BACKGROUND

Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIalpha and TFPIbeta. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIbeta) and indirect (TFPIalpha) glycosylphosphatidyl-inositol (GPT)-mediated anchorage.

OBJECTIVE

Compare the structure and properties of TFPIalpha and TFPIbeta.

METHODS

TFPIalpha and TFPIbeta, with protein molecular masses of 36 and 28 kDa, respectively, migrate similarly (46 kDa) on SDS-PAGE. Experiments using specific glycosidases were carried out to determine the different glycosylation pattern of the two forms. ECV304 cells, a cell line with some endothelial properties, were stimulated with IL-lbeta, LPS, and TNFalpha for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIalpha, TFPIbeta or both were produced using a plasmid-based small-interfering RNA technique. Surface TFPI activity was determined by a two-stage chromogenic assay based on the ability of each form to inhibit FXa activation by FVIIa on cells with comparable amount of tissue factor (TF).

CONCLUSIONS

The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIbeta. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIalpha comprises 80% of the surface-TFPI, TFPIbeta was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIalpha.

BACKGROUND

Although a large body of literature exists on cognitive functioning in alcohol-exposed children, it is unclear if there is a signature neuropsychological profile in children with Fetal Alcohol Spectrum Disorders (FASD). This study assesses cognitive functioning in children with FASD from several American Indian reservations in the Northern Plains States, and it applies a hierarchical model of simple versus complex information processing to further examine cognitive function. We hypothesized that complex tests would discriminate between children with FASD and culturally similar controls, while children with FASD would perform similar to controls on relatively simple tests.

METHODS

Our sample includes 32 control children and 24 children with a form of FASD [fetal alcohol syndrome (FAS) = 10, partial fetal alcohol syndrome (PFAS) = 14]. The test battery measures general cognitive ability, verbal fluency, executive functioning, memory, and fine-motor skills.

RESULTS

Many of the neuropsychological tests produced results consistent with a hierarchical model of simple versus complex processing. The complexity of the tests was determined "a priori" based on the number of cognitive processes involved in them. Multidimensional scaling was used to statistically analyze the accuracy of classifying the neurocognitive tests into a simple versus complex dichotomy. Hierarchical logistic regression models were then used to define the contribution made by complex versus simple tests in predicting the significant differences between children with FASD and controls. Complex test items discriminated better than simple test items. The tests that conformed well to the model were the Verbal Fluency, Progressive Planning Test (PPT), the Lhermitte memory tasks, and the Grooved Pegboard Test (GPT). The FASD-grouped children, when compared with controls, demonstrated impaired performance on letter fluency, while their performance was similar on category fluency. On the more complex PPT trials (problems 5 to 8), as well as the Lhermitte logical tasks, the FASD group performed the worst.

CONCLUSIONS

The differential performance between children with FASD and controls was evident across various neuropsychological measures. The children with FASD performed significantly more poorly on the complex tasks than did the controls. The identification of a neurobehavioral profile in children with prenatal alcohol exposure will help clinicians identify and diagnose children with FASD.

The tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and teleocidin markedly enhanced the transformation of C3H 10T1/2 mouse fibroblasts when these cells were transfected with the cloned human bladder cancer c-rasH oncogene. Transfection studies with the drug resistance marker gpt and time course studies indicate that this enhancement is not simply an effect on the process of DNA transfection. These findings, together with parallel studies with NIH 3T3 fibroblasts, also indicate that the competence of animal cells for DNA transfection is a function of the recipient cell line, the transfected marker, and the growth conditions. Our findings suggest that during multistage carcinogenesis tumor promoters may complement the function of activated cellular oncogenes.

