In this study a binary Mg-Zn magnesium alloy was researched as a degradable biomedical material. An Mg-Zn alloy fabricated with high-purity raw materials and using a clean melting process had very low levels of impurities. After solid solution treatment and hot working the grain size of the Mg-Zn alloy was finer and a uniform single phase was gained. The mechanical properties of this Mg-Zn alloy were suitable for implant applications, i.e. the tensile strength and elongation achieved were approximately 279.5MPa and 18.8%, respectively. The results of in vitro degradation experiments including electrochemical measurements and immersion tests revealed that the zinc could elevate the corrosion potential of Mg in simulated body fluid (SBF) and reduce the degradation rate. The corrosion products on the surface of Mg-Zn were hydroxyapatite (HA) and other Mg/Ca phosphates in SBF. In addition, the influence caused by in vitro degradation on mechanical properties was studied, and the results showed that the bending strength of Mg-Zn alloy dropped sharply in the earlier stage of degradation, while smoothly during the later period. The in vitro cytotoxicity of Mg-Zn was examined. The result 0-1 grade revealed that the Mg-Zn alloy was harmless to L-929 cells. For in vivo experiments, Mg-Zn rods were implanted into the femoral shaft of rabbits. The radiographs illustrated that the magnesium alloy could be gradually absorbed in vivo at about 2.32mm/yr degradation rate obtained by weight loss method. Hematoxylin and eosin (HE) stained section around Mg-Zn rods suggested that there were newly formed bone surrounding the implant. HE stained tissue (containing heart, liver, kidney and spleen tissues) and the biochemical measurements, including serum magnesium, serum creatinine (CREA), blood urea nitrogen (BUN), glutamic-pyruvic transaminase (GPT) and creatine kinase (CK) proved that the in vivo degradation of Mg-Zn did not harm the important organs. Moreover, no adverse effects of hydrogen generated by degradation had been observed and also no negative effects caused by the release of zinc were detected. These results suggested that the novel Mg-Zn binary alloy had good biocompatibility in vivo.

The third hypervariable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been proposed to play an important role in mediating viral entry. Antibodies to the V3 domain block HIV-1 infection but not virus binding to CD4. At the center of the V3 domain is a relatively conserved sequence of amino acids, GPGRA. It has previously been shown that mutation of some of these amino acids reduced the ability of gp160 expressed on the surface of cells to induce fusion with CD4-bearing cells. In order to analyze the role of V3 domain sequences in mediating HIV entry, we introduced several amino acid substitution mutations in the GPGRA sequence of gp160 derived from HIV-1 strain HXB2 and in the analogous sequence of strain SF33, GPGKV. Virus was generated by cotransfecting the env constructs and a selectable env-negative HIV vector, HIV-gpt. When complemented with a retrovirus env gene, infectious virus capable of a single round of replication was produced. The viral particles produced were analyzed biochemically for core and envelope proteins and for infectious titer. The transfected envs were also analyzed for ability to bind to CD4 and mediate cell fusion. Several of the amino acid substitutions resulted in moderate to severe decreases in virus infectivity and fusion activity. Envelope glycoprotein assembly onto particles and CD4 binding were not affected. These results provide evidence that V3 sequences are involved in mediating the fusion step of HIV-1 entry.

Homologous integrations into a nonselectable target locus have been highly enriched for following DNA transfections into mammalian cells. The target gene, the SV40 early region in COS1 cells, provides transcription signals to activate a defective selectable marker, the gpt gene. We find that nearly half of the selected clones have integrated the gpt gene at the homologous sequence in the COS1 genome. This is an estimated 100-fold enrichment for homologous events compared with transfections in which the gpt gene is transcriptionally active. As shown for yeast integration events, a double-strand break at a position of homology between the transfected DNA and the genomic target is necessary to achieve a high frequency of homologous integrations. Furthermore, the arrangement of sequences at the integration site includes a repair of the double-strand gap, which was present on the transfected DNA, suggesting that similarities exist between yeast and mammalian integrations. The experimental design, in which a defective marker is activated following a homologous integration, may have general applications for gene targeting in mammalian cells.

