver dysfunction. Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine significantly attenuated the liver dysfunction caused by LPS (P < 0.05, n = 10). Injection of LPS also caused a rapid (almost maximal at 2 h) increase in the serum levels of urea and creatinine, and hence, renal dysfunction. This renal dysfunction was not affected by 1-amino-2-hydroxy-guanidine (P>> 0.05; n = 10). Endotoxaemia also caused a dysfunction of pancreas (rise in serum levels of lipase) as well as a metabolic acidosis (falls in PCO2, HCO3 and base excess). Both pancreatic dysfunction and metabolic acidosis were largely attenuated by treatment of LPS-rats with 1-amino-2-hydroxy-guanidine. In rats infused with saline rather than LPS, 1-amino-2-hydroxy-guanidine had no effect on liver, renal or pancreatic function (n = 4). 5. Endotoxaemia for 6 h resulted in a rise in the serum levels of nitrite (11.0 +/- 0.8 microM, P < 0.01, n = 10), which was significantly reduced by 1-amino-2-hydroxy-guanidine (6.5 +/- 0.7 microM, P < 0.05, n = 10). Endotoxaemia for 6 h was also associated with a significant increase in iNOS activity in lung and liver, which was significantly reduced in lung or liver homogenates obtained from LPS-rats treated with 1-amino-2-hydroxy-guanidine. In addition, endotoxaemia for 6 h resulted in a significant increase in myeloperoxidase activity (MPO), an indicator of neutrophil infiltration, in the liver. Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine did not affect the rise in MPO-activity in the liver caused by endotoxin. 6. Thus, 1-amino-2-hydroxy-guanidine is a potent inhibitor of iNOS activity in macrophages or RASM in culture as well as in rats with endotoxic shock. Inhibition of iNOS activity with 1-amino-2-hydroxy-guanidine prevents the delayed circulatory failure and attenuates the dysfunction of liver, and pancreas, as well as the metabolic acidosis caused by endotoxaemia.

Depletion of the stratospheric ozone layer leads to an increase in ambient UV loads, which are expected to raise skin cancer incidences. Tumor development in the skin could be a multistep process in which various genetic alterations, such as point mutations and deletions, occur successively. Here, we demonstrate that UVB irradiation efficiently induces deletions in the epidermis using a novel transgenic mouse, gpt delta. In this mouse model, deletions in lambda DNA integrated in the chromosome are preferentially selected as Spi(-) (sensitive to P2 interference) phages, which can then be subjected to molecular analysis. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2). After 4 weeks, lambda phage was rescued from the genomic DNA of the epidermis by in vitro packaging reactions. The mutant frequencies of Spi(-) with large deletions in the epidermis increased >15-fold at a UVB dose of 0.5 kJ/m(2) over the control. Molecular sizes of most of the large deletions were >1000 bp. More than one-half of the large deletions occurred between short direct-repeat sequences from 1 to 6 bp, and the remainder had flush ends. In the unirradiated mouse, almost all of the Spi(-) mutants were 1-bp frameshifts in runs of identical bases. These results suggest that UVB irradiation induces deletions in the murine epidermis, and most of the deletions are generated through end-joining of double strand breaks in DNA.

Hepatoprotective effect of Tamra bhasma has been studied on cumene hydroperoxide (CHP) induced peroxidation, reduced glutathione content and superoxide dismutase (SOD)-in rat liver homogenate. The drug was orally given for 8 days which showed significant reduction in the level of malondialdehyde (MDA) production at different concentrations of cumene hydroperoxide in vitro. Glutathione content was maintained upto seventy minutes and SOD activity was enhanced to 166%. These animals did not show any rise in serum GOT and GPT. On similar doses no histological changes were observed in liver. The results suggested that Tamra bhasma is a strong antioxidant drug and could be used in the management of lipid peroxidation with no detectable adverse effect.