Ischaemic stroke is associated with an excessive release of glutamate in brain. GOT (glutamate-oxaloacetate transaminase) and GPT (glutamate-pyruvate transaminase) are two enzymes that are able to metabolize blood glutamate facilitating the lowering of extracellular levels of brain glutamate. Our aim was to study the association between blood levels of both enzymes and stroke outcome in patients with acute ischaemic stroke. We prospectively studied 365 patients with first ischaemic stroke<12 h. Glutamate, GOT and GPT levels were determined in blood samples obtained at admission. We considered functional outcome at 3 months [good outcome: mRS (modified Rankin Scale)≤2; poor outcome mRS >2], END (early neurological deterioration) in the first 72 h [increment ≥4 points in NIHSS (National Institutes of Health Stroke Scale)] and infarct volume [CT (computed tomography) at 36-72 h] as end points. We have found an inverse correlation between GOT and GPT levels and blood glutamate levels. Patients with poor outcome showed lower levels of GOT (11.9±8.2 compared with 22.7±10.2 m-units/ml, P<0.0001) and GPT (19.5±14.3 compared with 24.7±20.3 m-units/ml; P=0.004). A negative correlation has been found between GOT (Pearson coefficient=-0.477, P<0.0001) and GPT (Pearson coefficient=-0.116; P=0.027) levels and infarct volume. Patients with END showed higher levels of blood glutamate (381.7±97.9 compared with 237.6±114.0 μmol/l, P<0.0001) and lower levels of GOT (10.8±6.7 compared with 18.1±10.8 m-units/ml; P<0.0001). This clinical study shows an association between high blood GOT and GPT levels and good outcome in ischaemic stroke patients, this association being stronger for GOT than GPT levels.

We have studied the molecular mechanisms of spontaneous mutations in mouse cells carrying a selectable bacterial gene. The mouse cells carry the Escherichia coli xanthine (guanine) phosphoribosyltransferase (gpt) gene in a retroviral shuttle vector integrated into chromosomal DNA in a proviral form. Cells with spontaneous mutations in the gpt gene were selected as resistant to 6-thioguanine and then were fused with COS cells for recovery of the mutant genes. Out of a total of 77 independent 6-thioguanine-resistant cell lines isolated in this study, vector sequences could be rescued from 43 of the mutant lines, and the base sequences were determined for the gpt genes in all 43 of these lines. There was a variety of mutational events among the mutant gpt genes sequenced. The most frequent mutational event was a deletion (in 29 of the 43 mutant genes), and the next most frequent event was a base substitution mutation (in 11 of the 43 mutant genes). Among the deletion mutants, the great majority represent deletions of less than 10 base pairs. In fact, 19 of the 29 deletion mutants had deletions of 3 base pairs, and among the mutants with 3-base-pair deletions, there was a very strong deletion hot spot appearing in 16 independent mutants. All 19 of the 3-base-pair deletions resulted in the "in frame" loss of an aspartic acid codon. Among the base substitution mutations, transitions and transversions occurred with approximately equal frequency. Our results raise the possibility that small deletions represent the predominant mechanisms by which spontaneous mutations occur in mammalian cells.

The nature of the interaction of glucose with toluene-treated cells of Escherichia coli leading to inhibition of adenylate cyclase was examined by the use of analogues. Those analogues with variations of the substituents about carbon atoms 1 or 2 (e.g. alpha-methylglucoside or 2-deoxyglucose) are inhibitory, and they are also substrates of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Analogues with changes in other parts of the molecule (e.g. 3-O-methylglucose or galactose), L-glucose and several disaccharides and pentoses, do not inhibit adenylate cyclase and are not substrates of the phosphotransferase system. This correlation suggests some functional relationship between the adenylate cyclase and phosphotransferase systems. Further studies were done with mutants defective in glucose enzymes II of the phosphotransferase system (designated GPT and MPT); these two activities are measured by phosphorylation of alpha-methyl-glucoside and 2-deoxyglucose, respectively. The wild-type parent phosphorylates both analogues, and both inhibit adenylate cyclase. In the GPT- mutant, alpha-methylglucoside does not inhibit adenylate cyclase and is not phosphorylated, while 2-deoxyglucose is inhibitory and phosphorylated. In the GPT- MPT- double mutant, adenylate cyclase activity is present, but neither alpha-methylglucoside nor 2-deoxyglucose inhibits adenylate cyclase, and neither sugar is phosphorylated. These studies demonstrate that glucose inhibition of adenylate cyclase in toluene-treated cells requires an interaction of this sugar with either the GPT or mpt enzyme II of the phosphotransferase system.