We recently identified a Bacillus anthracis glycoprotein which is a structural constituent of the exosporium filaments (P. Sylvestre, E. Couture-Tosi, and M. Mock, Mol. Microbiol. 45:169-178, 2002). This Bacillus collagen-like protein (BclA) contains an internal collagen-like region (CLR) of GXX repeats which includes a large proportion of GPT triplets. Here, we report that the polymorphic marker Ceb-Bams13, for which there are nine alleles (P. Le Flèche et al., BMC Microbiol. 1:2, 2001), maps within the open reading frame encoding BclA. The bclA gene in 11 B. anthracis strains representative of seven Ceb-Bams13 alleles was sequenced and compared to the Ames bclA gene sequence. The amino- and carboxy-terminal sequences surrounding the CLR are conserved. The CLR itself is highly polymorphic: it contains between 17 and 91 GXX repeats and one to eight copies of the 21-amino-acid sequence (GPT)(5)GDTGTT, named the BclA repeat. The length of the filament on the spore surface differed between the strains. We exchanged the bclA gene between strains with different CLRs and examined the spore surfaces by electron microscopy analysis. The length of the BclA CLR is responsible for the variation in filament length.

Affinity maturation of antibodies is characterized by localized hypermutation of the DNA around the V segment. Here we show, using mice containing single or multiple transgene constructs, that an immunoglobulin V kappa segment can be replaced by human beta-globin or prokaryotic neo or gpt genes without affecting the rate of hypermutation; the V gene itself is not necessary for recruiting hypermutation. The ability to target hypermutation to heterologous genes in vivo could find more general applications in biology.

We have introduced the chicken genes for cytoplasmic beta-actin, cardiac alpha-actin, and skeletal alpha-actin into C2 cells, a murine myogenic cell line, and into L cells by using the simian virus 40-derived vector PSV2 -gpt. In each selection, the entire population of transformed cells was analyzed for the expression and regulation of the actin genes by nuclease S1 assay and primer extension. This was compared to the expression of the vector marker Eco-gpt. The beta-actin gene is transcribed accurately and efficiently both in L-cells and in undifferentiated C2 cells. In fused C2 cells, beta-actin transcripts decrease significantly in parallel with the endogenous level of mouse beta-actin mRNA. Eco-gpt RNA levels remain essentially constant during myogenesis. The alpha-actin genes are correctly expressed at low levels in L cells but at significantly higher levels in the C2 cell background. Unlike the endogenous mouse alpha-actin gene, this level of expression does not change measurably with myogenesis. The skeletal alpha-actin gene is expressed poorly in pre- and post-fusion C2 cells, displaying no induction with differentiation. These results suggest that the tissue specificity of expression is maintained but the pattern of gene regulation for the sarcomeric actins is not. Factors in addition to the sequences flanking these genes are important for modulating gene expression during development. The decrease in the levels of beta-actin RNA during C2 cell differentiation provides a model system in which to study gene repression during development.

Soluble glutamic-pyruvic transaminase (GPT) has three common phenotypes, each representing the homozygous and heterozygous expression of two alleles, Gpt(1) and Gpt(2) at an autosomal locus. The frequencies of these alleles vary considerably from one population to another.

The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.

The origin of modern wheats involved alloploidization among related genomes. To determine if Aegilops speltoides was the donor of the B and G genomes in AABB and AAGG tetraploids, we used a 3-tiered approach. Using 70 amplified fragment length polymorphism (AFLP) loci, we sampled molecular diversity among 480 wheat lines from their natural habitats encompassing all S genome Aegilops, the putative progenitors of wheat B and G genomes. Fifty-nine Aegilops representatives for S genome diversity were compared at 375 AFLP loci with diploid, tetraploid, and 11 nulli-tetrasomic Triticum aestivum wheat lines. B genome-specific markers allowed pinning the origin of the B genome to S chromosomes of A. speltoides, while excluding other lineages. The outbreeding nature of A. speltoides influences its molecular diversity and bears upon inferences of B and G genome origins. Haplotypes at nuclear and chloroplast loci ACC1, G6PDH, GPT, PGK1, Q, VRN1, and ndhF for approximately 70 Aegilops and Triticum lines (0.73 Mb sequenced) reveal both B and G genomes of polyploid wheats as unique samples of A. speltoides haplotype diversity. These have been sequestered by the AABB Triticum dicoccoides and AAGG Triticum araraticum lineages during their independent origins.

