We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells. A transgenic cell line G12 containing a single copy of the E. coli gpt gene was developed in this laboratory from Chinese hamster V79 cells. The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells. We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells. Only at a toxic dose is lead acetate significantly mutagenic to G12 cells. Lead nitrate is not significantly mutagenic at any dose. Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium. A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA. Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA. At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light. DNA damage by ultraviolet light is not enhanced by lead ions in vitro. Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair. Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen.

The aim of this study was to investigate the hepatoprotective action of the protein fraction of Phyllanthus niruri against acetaminophen (APAP) hepatotoxicity. The partially purified protein fraction of P. niruri was injected intraperitoneally in mice either prior to (preventive) or after the induction of toxicity (curative). Levels of different liver marker enzymes in serum and different antioxidant enzymes, as well as lipid peroxidation in total liver homogenates were measured in normal, control (toxicity induced) and P. niruri protein fraction-treated mice. P. niruri significantly reduced the elevated glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) levels in the sera of toxicity induced mice, compared with the control group. Lipid peroxidation levels were also reduced in mice treated with P. niruri protein fraction compared with the APAP treated control group. Among the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) levels were restored to almost normal levels compared with the control group. P. niruri treatment also enhanced reduced hepatic glutathione (GSH) levels caused by APAP administration. The results demonstrated that the protein fraction of P. niruri protected liver tissues against oxidative stress in mice, probably acting by increasing antioxidative defense.

The present study is aiming at evaluating the hepatoprotective and antioxidant effects of Astragalus polysaccharide (APS) on the carbon tetrachloride (CCl(4))-induced hepatocyte and liver injury in common carp in vitro and in vivo. In vitro, APS (200, 400 and 800 μg/ml) was added to the carp primary hepatocytes before (pre-treatment), after (post-treatment) and both before and after (pre- and post-treatment) the incubation of the hepatocytes with CCl(4) at 8 mM in the culture medium. APS at concentrations of 200, 400 and 800 μg/ml significantly improved cell viability and inhibited the elevation of glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), lactate dehydrogenase (LDH) and malondialdehyde (MDA) and significantly increased the reduced level of superoxide dismutase (SOD). In vivo administration of APS at the doses of 1.5 and 3 g/kg in the diet for 60 days prior to CCl(4) intoxication significantly reduced the elevated activities of GPT, GOT and LDH and increased the reduced levels of total protein and albumin in the serum; meanwhile, the reduced levels of SOD, glutathione and total antioxidant capacity (T-AOC) were markedly increased and the MDA formation was significantly inhibited in liver tissue. Overall results proved the hepatoprotective action of APS, which is likely related to its antioxidant activity. The results support the use of APS as a hepatoprotective and antioxidant agent in fish.

Fibrates modify the expression of genes implicated in lipoprotein and fatty acid metabolism via the peroxisome proliferator-activated receptor alpha(PPARalpha), leading to reductions in serum triglycerides and cholesterol. The expression of certain genes regulated by PPARalpha have been shown to be modified in a species dependent manner. Aspartate aminotransferase (AspAT or GOT) and alanine aminotransferase (AlaAT or GPT) are enzymes involved in intermediate metabolism in all cells and in hepatic gluconeogenesis. These enzymes are also widely used as serum markers of possible tissue damage. This study investigated whether fenofibrate could modify the expression of liver AspAT and/or AlaAT and thus possibly alter transaminase levels independently of a cytotoxic effect. In human Hep G2 cells, fenofibrate increased cytosolic AspAT (cAspAT) activity by 40% and AlaAT activity by 100%, as well as both mRNAs. Nuclear run on assays showed that this effect was, at least in part, transcriptional. Increases in mRNA were also observed in human hepatocyte cultures at concentrations of the drug attained in patients. In C57BL/6 mice, fenofibrate decreased cAspAT and cAlaAT mRNA, while these effects were abolished in PPARalpha knock-out mice. In conclusion, fenofibrate has been shown to modify cAspAT and AlaAT gene expression in a species and PPARalpha dependent manner. This is the first demonstration that cAspAT and AlaAT activities may be pharmacologically altered, independently of a toxic phenomenon.