Helicobacter pylori is a human carcinogen, but the mechanisms evoked in carcinogenesis during this chronic inflammatory disease remain incompletely characterized. We determined whether chronic H. pylori infection induced mutations in the gastric mucosa of male and female gpt delta C57BL/6 mice infected for 6 or 12 mo. Point mutations were increased in females infected for 12 mo. The mutation frequency in this group was 1.6-fold higher than in uninfected mice of both sexes (P < 0.05). A:T-to-G:C transitions and G:C-to-T:A transversions were 3.8 and 2.0 times, respectively, more frequent in this group than in controls. Both mutations are consistent with DNA damage induced by oxidative stress. No increase in the frequency of deletions was observed. Females had more severe gastric lesions than males at 6 mo postinfection (MPI; P < 0.05), but this difference was absent at 12 MPI. In all mice, infection significantly increased expression of IFNgamma, IL-17, TNFalpha, and iNOS at 6 and 12 mo, as well as H. pylori-specific IgG1 levels at 12 MPI (P < 0.05) and IgG2c levels at 6 and 12 MPI (P < 0.01 and P < 0.001). At 12 MPI, IgG2c levels in infected females were higher than at 6 MPI (P < 0.05) and also than those in infected males at 12 MPI (P < 0.05). Intensity of responses was mediated by sex and duration of infection. Lower H. pylori colonization indicated a more robust host response in females than in males. Earlier onset of severe gastric lesions and proinflammatory, Th1-biased responses in female C57BL/6 mice may have promoted mutagenesis by exposing the stomach to prolonged oxidative stress.

Epigenetic gene silencing by aberrant DNA methylation of gene promoter regions is a nonmutagenic but heritable epigenetic mechanism that may mistakenly cause the silencing of important cancer-related tumor suppressor genes. Using a transgenic, V79-derived, mammalian cell line (G12) that contains a bacterial gpt reporter gene in its DNA, we can study carcinogen-induced gene inactivation by mutagenic as well as epigenetic DNA methylation mechanisms. Whereas numerous carcinogens have previously been shown to be mutagenic in these cells, a few carcinogens, including nickel, diethylstilbestrol, and X-rays, are also capable of silencing the G12 cell gpt transgene by aberrant DNA methylation. Here we report for the first time that carcinogenic potassium chromate salts can also induce aberrant DNA methylation in this system. In contrast insoluble barium chromate produced significant level of mutations in these cells but did not cause DNA methylation changes associated with transgene expression.

We have studied the mutagenicity (by selecting for mutants resistant to 6-thioguanine) and cytotoxicity (by determining cellular cloning efficiency) of physical and chemical agents in Chinese hamster ovary (CHO) cells, clone CHO-K1-BH4 (K1-BH4), and its radiation-hypersensitive transformant, AS52. AS52 cells contain a single functional copy of a bacterial gene, the xanthine/guanine phosphoribosyltransferase (gpt) gene instead of its mammalian equivalent, the hypoxanthine/guanine phosphoribosyltransferase (hprt) gene. We found that x-ray and neutron irradiations are equally toxic to both cell types; however, these physical agents are approximately equal to 10 times more mutagenic to AS52 cells than to K1-BH4 cells. Our earlier studies using Southern blot analysis showed that x-irradiation produces mostly or exclusively deletion mutations in both cell types. If reactive oxygen species mediate the mutagenic effects of radiations and chemicals, then radiomimetic compounds such as streptonigrin and bleomycin, which exert their biological effects via reactive oxygen species, and oxidizing compounds such as potassium superoxide and hydrogen peroxide should elicit a similar differential mutagenic response in both cell types. On the other hand, agents such as ethyl methanesulfonate, ICR 191, and UV light, which do not produce reactive oxygen species, should not elicit differential mutagenicity. Our results fulfill such predictions. The apparent hypermutability of AS52 cells probably results from a higher recovery of multilocus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants.