The efficacy of gabapentin (GPT) and pregabalin (PGB) in the treatment of fibromyalgia syndrome (FMS) was assessed. We screened MEDLINE, PsycINFO, SCOPUS, www.clinicaltrials.org, the Cochrane Library (through October 2008), and the reference sections of original studies on GPT/PGB in FMS. Randomized controlled trials (RCTs) on the treatment of FMS with GPT and PGB were analyzed. Six out of 127 RCTs studying 2422 subjects on treatment with GPT (one study) or PGB (five studies) and 1056 subjects on placebo with a median treatment duration of 11 weeks were included into the systematic review. Five studies were suitable for meta-analysis. Effects were summarized using standardized mean differences (SMD). There was strong evidence for a reduction of pain (SMD -0.28, 95% CI -0.36, -0.20; p<0.001), improved sleep (SMD -0.39, 95% CI -0.48, -0.39; p<0.001), and improved health-related quality of life (HRQOL) (SMD -0.30, 95% CI -0.46, -0.15; p<0.001), but not for depressed mood (SMD -0.12, 95% CI -0.30, 0.06; p=0.18). There was strong evidence for a non-substantial reduction of fatigue (SMD -0.16, 95% CI -0.23, -0.09, p<0.001) and of anxiety (SMD -0.18, 95% CI -0.27, -0.10; p<0.001). The external validity of the studies was limited because patients with severe somatic and mental disorders were excluded.

The protein encoded by the US28 gene of human cytomegalovirus (HCMV) has homology to G protein-coupled receptors (GCR). Previous studies demonstrated that recombinant US28 protein can bind the beta class of chemokines (K. Neote, D. DiGregorio, J. Y. Mak, R. Horuk, and T. J. Schall, Cell 72:415-425, 1993) and induce a rise in intracellular calcium after the binding of chemokines (J. L. Gao and P. M. Murphy, J. Biol. Chem. 269:28539-28542, 1994). In order to investigate the function of the US28 protein in virus-infected cells, a recombinant HCMV (HV5.8) was constructed, with the US28 open reading frame disrupted by the insertion of the Escherichia coli gpt gene and the gene for the green fluorescent protein. The US28 gene is not required for growth in human fibroblasts (HF). HF infected with wild-type HCMV bound RANTES at 24 h postinfection and demonstrated an intracellular calcium flux induced by RANTES. In cells infected with HV5.8, RANTES did not bind or induce a calcium flux, demonstrating that US28 is responsible for the beta-chemokine binding and induced calcium signaling in HCMV-infected cells. The ability of the US28 gene to bind chemokines was shown to cause a significant reduction in the concentration of RANTES in the medium of infected cells. Northern analysis of RNA from infected cells showed that US28 is an early gene, while US27 (another GCR) is a late gene.

The frequency and distribution of the rare dinucleotide CpG was examined in 15 mammalian genes. CpG is highly methylated at cytosine in mammalian DNA (1,2) and 5-methylcytosine (5mC) is thought to undergo a transition mutation via deamination to produce thymine (3). This would result in the accumulation of TpG and CpA and depletion of CpG during evolution (4). Consistent with this hypothesis, the gene sample of 26,541 dinucleotides contained CpG at 40% the frequency expected by base composition and the CpG transition products, TpG+CpA, were significantly elevated at 124% of expected random frequency. However, because CpG occurs at only 25% of expected random frequency in the genome, the sampled genes were considerably enriched in this dinucleotide. CpGs were asymmetrically distributed in sequences flanking the genes. 5'-flanking sequences were enriched in CpG at 135% of the frequency expected assuming a symmetrical distribution of all the CpGs in the sampled genes (p less than 0.01), while 3'-flanking regions were depleted in CpG at 40% of expected values (p less than 0.0001). This asymmetry may reflect the role of 5-methylcytosine in gene expression. In contrast the frequencies of GpC and GpT+ ApC did not differ significantly from that predicted by base composition and these dinucleotides were not asymmetrically distributed.

The relationship of T cell activation to HIV entry and generation of viral DNA intermediates was studied in freshly isolated CD4+ T lymphocytes. Unstimulated cells exposed to infectious virus for up to 48 h did not synthesize any detectable unintegrated HIV DNA duplex forms or integrated genomic provirus. However, activation of these cells with either PHA or OKT3 (anti-CD3) mAb before viral exposure resulted in the generation of unintegrated HIV DNA after 6 h and integrated copies after 24 h. Cell-to-cell fusion studies showed significantly attenuated fusion between freshly isolated resting T cells and T cells constitutively expressing high levels of HIV envelope glycoprotein (HXB/gpt) compared with T cells first stimulated with either PHA or OKT3 mAb. The baseline fusion observed with resting T cells is believed to be a consequence of allogeneic stimulation by the HXB/gpt cell line. These results provide evidence that HIV entry and HIV envelope-dependent cell-to-cell fusion require T cell activation.