Rat 3Y1 cells were transfected with recombinant gARC ( pSV2gpt carrying the adenovirus 12 early region 1 [E1] gene), and focus formation was observed in monolayer cultures after culture of cells in gpt-selective medium (Eagle medium containing 10% fetal calf serum, xanthine, thymidine, aminopterin, and mycophenolic acid) for 10 days, followed by focus formation. Transformed E1Y cell lines were then established from these foci. The E1Y cells were transformed morphologically similarly to cells transformed with intact adenovirus 12 DNA but formed no colonies in soft-agar culture and induced tumors in transplanted rats only after a long incubation period. For the establishment of completely transformed cells, 3Y1 cells were transformed with combinations of gARC , pE3 (pBR322 carrying the adenovirus 12 E3 gene), and gE4 ( pSV2gpt carrying the adenovirus 12 E4 gene) DNA. E1- 3Y cells (3Y1 cells transformed with gARC and pE3 DNA), E1- 4Y cells (3Y1 cells transformed with gARC and gE4 DNA), and E1-3- 4Y cells (3Y1 cells transformed with gARC , pE3 , and gE4 DNA) were established. These transformed cell lines were compared for growth in Eagle medium with 2 or 10% fetal calf serum, colony formation in soft-agar culture, and tumor growth in rats transplanted with the transformed cells. Several transformed cell lines of E1- 4Y and E1-3- 4Y cells showed colony formation in soft-agar culture and abundant expression of the E1B gene. T antigen f was seen by immunofluorescence as flecks in these cells, in which the E4 gene was transcribed, but was not seen in E1Y cells, suggesting that T antigen f was encoded by the E4 gene. The suggestion was confirmed by the observation that T antigen f was detected in COS-1 cells transfected singly with gE4 DNA by immunofluorescence with polyclonal and monoclonal antibodies. Transcription of the E4 gene was confirmed in gE4 -transfected COS-1 cells. T antigen f, one of the E4 gene products, was identified as a polypeptide of molecular weight 11,000 (E4- 11K ) by immunoprecipitation with monoclonal antibodies. The above results also suggest that expression of the E4 gene gives cells the advantage of forming colonies in soft-agar culture. A tendency was noticed for E1B gene expression to be enhanced by E4 gene expression. The relationship between enhancement of colony formation in soft-agar culture and enhancement of E1B gene expression is discussed.

The present study was conducted to determine the concentration of amino acids in the cerebrospinal spinal fluid (CSF) and the activities of two tramsaminases: glutamic oxaloacetate transaminase (GOT) and glutamic pyruvate transaminase (GPT) in human Alzheimer disease (AD) and normal brain. L-glutamic acid, L-glutamine and L-alanine are the most abundant amino acids in the CSF (50-55% of total amino acids). L-glutamine occurs at much higher levels in Alzheimer CSF compared to the normal CSF (229+/-91.8 nmol/ml in AD versus 107+/-47.2 nmol/ml in normal; P=0.0041). In contrast, L-aspartate occurs at significantly lower concentrations in Alzheimer CSF than normal CSF (46.1+/-25.7 nmol/ml in Alzheimer versus 95.2+/-52.6 nmol/ml in normal; P=0.020). In Alzheimer brain (frontal, parietal and occipital cortices) GOT is present at significantly higher activities than in normal brain cortices (about 1.5 times higher; P<0.01). No significant differences for GPT activity occurred between normal and AD brain. Since CSF receives amino acids from brain tissues, and since GOT catalyzes the conversion of L-aspartate to L-glutamate, the higher concentrations of L-glutamine (which is derived from L-glutamate), and the lower concentrations of L-aspartate found in Alzheimer CSF could be considered as a consequence of the higher activity of GOT that occurs in Alzheimer brain.

Recently, Fryxell and Moon (2005) examined methylation-dependent transition rates (5mC deamination rates), which were calculated by the difference between the CpG transition and GpC transition rates, using 4,437 transition mutations in CpG or GpC dinucleotides. They concluded that 5mC deamination rates were highly dependent on local GC content but not on local sequence lengths over which GC content was calculated or the genomic regions where the mutations occurred. Here, we reexamined these statements by using 292,216 CpG->>TpG/CpA and GpC->>GpT/ApC mutations, an increase of 66 times as much data. Contrary to Fryxell and Moon's conclusions, our analysis indicated that 5mC deamination rates in the human genome were dependent on both the local sequence length and the genomic region. Some explanations for their conclusions were provided.