Preeclampsia (PE), a specific syndrome of pregnancy, can be classified into early and late onset, depending on whether clinical manifestations occur before or after 34 weeks' gestation. We determined whether plasma concentrations of Hsp60 and Hsp70 were related to circulating cytokine levels, as well as kidney and liver functions, in early- and late-onset PE. Two hundred and thirty-seven preeclamptic women (95 with early- and 142 with late-onset PE) were evaluated. Plasma levels of Hsp60, Hsp70, and their specific antibodies, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-10, IL-12, and soluble TNF-α-receptor I (sTNFRI) concentrations, were determined by enzyme-linked immunosorbent assay (ELISA). Concentrations of Hsp70, TNF-α, IL-1β, IL-12, and sTNFRI were significantly elevated in patients with early-onset PE compared with women with late-onset PE; IL-10 levels were significantly lower in the early-onset PE group. Concentrations of urea, uric acid, proteinuria, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and lactate dehydrogenase (LDH) were also significantly higher in early-onset PE. The percentage of infants with intrauterine growth restriction was also significantly higher in women with early-onset PE. There were positive correlations between Hsp70 levels and TNF-α, TNFRI, IL-1β, IL-12, GOT, GPT, LDH, and uric acid concentrations in early-onset PE group. Thus, early-onset PE was associated with greater maternal and fetal impairment. There are differences in pathophysiology between early- and late-onset PE, highlighting by the difference in Hsp70 levels.

OBJECTIVE

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder with high prevalence in adulthood. There is a recognized need to assess the efficacy of psychotherapy in adult ADHD.

OBJECTIVE

To evaluate the efficacy of cognitive behavioral group psychotherapy (GPT) compared with individual clinical management (CM) and that of methylphenidate hydrochloride compared with placebo.

METHODS

Prospective, multicenter, randomized clinical trial of 18- to 58-year-old outpatients with ADHD from 7 German study centers. Patients were recruited between January 2007 and August 2010, treatment was finalized in August 2011, and final follow-up assessments occurred in March 2013.

METHODS

Sessions of GPT and CM were held weekly for the first 12 weeks and monthly thereafter (9 months). Patients received either methylphenidate or placebo for 1 year.

METHODS

The primary outcome was the change in the ADHD Index of the Conners Adult ADHD Rating Scale from baseline to the end of the 3-month intensive treatment (blinded observer ratings). Secondary outcomes included ADHD ratings after 1 year, blinded observer ratings using the Clinical Global Impression Scale, and self-ratings of depression.

RESULTS

Among 1480 prescreened patients, 518 were assessed for eligibility, 433 were centrally randomized, and 419 were analyzed as randomized. After 3 months, the ADHD Index all-group baseline mean of 20.6 improved to adjusted means of 17.6 for GPT and 16.5 for CM, with no significant difference between groups. Methylphenidate (adjusted mean, 16.2) was superior to placebo (adjusted mean, 17.9) (difference, -1.7; 97.5% CI, -3.0 to -0.4; P = .003). After 1 year, treatment effects remained essentially stable. Descriptive analyses showed that methylphenidate was superior to placebo in patients assigned to GPT (difference, -1.7; 95% CI, -3.2 to -0.1; P = .04) or CM (difference, -1.7; 95% CI, -3.3 to -0.2; P = .03). Regarding depression, no significant differences were found. In contrast, GPT was superior to CM for all visits in the Clinical Global Impression global assessment of effectiveness.

CONCLUSIONS

Highly structured group intervention did not outperform individual CM with regard to the primary outcome. Psychological interventions resulted in better outcomes during a 1-year period when combined with methylphenidate as compared with placebo.

BACKGROUND

isrctn.org Identifier: ISRCTN54096201.