Dideoxy chain-termination DNA sequencing was used to determine the specific DNA base changes induced after in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. The resultant mutation spectra were compared with the levels of O6-alkylguanine and O4-alkylthymidine in genomic DNA immediately after exposure. All (39/39) of the MNU-induced mutations were G X C----A X T transitions. In contrast, 24/33 point mutations isolated following ENU treatment were G X C----A X T transitions, 7/33 were A X T----G X C transitions, 1/33 was a G X C----C X G transversion, and 1/33 was an A X T----C X G transversion. Three large insertions, probably of spontaneous origin, were also isolated. O4-alkylthymidine/O6-alkylguanine ratios were 0.014 for MNU and 0.28 for ENU. These data suggest that the difference in the mutation spectrum of MNU versus ENU may be attributed, in part, to the different ratio of O6-alkylguanine versus O4-alkylthymidine produced in the DNA. Of the G X C----A X T transitions, 82% of the MNU- and 71% of the ENU-induced mutations occurred at the middle guanine of the sequence 5'-GG(A or T)-3'.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.

Genes coding for enzymes functioning in purine salvage pathways have been located on the chromosome of Escherichia coli. The gene add encoding adenosine deaminase was located by transduction at 31 min, the gene order was established to be man-uidA-add-aroD. A deletion covering man-uidA-add was obtained. The gene gsk encoding guanosine kinase was cotransducible with purE and shown to be located at 13 min. The gene hpt encoding hypoxanthine phosphoribosyltransferase was cotransducible with tonA indicating a location at 3 min. The location of the gene gpt encoding guanine (xanthine) phosphoribosyltransferase in the proA-proB region was confirmed.

The objective of the present study was to assess the toxicology of melatonin (10 mg), administered for 28 days to 40 volunteers randomly assigned to groups receiving either melatonin (N = 30) or placebo (N = 10) in a double-blind fashion. The following measurements were performed: polysomnography (PSG), laboratory examinations, including complete blood count, urinalysis, sodium, potassium and calcium levels, total protein levels, albumin, blood glucose, triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), urea, creatinine, uric acid, glutamic-oxalacetic transaminase (GOT), glutamic-pyruvate transaminase (GPT), bilirubin, alkaline phosphatase, gama-glutamic transaminase (GGT), T3, T4, TSH, LH/FSH, cortisol, and melatonin serum concentrations. In addition, the Epworth Somnolence Scale (ESS) and a sleep diary (SD) were also applied to the volunteers 1 wk before each PSG. In addition, the volunteers were asked about possible side effects (SE) that appeared during the treatment. The study was carried out according to the following timetable: Visit 0, filling out the term of consent and inclusion criteria; Visit 1, PSG, laboratory examinations, ESS, SD, melatonin serum concentrations; Visit 2, SD, melatonin serum concentrations, SE; Visit 3, melatonin serum concentrations, PSG, ESS, SE; Visit 4, laboratory examinations, SE, melatonin serum concentrations, SD; and Visit 5, PSG, ESS, SE. Analysis of the PSG showed a statistically significant reduction of stage 1 of sleep in the melatonin group. No other differences between the placebo and melatonin groups were obtained. In the present study we did not observe, according to the parameters analyzed, any toxicological effect that might compromise the use of melatonin at a dose of 10 mg for the period of time utilized in this study.

The energy status of plant cells strongly depends on the energy metabolism in chloroplasts and mitochondria, which are capable of generating ATP either by photosynthetic or oxidative phosphorylation, respectively. Another energy-rich metabolite inside plastids is the glycolytic intermediate phosphoenolpyruvate (PEP). However, chloroplasts and most non-green plastids lack the ability to generate PEP via a complete glycolytic pathway. Hence, PEP import mediated by the plastidic PEP/phosphate translocator or PEP provided by the plastidic enolase are vital for plant growth and development. In contrast to chloroplasts, metabolism in non-green plastids (amyloplasts) of starch-storing tissues strongly depends on both the import of ATP mediated by the plastidic nucleotide transporter NTT and of carbon (glucose 6-phosphate, Glc6P) mediated by the plastidic Glc6P/phosphate translocator (GPT). Both transporters have been shown to co-limit starch biosynthesis in potato plants. In addition, non-photosynthetic plastids as well as chloroplasts during the night rely on the import of energy in the form of ATP via the NTT. During energy starvation such as prolonged darkness, chloroplasts strongly depend on the supply of ATP which can be provided by lipid respiration, a process involving chloroplasts, peroxisomes, and mitochondria and the transport of intermediates, i.e. fatty acids, ATP, citrate, and oxaloacetate across their membranes. The role of transporters involved in the provision of energy-rich metabolites and in pathways supplying plastids with metabolic energy is summarized here.