Identification of novel tumor-associated antigens (TAA) capable of eliciting T-cell responses has renewed interest in the development of anti-tumor vaccines. The insertion of genes encoding specific TAA into a vaccinia virus (rVV) is one approach to vaccination since large amounts of foreign DNA can be stably integrated into the poxvirus genome. Recent reports have documented an increased therapeutic effectiveness of poxvirus-based vaccines when additional treatment with cytokines, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) were used, but the combination of these cytokines as adjuvants for a rVV encoding TAA have not been previously reported. The combination of IL-2 and IL-12 at single regimen systemic doses was toxic and sometimes fatal, manifesting largely as segmental epithelial apoptosis of the large bowel. To explore the local delivery of both cytokines to the site of vaccination, the genes encoding IL-2 and IL-12 were inserted into vaccinia virus along with a model tumor antigen gene. This construct contained five heterologous genes: LacZ (the model antigen), gpt (reporter gene), IL-2, and the two IL-12 subunit genes (p35 and p40). Treatment with this recombinant virus resulted in a reduced number of pulmonary metastases, improved survival, and minimal toxicity in a murine tumor model. The use of vaccinia virus for the insertion of other heterologous gene combinations may provide a powerful and less toxic approach for novel vaccination strategies in the treatment and prevention of cancer.

The possible involvement of oxidative damage in the progression of atherosclerosis has been suggested. There is some evidence that antioxidant therapy may be beneficial for the prevention of coronary heart disease. In this study, we investigated the relationship between coronary artery disease (CAD) and serum antioxidative status by measuring the total antioxidant status (TAS). Other relevant antioxidants, such as retinol, alpha, gamma-tocopherol, ascorbic acid, alpha, beta-carotenoids, erythrocyte glutathione peroxidase (GSH-Px) and oxidative products, were also determined in 31 male CAD patients with angiographically defined CAD and 66 male controls, aged 40-70 years, in a case-control study. The TAS levels, ratio and the concentrations of retinol, albumin, total protein and HDL cholesterol were significantly lower in the CAD patients than in the controls (p<0.01), and alpha-tocopherol and alpha/gamma-tocopherol were significantly higher in the CAD patients than in the controls. The TAS level correlated positively with gamma-GTP, GPT, GOT and uric acid (p<0.01). A multiple regression analysis in the CAD patients revealed that the TAS levels correlated most negatively with the number of diseased vessels. The concentrations of carotenoids and GSH-Px, as well as the alpha/gamma-tocopherol ratio were also significantly associated. Although conditional logistic regression analysis suggested low levels of HDL-cholesterol to be a significant coronary risk factor (OR=5.1, 95% CI=1.09-24.3), the TAS level showed no significant independent contribution to CAD. This study demonstrated an association of antioxidant parameters with the atherosclerosis progression, however, it did not confirm antioxidants as an independent risk factor for CAD event.

BACKGROUND

Previous reports have revealed that fatty liver involving obesity is closely related to insulin resistance or hyperinsulinemia in adulthood. This study investigates the importance of hyperinsulinemia in obese children with fatty liver.

METHODS

The subjects were 228 obese children 6 to 15 years old in Niigata Prefecture, Japan. The serum level of glutamic pyruvic transaminase (GPT) was evaluated as an indicator of fatty liver. The effects of their percent obesity (percent over ideal body weight), Rohrer index (g/cm3), skinfold thickness, percent body fat measured by bioelectrical impedance analysis, total cholesterol (TC), high-density lipoprotein cholesterol, triglyceride (TG), blood glucose, and immuno-reactive insulin (IRI) on GPT were evaluated using regression analyses.

RESULTS

The incidence of fatty liver (GPT over 35 IU/I) was 24.1% in this study. In simple regression analyses, percent obesity, Rohrer index, skinfold thickness, TC, TG, blood glucose, and IRI correlated positively with GPT (p < 0.05). In stepwise regression analysis including these seven variables, the predictive equation for GPT as a function of IRI alone accounted for 24.2% of the total variance of GPT. The addition of TC alone, and TC and percent obesity together increased the coefficients of determination to 28.4% and 29.9%, respectively. Rohrer index, skinfold thickness, Tg, and blood glucose were not taken as related variables with GPT.

CONCLUSIONS

Hyperinsulinemia is an important contributor to the development of fatty liver, apparently more than anthropometric data, blood glucose, or serum lipids in childhood obesity.

METHODS

Two unrelated women, aged 39 and 42 years, had been admitted (at different times) to hospital because of "recurrence of an aetiologically uncertain acute hepatitis". Both patients had a history of acute hepatitis with GPT concentration of 796 and 755 U/l, respectively. Each of them had experienced recurrences of hepatitis, each of them preceded by taking herbal remedies as alternative medication, containing kava or common (or lesser) celandine, respectively. In each patient physical examination had been unremarkable.