The purpose of this study was to investigate the effect of melatonin, at pharmacological doses, on serum lipids of rats fed with a hypercholesterolemic diet. Therefore, different groups of animals were fed with either the regular Sanders Chow diet or a diet enriched in cholesterol. Moreover, animals were treated with or without melatonin in the drinking water for 3 months. We show that melatonin treatment did not affect the levels of cholesterol or triglycerides in rats fed with a regular diet. However, the increase in total cholesterol and low-density lipoprotein (LDL)-cholesterol induced by a cholesterol-enriched diet was reduced significantly by melatonin administration. On the other hand, melatonin administration prevented the decrease in high-density lipoprotein (HDL)-cholesterol induced by the same diet. No differences in the levels of very low-density lipoprotein (VLDL)-cholesterol and triglycerides were found. We also found that melatonin administration slightly decreased serum uric, bilirubin and increased serum glucose levels. Other biochemical parameters, including total proteins, creatinine, urea, phosphorus, calcium, glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), gamma-glutamyltranspeptidase (gamma-GT), acetyl cholinesterase (AcCho), and alkaline phosphatase (ALP) were not modified by melatonin treatment. Finally, lipid peroxidation (LPO) was studied in membranes of liver, brain, spleen, and heart as an index of membrane oxidative damage. Results show that hypercholesterolemic diet did not modify the LPO status in any of the tissues studied. However, chronic melatonin administration significantly decreased LPO. Results confirm that melatonin participates in the regulation of cholesterol metabolism and in the prevention of oxidative damage to membranes.

Various series have reported similar survival and recurrence rates after resection of colorectal liver metastases (CRLM). If outcomes were predictable, indications for surgery could be improved. This hypothesis was tested in 135 consecutive patients with CRLM who underwent "curative" resection from 1977 to 1997. Among the 132 patients available for follow-up, three groups were identified on the basis of outcome: (1) survival of more than 5 years disease-free (n = 32; 24%); (2) diffuse recurrences within the first 6 months (n = 24; 18%); and (3) discrete recurrences for which reresection was performed (n = 16; 12%). As our results are similar to those reported in the literature, we assumed that about 50% of patients with resectable lesions have recognizable patterns of recurrence. At multivariate analysis, factors significant for disease-free survival (DFS) were the percentage of liver invasion, metastases to lymph nodes at the primary site, number of metastases, preoperative glutamic pyruvic transaminase (GPT) level, and type of liver resection. On the basis of the relative risk (RR) expressed by significant prognostic factors, a score model was developed, and three prognostic groups were defined: Group A, with the best prognostic score, included 23 of 32 (72%) patients who survived more than 5 years, and that with the worst prognostic score (group C) included 22 of 24 (92%) patients with early diffuse recurrences. Extreme (especially unfavorable) outcomes can therefore be predicted. By using improved models of outcome analysis, many patients could be spared surgery as first-line treatment, and stratification criteria could be worked out for future trials.

Tu-Si-Zi, the seeds of Cuscuta chinensis Lam. (Convolvulaceae), is a traditional Chinese medicine that is commonly used to nourish and improve the liver and kidney conditions in China and other Asian countries. As oxidative stress promotes the development of acetaminophen (APAP)-induced hepatotoxicity, the aim of the present study was to evaluate and compare the hepatoprotective effect and antioxidant activities of the aqueous and ethanolic extracts of C chinensis on APAP-induced hepatotoxicity in rats. The C chinensis ethanolic extract at an oral dose of both 125 and 250mg/kg showed a significant hepatoprotective effect relatively to the same extent (P<0.05) by reducing levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and alkaline phosphatase (ALP). In addition, the same ethanolic extract prevented the hepatotoxicity induced by APAP-intoxicated treatment as observed when assessing the liver histopathology. Regarding the antioxidant activity, C chinensis ethanolic extract exhibited a significant effect (P<0.05) by increasing levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and by reducing malondialdehyde (MDA) levels. In contrast, the same doses of the aqueous extract of C chinensis did not present any hepatoprotective effect as seen in the ethanolic extract, and resulted in further liver deterioration. In conclusion, these data suggest that the ethanolic extract of Cuscuta chinensis can prevent hepatic injuries from APAP-induced hepatotoxicity in rats and this is likely mediated through its antioxidant activities.