Background/Introduction: Wild bitter gourd (Momordica charantia L. var. abbreviate Seringe) common vegetable in Asia, is used in traditional medicine to treat various diseases, including inflammation. Extant literature indicates that wild bitter gourds have components that activate PPARα and PPARγ. This research probed influence of adding wild bitter gourd to diets on inflammation responses in mice with sepsis. Purpose: This study evaluated influence of eating wild bitter gourd on inflammation responses in mice with sepsis. Methods: We injected intraperitoneal LPS to induce sepsis. Male BALB/c mice were divided normal, sepsis, positive control, and three experimental groups. The latter ate diets with low (1%), moderate (2%), and high (10%) ratios of wild bitter gourd lyophilized powder. Before mice were sacrificed, with the exception of the normal group, intraperitoneal injection of LPS induced sepsis in each group; positive control group was injected with LPS after PDTC. Results: This experiment revealed weights in groups with added wild bitter gourd starkly lower than those of the remaining groups. Blood lipids (TG, cholesterol, and NEFA) were also lower in comparison to the sepsis group, and blood glucose concentrations recovered and approached normal levels. Blood biochemistry values related to inflammation reactions indicated GOT, GPT, C-RP, and NO concentrations of groups with wild bitter gourd added all lower than that of the sepsis group. Secretion levels of the spleen pro-inflammatory cytokines IL-1, IL-6, and TNF-α tallied significantly lower in comparison to the sepsis group, whereas secretion levels of IL-10 anti-inflammatory cytokine increased. Expression level of proteins NF-κB, iNOS, and COX-2 were inhibited significantly. Conclusion: Wild bitter gourd in diets promoted lipid metabolism, improved low blood glucose in sepsis, and attenuated inflammatory stress. These findings suggested that this plant food might provide medical benefits for certain persons.

Natural remedies from medicinal plants are considered to be effective and safe alternative treatment for diabetes mellitus. The aim of the present study was to investigate the hypoglycemic activity of the crude tea leaves extract on streptozotocin (STZ)-induced diabetic mice. The average body weight of animals with diabetes and their percentage changes of body weight gain after 15 and 30 days were significantly lower than that of the normal control mice. In diabetic mice, supplementation with tea leaves extract decreased the loss of body weight. After 15 and 30 days, significant increases in the levels of serum glucose, triglycerides, cholesterol, creatinine, urea, uric acid, glutamic pyruvic acid transaminase (GPT) and glutamic oxaloacetic acid transaminase (GOT) were noted in STZ-diabetic mice fed with normal diet. Also, the values of total protein in this group were statistically declined after 15 and 30 days. The levels of serum glucose and GPT were significantly elevated after 15 and 30 days in diabetic mice supplemented with tea leaves extract. Moreover, the level of serum GOT was notably increased after 30 days. Insignificant alterations were observed in the levels of serum triglycerides, cholesterol, total protein, creatinine, urea and uric acid in diabetic mice supplemented with tea leaves extract. Thus, the present results have shown that tea leaves extract has the antihyperglycemic, antihyperlipidemic, and antihyperproteinemic effects and consequently may alleviate liver and kidney damage associated with STZ-induced diabetes in mice.

The antimutagenic activity of the methanolic extract of the fruiting bodies of Ganoderma lucidum (Fr.) P. Krast. occurring in South India was investigated. The activity was assayed by Ames Salmonella mutagenicity test using histidine mutants of Salmonella typhimurium tester strains, TA98, TA100 and TA102. The methanolic extract of the mushroom significantly inhibited (P<0.001) the in vitro sodium azide (NaN(3)), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitro-o-phenylenediamine (NPD), and benzo[a]pyrene (B[a]P) induced his(+) revertants in a dose dependent manner. In vivo antimutagenic activity of extract was also assayed by determining the mutagenicity of the urine of rats administrated with B[a]P as a mutagen. The prior administration of extract markedly inhibited mutagenicity induced by B[a]P. The results indicated that the methanolic extract of Ganoderma lucidum occurring in South India possessed significant antimutagenic activity. The effect of B[a]P on hepatic enzymes, such as serum glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and alkaline phosphtase (ALP), were also evaluated. The extract prevented the increase of SGOT, SGPT, and ALP activities consequent to B[a]P challenge, and enhanced the levels of reduced glutathione (GSH) and activities of glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT). The extract also profoundly inhibited lipid peroxidation induced by B[a]P. The results revealed that Ganoderma lucidum extract restored antioxidant defense and prevented hepatic damage consequent to the challenge by B[a]P.