METHODS

Maximal values of GPT in the two patients were 422 and 350 U/l, respectively. Viral, autoimmune and metabolic causes of the hepatitis were excluded. In each of them liver biopsy revealed the picture of acute necrotizing hepatitis.

METHODS

As it was suspected that the hepatitis was medication-induced, the intake of the mentioned herbal preparations was stopped. The liver function tests quickly became normal.

CONCLUSIONS

In view of the rapid response to their withdrawal, a causal connection between intake of the herbal preparations and the recurrences of acute hepatitis is the most likely explanation in both cases.

BACKGROUND

Atherosclerosis may be associated with cognitive function; however the studies are few, especially among midlife adults.

METHODS

Participants in the beaver dam offspring study who had cognitive test data and gradable carotid artery ultrasound scans were included (n=2794, mean age: 49 years). Atherosclerosis was measured by carotid intima-media thickness (IMT) and the presence of plaque. Cognitive function was measured by the trail making test (TMT), grooved pegboard test (GPT) and mini-mental state examination (MMSE). Generalized cognitive function was defined by a summary score calculated from the TMT and GPT. Linear regression was used to evaluate the associations between carotid atherosclerosis and cognitive function tests.

RESULTS

Larger IMT was associated with lower GPT, MMSE and the summary score adjusting for multiple factors, the coefficients were: 13.8s (p<0.0001), -0.6 (p=0.007), and 0.47 (p=0.01), respectively for 1mm increase in IMT. Plaque scores were significantly associated with TMT-B, GPT, MMSE, and the summary score adjusting for age, sex and education. The associations remained statistically significant after further adjustments except for the association with TMT-B, which was attenuated and no longer significant.

CONCLUSIONS

Our results show the significant associations between markers of carotid atherosclerosis and cognitive function in a cohort of persons aged 21-84 years. Longitudinal studies are needed to further examine these associations.

Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1-/-) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1-/- and Parp-1+/+ mice (P>0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1-/- mice was 1.6-fold higher than that in Parp-1+/+ mice (P<0.05). Categorization of the mutations revealed that deletions larger than 1 kb or those accompanying 1-5 bp insertions at the deletion junctions, as well as rearrangements, were more frequently observed in Parp-1-/- than in Parp-1+/+ mice (P<0.05, respectively). In contrast, mutant frequencies of the gpt gene in the livers of Parp-1(-/-) and Parp-1(+/+) mice after BHP treatment were both elevated and there was no significant difference between the genotypes. These results indicate that Parp-1 is implicated in suppressing deletion mutations in vivo, especially those accompanying small insertions or rearrangements.

This study investigated the possible protective effects and mechanism of rhein on Acetaminophen (APAP)-induced hepatotoxicity and nephrotoxicity in rats. Treatment of rats with APAP resulted in severe liver and kidney injuries, as demonstrated by drastic elevation of serum glutamate-pyruvate transaminase (GPT), glutamate-oxaloacetic transaminase (GOT), total bilirubin (TBIL), creatinine (CREA), urea nitrogen (UREA) levels and typical histopathological changes including necrosis, phlogocyte infiltration and fatty degeneration in liver, tubules epithelium swelling and severe vacuolar degeneration in kidney. APAP caused oxidative stress, as evidenced by increased reactive oxygen species (ROS) production, nitric oxide (NO) and malondiadehyde (MDA) levels, together with depleted glutathione (GSH) concentration in the liver and kidney of rats. However, rhein can attenuate APAP-induced hepatotoxicity and nephrotoxicity in a dose-dependent manner. Our results showed that GPT, GOT, UREA and CREA levels and ROS production were reduced dramatically, NO, MDA, GSH contents were restored remarkedly by rhein administration, as compared to the APAP alone treated rats. Moreover, the histopathological damage of liver and kidney were also significantly ameliorated by rhein treatment. These findings suggested that the protective effects of rhein against APAP-induced liver and kidney injuries might result from the amelioration of APAP-induced oxidative stress.

Epigenetics refers to heritable patterns of gene expression that do not depend on alterations of the genomic DNA sequence. Nickel compounds have demonstrated carcinogenicity without any associated mutagenesis, suggesting that its mechanism of carcinogenesis is epigenetic in nature. One such potential mechanism is the heterochromatinization of chromatin within a region of the genome containing a gene sequence, inhibiting any further molecular interactions with that underlying gene sequence and effectively inactivating that gene. We report here the observation, by atomic force microscopy and circular dichroism spectropolarimetry, that nickel ion (Ni(2+)) condenses chromatin to a greater extent than the natural divalent cation of the cell, magnesium ion (Mg(2+)). In addition, we use a model experimental system that incorporates a transgene, the bacterial xanthine guanine phosphoribosyl transferase gene (gpt), differentially near, and far from, a heterochromatic region of the genome, in two cell lines, the Chinese hamster V79-derived G12 and G10 cells, respectively, to demonstrate by a DNase I protection assay that nickel treatement protects the gpt gene sequence from DNase I exonuclease digestion in the G12 cells, but not in the G10 cells. We conclude that condensation of chromatin by nickel is a potential mechanism of nickel-mediated gene regulation.