Purpose: Despite promising clinical activity, T-cell-engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug.Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug.Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell-binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD.Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785-97. ©2018 AACR See related commentary by Prakash and Diefenbach, p. 4631.

The Ly-6 locus is now regarded as a gene complex consisting of at least five closely linked loci (Ly-6A-Ly-6E) whose polymorphic products are identified by monoclonal antibodies and distinguished by different tissue distributions. Ly-6 has been assigned by other investigators to chromosome (Chr) 9 (linked to Thy-1) or to Chr 2. We report that the Ly-6 gene complex, together with the Xp-14 and Gdc-1 loci, is situated on Chr 15 linked to Gpt-1. These new linkage data are derived from four sources: (1) three separate crosses that failed to demonstrate linkage of Ly-6 to either Thy-1 on Chr 9 or to any of five genes present on Chr 2; (2) the NXSM recombinant inbred strains, which suggested the linkage of Ly-6 and Xp-14 to Gpt-1 on Chr 15; (3) several Gpt-1 and Gdc-1 congenic strains that confirmed the assignment of Ly-6 and Xp-14 to Chr 15; and (4) backcrosses that further confirmed the linkage of Ly-6, Gpt-1, Gdc-1, and Xp-14, the probable gene order being Gpt-1/Ly-6-Xp-14-Gdc-1.

We have demonstrated genetic transposition in human cells. An experimental system was established in which the Ecogpt (gpt) gene was employed as a target for inactivation. The human lung carcinoma cell line A549 containing this target was fused to UV-irradiated A549 cells that did not contain the target. From the fusion products, sublines carrying an inactivated gpt gene were analyzed. UV irradiation increased the frequency of inactivated gpt genes in the fusion cells by 100-fold. One subline was found to contain a complete Alu sequence in the coding region of the gpt gene. The inserted element differed from the Blur8 sequence by only 7 out of the 270 nucleotides. The insertion of this Alu element created a 5 bp insertion site duplication.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.

Mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (Mo-MuLV) at a single locus have been derived previously by infection of preimplantation embryos. Here we explore the potential of retroviral vectors for transferring nonviral genes into the germ line of mice. Preimplantation mouse embryos were cocultivated with a cell line that produces a recombinant retrovirus whose genome carries the Escherichia coli gene gpt. We show that the vector sequence was inserted into the genome of the embryo and into the germ line at a frequency similar to that for the Mo-MuLV-helper sequence. A new mouse strain, Mgpt-1, was developed that is homozygous for a single MSVgpt proviral genome. The proviral sequences were highly methylated and not expressed in tissues of Mgpt-1 mice. When cells derived from transgeneic animals were treated with 5-azacytidine, the proviral sequences were not methylated and were transcriptionally activated. These results indicate that nonviral genes that are under the control of the viral long terminal repeat are inactivated when transferred into the germ line of animals.

Transgenic potato (Solanum tuberosum) plants simultaneously over-expressing a pea (Pisum sativum) glucose-6-phosphate/phosphate translocator (GPT) and an Arabidopsis thaliana adenylate translocator (NTT1) in tubers were generated. Double transformants exhibited an enhanced tuber yield of up to 19%, concomitant with an additional increased starch content of up to 28%, compared with control plants. The total starch content produced in tubers per plant was calculated to be increased by up to 44% in double transformants relative to the wild-type. Single over-expression of either gene had no effect on tuber starch content or tuber yield, suggesting that starch formation within amyloplasts is co-limited by the import of energy and the supply of carbon skeletons. As total adenosine diphosphate-glucose pyrophosphorylase and starch synthase activities remained unchanged in double transformants relative to the wild-type, they cannot account for the increased starch content found in tubers of double transformants. Rather, an optimized supply of amyloplasts with adenosine triphosphate and glucose-6-phosphate seems to favour increased starch synthesis, resulting in plants with increased starch content and yield of tubers.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

We have developed a procedure for the selection of recombinant vaccinia viruses with applicability to poxvirus mutagenesis studies and to the use of vaccinia virus as an expression vector. The method depends on the specific inability of a recombinant vaccinia virus expressing the Escherichia coli guanine phosphoriboxyltransferase gene (gpt) to form plaques on a hypoxanthine-guanine phosphoribosyltransferase-negative line of mouse fibroblasts in the presence of 6-thioguanine. Recombinant viruses that have the gpt removed can form plaques under selection conditions, thus providing a simple and efficient selection protocol. We have demonstrated the method by isolating a pseudo-wild type revertant virus and a simple deletion mutant virus from a recombinant vaccinia virus with gpt inserted into the vaccinia virus gene encoding the major 35,000-Da secretory protein.