BACKGROUND

Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen.

METHODS

We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation.

RESULTS

Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12.

CONCLUSIONS

This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.

The influence of hemodialysis on plasma fenofibric acid kinetics has been investigated in patients with chronic renal failure given 300 mg of fenofibrate in a single oral dose. A very pronounced lengthening of the fenofibric acid plasma decay was observed in both hemodialyzed (n = 6) and nonhemodialyzed (n = 9) patients. Hemodialysis did not modify the plasma levels and the ultrafiltrates contained very small amounts of fenofibric acid. The repeated daily administration of 100 mg of fenofibrate during 2 weeks in 5 renal patients on regular hemodialysis resulted in increasing plasma levels and led to progressive cumulation of fenofibric acid. Plasma fenofibric acid conjugates could not be detected. No particular clinical side effects or increase of CPK, GOT, GPT were be observed.

Fructus Schizandrae sinensis Baill, a traditional Chinese medicine, used as tonic and sedative, has been shown at the beginning of 70's to lower the elevated serum glutamic-pyruvic transaminase (SGPT) levels of patients suffering from chronic viral hepatitis. During past 20 years, a series of neolignans have been isolated and identified as effective principles. Pharmacological studies revealed that they increased liver protein and glycogen synthesis, antagonized liver injuries from CCl4 and thioacetamide. The mechanism of SGPT lowering was considered as a hepato-protective and membrane stabilize action, although inhibition of the activity of liver GPT may also be existed. It was found that some principles of Schizandrae have an inducing effect on hepatic microsomal drug-metabolizing enzyme system P-450, thus explained their anti-toxic, anti-carcinogenic and anti-mutagenic effects. A synthetic derivative compound of Schisandrin called DDB has most of the above mentioned actions now used widely in China as a hepato-protective drug with high effectiveness in normalizing liver functions and very low side effects. From natural Schisandrin to synthesized DDB, pointed out a successful way in the development of new drugs from natural products.

We investigated the interaction with DNA of two synthetic derivatives of the antitumor antibiotic rebeccamycin: R-3, which is a potent topoisomerase I inhibitor and contains a methoxyglucose moiety appended to the indolocarbazole chromophore, and its aglycone, R-4. Spectroscopic measurements indicate that R-3 intercalates into DNA and that its carbohydrate domain contributes significantly to reinforce the affinity for DNA. Two complementary ligation assays concur that R-3, but not its aglycone counterpart, exerts a significant effect on the curvature and/or the flexibility of DNA. The sugar moiety may be responsible for preferential binding of R-3 to circular (or bent) DNA molecules as opposed to linear DNA fragments. The sequence selectivity of binding to DNA has been studied thoroughly by footprinting with DNase I and two other nucleases. The glycosylated compound is highly selective for nucleotide sequences containing GpT (ApC) and TpG (CpA) steps. The derivative lacking the sugar moiety on the indolocarbazole chromophore binds at essentially identical sites but with considerably lower affinity, so it seems that the chromophore rather than the carbohydrate is responsible for the preferential binding to sequences surrounding GpT and TpG steps. The influence of the exocyclic substituents present on the bases at the recognition sites (i.e., the 2-amino group of guanine and the 5-methyl group of thymine) was evaluated using two series of modified DNA molecules prepared by polymerase chain reaction containing inosine and/or 2,6-diaminopurine and uridine and/or 5-methylcytosine residues. The introduction of the amino group onto purine residues or the addition of a methyl group to pyrimidine residues suffices to create new drug binding sites. Therefore, unlike most DNA-binding small molecules, the rebeccamycin analogue seems to be highly sensitive to any modification of the exocyclic substituents on the bases in both the major and minor grooves of the double helix. The footprinting profiles with the different DNA fragments bear a remarkable resemblance to those determined for nogalamycin and bisnaphthalimide compounds known to recognize their preferred GpT and TpG sites via intercalation from the major groove. The unique DNA binding characteristics of the rebeccamycin analogue correlate well with its inhibitory effects on topoisomerase I.