Lead (Pb(2+)) toxicity is the most common form of heavy metal intoxication in humans and animals. Therefore, the current study was conducted to evaluate the potential ameliorative effects of curcumin on lead acetate (LA)-induced deleterious effects in the liver and kidney. Forty male Wistar rats were divided into four equal groups; first group was used as a control and given both corn oil orally and vehicle of lead acetate intraperitoneally (i.p). Groups from 2-4 were treated with lead acetate (LA; 50 mg/kg BW i.p), curcumin (200 mg/kg BW orally), and curcumin plus lead acetate, respectively. Curcumin was administered 3 weeks before LA injection for 7 days. Pb(2+)-intoxicated rats have higher Pb(2+) levels compared to other treated groups. Results revealed that lead acetate significantly increased the serum levels of hepatic transaminases (GPT and GOT), urea and creatinine, while albumin was significantly decreased. In parallel, serum IgG, IgM, and IgA were significantly decreased in LA-injected rats. LA groups showed decrease in messenger RNA (mRNA) expression of catalase, SOD, GST, GPx, and alpha-1 acid glycoprotein (AGP), while the gene expression of desmin, vimentin, transforming growth factor-β1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), and alpha-2 macroglobulin (α-2M) was increased. Prior and coadministration of curcumin with LA for 7 days significantly improved the ameliorated changes in liver and kidney, immunoglobulins, and mRNA expression. Moreover, curcumin ameliorated LA-induced congestion of hepatic and renal blood vessels and decreased fibrous tissue proliferation and necrosis of hepatocytes. In the kidney, LA-induced degeneration in tubular epithelium and intraluminal hyaline casts and prior curcumin administration restored normal renal structure with mild congestion of renal blood vessels. The results clarify the potential of curcumin to counteract the immunosuppressive alteration in gene expression as well as hepatic and renal damage occurred after Pb(2+) intoxication.

Due to the intensive commercial application of silver nanoparticles (Ag-NPs), their health risk assessment is of great importance. For acute toxicity evaluation of orally administered Ag-NPs, induction of reactive oxygen species (ROS), activity of liver function enzymes [(alanine (ALT/GPT), aspartate (AST/GOT), alkaline phosphatase (ALP)], concentration of lipid hydroperoxide (LHP), comet assay, and histopathology of liver in the rat model were performed. Four groups of five male rats were orally administered Ag-NPs, once a day for five days with doses of 5, 25, 50, 100 mg/kg, body weight. A control group was also made of five rats. Blood and liver were collected 24 h after the last treatment following standard protocols. Ag-NPs exposure increased the induction of ROS, activities of the liver enzymes (ALT, AST, ALP), concentration of lipid hydroperoxide (LHP), tail migration, and morphological alterations of the liver tissue in exposed groups compared to control. The highest two doses, 50 and 100 mg/kg showed statistically significant (p < 0.05) increases in ROS induction, ALT, AST, ALP activity, LHP concentration, DNA damage, and morphological alterations of liver compared to control. Based on these results, it is suggested that short-term administration of high doses of Ag-NP may cause organ toxicity and oxidative stress.

Genetic and epigenetic factors may predispose women to polycystic ovary syndrome (PCOS), a common heritable disorder of unclear etiology. Here we investigated differences in genome-wide gene expression and DNA methylation in adipose tissue from 64 women with PCOS and 30 controls. In total, 1720 unique genes were differentially expressed (Q < 0.05). Six out of twenty selected genes with largest expression difference (CYP1B1, GPT), genes linked to PCOS (RAB5B) or type 2 diabetes (PPARG, SVEP1), and methylation (DMAP1) were replicated in a separate case-control study. In total, 63,213 sites (P < 0.05) and 440 sites (Q < 0.15) were differently methylated. Thirty differentially expressed genes had corresponding changes in 33 different DNA methylation sites. Moreover, a total number of 1913 pairs of differentially expressed "gene-CpG" probes were significantly correlated after correction for multiple testing and corresponded with 349 unique genes. In conclusion, we identified a large number of genes and pathways that are affected in adipose tissue from women with PCOS. We also identified specific DNA methylation pathways that may affect mRNA expression. Together, these novel findings show that women with PCOS have multiple transcriptional and epigenetic changes in adipose tissue that are relevant for development of the disease.