The antiviral activities of two substrate-based inhibitors of human immunodeficiency virus type 1 (HIV-1) protease, UK-88,947 and Ro 31-8959, were studied in acute infections. H9 and HeLaCD4-LTR/beta-gal cells were infected either with HIV-1IIIB or a replication-defective virus, HIV-gpt(HXB-2). Both inhibitors were capable of blocking early steps of HIV-1 replication if added to cells prior to infection. Partial inhibition was also obtained by addition of inhibitor at the time of or as late as 15 min after infection. The inhibitors were ineffective if added 30 min postinfection. The inhibitory effects were studied by cDNA analysis with PCR followed by Southern blot hybridization and by infectivity assays allowing quantitation of HIV-1 in a single cycle of replication. When UK-88,947-treated H9 cells were coinfected with HIV-1 and human T-cell leukemia virus type I only the replication of HIV-1 was inhibited, demonstrating viral specificity. Pretreating the infectious virus stocks with the inhibitors also prevented replication, indicating that the inhibitors block the action of the viral protease and not a cellular protease. A panel of primer sets was used to analyze cDNA from cell lysates by PCR amplification at 4 and 18 h postinfection. Four hours after infection, viral specific cDNA was detected with all of the four primer pairs used: R/U5, nef/U3, 5' gag, and long terminal repeat (LTR)/gag. However, after 18 h, only the R/U5 and nef/U3 primer pairs and not the 5' gag or LTR/gag primer pair were able to allow amplification of cDNA. The results suggest a crucial role of HIV-1 protease in the early phase of viral replication. Although it is not clear what early steps are affected by the protease, it is likely that the target is the NC protein, as referred from our previous reports of the in situ cleavage of the nucleocapsid (NC) protein by the viral protease inside lentiviral capsids. The results suggest that it is not the inhibition of initiation and progression of reverse transcription but the stability of full-size unintegrated cDNA which is affected in the presence of protease inhibitors. Alternatively, the cleavage of the NC protein may be required for the proper formation of preintegration complex and/or for its transport to the nucleus.

Ochratoxin A (OTA) can induce renal tumors that originate from the S3 segment of the proximal tubules in rodents, but the results of conventional mutagenicity tests have caused controversy regarding the role of genotoxic mechanisms in the carcinogenesis. Human exposure to OTA from various foods is unavoidable. Therefore, an understanding of OTA-induced renal carcinogenesis is necessary for accurate estimates of the human risk hazard. In the present study, a 13-week exposure of gpt delta rats to OTA at a carcinogenic dose induced karyomegaly and apoptosis at the outer stripe of the outer medulla (OM) of the kidney but failed to affect the reporter gene mutations in DNA extracted from whole kidneys. This site specificity resulting from the kinetics of specific transporters might be responsible for the negative outcome of in vivo mutagenicity. The kidney was then macroscopically divided, based on anatomical characteristics, into the cortex, the OM, and the inner medulla, each of which was histopathologically confirmed. Spi⁻ mutant frequencies (MFs) but not gpt MFs in the OM after a 4-week exposure to OTA were significantly higher than in controls despite the absence of cortical changes. There were also no changes in 8-hydroxydeoxyguanosine levels in kidney DNA. These results strongly suggest the involvement of a genotoxic mechanism, with the exception of oxidative DNA damage in OTA-induced renal carcinogenesis. In addition, the reporter gene mutation assay using DNA from target sites could be a more powerful tool to investigate in vivo genotoxicities.