Changes in CSF enzyme activity were studied after brain trauma for their prognostic value. Raised values of CPK and HBDH were demonstrated in the CSF of patients with severe brain injuries. Standardised cold lesions of the brain were induced in cats. The activities of the enzymes CPK, HBDH, LDH, GOT, GPT, and pseudocholinesterase were studied at half hour intervals in the cerebrospinal fluid and at hourly intervals in the serum. A statistically highly significant increase of all enzymes studied developed in the CSF. The greatest changes occurred within four hours of freezing. Large increases could occur in half an hour. Isoenzyme studies demonstrated that CPK and LDH were of cerebral origin. No consistently significant changes could be shown in the serum enzyme activity. It is concluded that after brain injuries, enzymes are released into the extracellular fluid of the brain and transported to the CSF. The limited value of a single enzyme estimation is emphasised. The results described seem to provide indirect evidence for transependymal flow of extracellular fluid in brain oedema.

Elevated plasma levels of xanthine oxidase and liver function parameters have been associated with inflammatory events in several human diseases. While xanthine oxidase provides in vitro protection against malaria, its pathophysiological functions in vivo and interactions with liver function parameters remain unclear. This study examined the interactions and plasma levels of xanthine oxidase (XO) and uric acid (UA), catalase (CAT) and liver function parameters GOT, GPT and bilirubin in asymptomatic (n=20), uncomplicated (n=32), and severe (n=18) falciparum malaria children aged 3-13 years. Compared to age-matched control (n=16), significant (p<0.05) elevation in xanthine oxidase by 100-550%, uric acid by 15.4-153.8%, GOT and GPT by 22.1-102.2%, and total bilirubin by 2.3-86% according to parasitaemia (geometric mean parasite density (GMPD)=850-87100 parasites/microL) was observed in the malarial children. Further comparison with control revealed higher CAT level (16.2+/-0.5 vs 14.6+/-0.4 U/L; p<0.05) lacking significant (p>0.05) correlation with XO, but lower CAT level (13.4-5.4 U/L) with improved correlations (r=-0.53 to -0.91; p<0.05) with XO among the asymptomatic and symptomatic malaria children studied. 75% of control, 45% of asymptomatic, 21.9% of uncomplicated, and none of severe malaria children had Hb level>11.0 g/dL. Multivariate analyses further revealed significant (p<0.05) correlations between liver function parameters and xanthine oxidase (r=0.57-0.64) only in the severe malaria group. We conclude that elevated levels of XO and liver enzymes are biochemical features of Plasmodium falciparum parasitaemia in Nigerian children, with both parameters interacting differently to modulate the catalase response in asymptomatic and symptomatic falciparum malaria.

8-Nitroguanosine is a nitratively modified nucleoside that is formed endogeneously under inflammatory conditions dependent on nitric oxide production, particularly associated with cancer risks. Here, we investigated the mutagenic potential of 8-nitroguanosine in mammalian cells. Treatment with 8-nitroguanosine (10-1000 microM) for 1h significantly increased (by 6-8 times) the mutation frequency of the xanthine-guanine phosphoribosyltransferase (gpt) gene in AS52 cells without cytotoxic effects. 8-Nitroguanosine treatment induced a G-to-T transversion in gpt gene at position 86. It also significantly increased levels of abasic sites in DNA. These observations suggest that formation of 8-nitroguanosine may contribute to the pathogenesis of inflammation-associated carcinogenesis.