In these studies we show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The loss of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by the analysis for DNA sequences specific for the donor chromosome. Our results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results we propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.

Some families with abnormalities of chromosome 9 have been combined with others from the literature to show that AK1 and ABO must lie near the end of that chromosome. Current evidence suggests that both lie in band 9q34. MNSs, GPT and Gc can be excluded from chromosome 9.

We have previously shown that strong epitopes recognized by anti-Friend virus (FV) cytotoxic T lymphocytes (CTL) in H-2b mice are encoded in both the env and gag/pol regions of the helper friend leukemia virus genome. Two approaches have been used to identify these epitopes. At the nucleic acid level, we have constructed env genes with either of two in-frame deletions: pKR2, an env gene with a 681-bp deletion in the gp70 region and inserted into the pSV2-gpt-1 expression vector; and pKR1, an env gene with an 81-bp deletion in the p15E region and inserted into pSV2-gpt-1. Cell clones were established by transfecting Fisher rat embryo cells with pDb (the H-2Db restriction element), pNEO (for G418 selection) and either pKR1 or pKR2. Db and env gene expression was monitored by immunoprecipitation with polyclonal antibodies or by detection of viral RNA on Northern blots. Expressor cell clones were tested for susceptibility to lysis by polyclonal anti-FV/Db CTL in 51Cr-release assays. Whereas cells expressing pKR1 were lysed to the same extent as cells expressing the intact env gene, cells expressing pKR2 were resistant to lysis, suggesting that all detectable env epitopes are encoded within the 681-bp deletion. Polypeptides representing the two most likely candidate epitopes encoded in this segment were synthesized and tested for their abilities to sensitize FRE cells expressing Db alone for lysis by the CTL. One 17-mer polypeptide, AGTGDRLLNLVQGAYQA [corrected], functioned as a strong CTL epitope in this assay, but the other 18-mer polypeptide was inactive. Studies of the role of this epitope in the immune response to candidate viral vaccines are in progress.

The traditional method of site-specific mutagenesis is to introduce predetermined mutations into an expression vector, which is then transferred to cells so that the relevant gene product and the effects of the mutations can be measured. A problem with this approach is that the expression of the transferred genes varies from transformant to transformant, presumably because the number of vector copies and the site of chromosomal integration vary among transformants. While it should be possible to avoid this variability by mutagenizing the chromosomal gene itself, the labor involved in introducing predetermined mutations by homologous recombination with a mutagenized vector is usually so intense that this has not been the favored method. We describe here a system for introducing mutations into the IgH locus of hybridoma cells. This system greatly reduces the labor that would usually be required to identify and recover the rare recombinants. This is a two-step, so-called "hit-and-run," method, whereby mutations are first introduced into the chromosomal locus by targeted vector integration, after which the vector is excised so as to leave the mutation in the chromosomal target. The first step employs an enhancer trap vector bearing an enhancerless gpt gene; using this vector the frequency of randomly inserted transformants which grow in mycophenolic acid containing selective medium is so low that approximately 25% of the selected transformants have integrated the vector into the IgH locus by homologous recombination. Properly targeted transformants can then be used to derive secondary recombinants that have excised the vector and thus become gpt-. This second step which involves selection of gpt- cells by their resistance to 6-thioxanthine is also efficient, in that approximately 75% of the treated cells have excised the gpt gene by homologous recombination. Overall the labor involved in mutagenizing the chromosomal locus is not much more than is needed to produce the traditional transformants expressing a mutagenized transferred gene.

The anti-stress and anti-fatigue effects of a hot water extract of fermented rice bran (FRB) were investigated with Saccharomyces cerevisae IFO 2346 on rats or mice. Oral administration (1 g/kg/day) of a hot water extract of FRB inhibited major changes in weight of the adrenal, thymus, spleen and thyroid, showing the anti-stress effect. A hot water extract of FRB also inhibited the increase of GPT and LDH activity, cholesterol and serum glucose levels. Administration (1 g/kg/day) for 2 weeks significantly prolonged the swimming time, resulting in an increase of the anti-fatigue effect. From these results, it can be considered that FRB has an anti-stress and anti-fatigue effect.