We describe the mutagenesis of the IRSI-US5 region of the human cytomegalovirus genome, demonstrating the potential of the E. coli guanosine phosphoribosyl transferase (gpt) gene as a selectable marker for insertion and deletion mutagenesis of high passage (AD169, Towne) as well as low passage (Toledo) strains of virus. Despite evidence suggesting that the US3 gene product may play a regulatory role, disruption of this gene with a gpt insert had no effect on growth of any of these strains of virus in resting or dividing human fibroblasts, or in human thymus plus liver implants in SCID-hu mice. Transcripts of the gpt gene, under control of the herpes simplex virus thymidine kinase promoter adjacent to the US3 enhancer in the viral genome, accumulated with delayed early (beta) kinetics. Mutants with deletions in the IRS1 and US3-US5 regions were isolated by back-selection against gpt with the drug 6-thioguanine by growing virus in human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyl transferase deficient) skin fibroblasts immortalized with human papillomavirus oncogenes. Thus, we demonstrate a dependable method for insertion and deletion mutagenesis that can be applied to any region of the viral genome.

A major problem in biology is to understand how complex tissue shapes may arise through growth. In many cases this process involves preferential growth along particular orientations raising the question of how these orientations are specified. One view is that orientations are specified through stresses in the tissue (axiality-based system). Another possibility is that orientations can be specified independently of stresses through molecular signalling (polarity-based system). The axiality-based system has recently been explored through computational modelling. Here we develop and apply a polarity-based system which we call the Growing Polarised Tissue (GPT) framework. Tissue is treated as a continuous material within which regionally expressed factors under genetic control may interact and propagate. Polarity is established by signals that propagate through the tissue and is anchored in regions termed tissue polarity organisers that are also under genetic control. Rates of growth parallel or perpendicular to the local polarity may then be specified through a regulatory network. The resulting growth depends on how specified growth patterns interact within the constraints of mechanically connected tissue. This constraint leads to the emergence of features such as curvature that were not directly specified by the regulatory networks. Resultant growth feeds back to influence spatial arrangements and local orientations of tissue, allowing complex shapes to emerge from simple rules. Moreover, asymmetries may emerge through interactions between polarity fields. We illustrate the value of the GPT-framework for understanding morphogenesis by applying it to a growing Snapdragon flower and indicate how the underlying hypotheses may be tested by computational simulation. We propose that combinatorial intractions between orientations and rates of growth, which are a key feature of polarity-based systems, have been exploited during evolution to generate a range of observed biological shapes.

Solanum nigrum L. (SN) is an herbal plant that has been used as hepatoprotective and anti-inflammation agent in Chinese medicine. In this study, the protective effects of water extract of SN (SNE) against liver damage were evaluated in carbon tetrachloride (CCl4)-induced chronic hepatotoxicity in rats. Sprague-Dawley (SD) rats were orally fed with SNE (0.2, 0.5, and 1.0 g kg(-1) bw) along with administration of CCl4 (20% CCl4/corn oil; 0.5 mL kg(-1) bw) for 6 weeks. The results showed that the treatment of SNE significantly lowered the CCl4-induced serum levels of hepatic enzyme markers (GOT, GPT, ALP, and total bilirubin), superoxide and hydroxyl radical. The hepatic content of GSH, and activities and expressions of SOD, GST Al, and GST Mu that were reduced by CCl4 were brought back to control levels by the supplement of SNE. Liver histopathology showed that SNE reduced the incidence of liver lesions including hepatic cells cloudy swelling, lymphocytes infiltration, hepatic necrosis, and fibrous connective tissue proliferation induced by CCl4 in rats. Therefore, the results of this study suggest that SNE could protect liver against the CCl4-induced oxidative damage in rats, and this hepatoprotective effect might be contributed to its modulation on detoxification enzymes and its antioxidant and free radical scavenger effects.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common manifestations of chronic liver disease worldwide. The aim of the present study was to assess the effect of resveratrol on liver fat accumulation, as well as on the activity of those enzymes involved in lipogenesis and fatty acid oxidation in fa/fa Zucker rats. A total of thirty rats were assigned to three experimental groups and orally treated with resveratrol for 6 weeks, or without resveratrol (C: control group; RSV15 group: 15 mg/kg body weight per d; RSV45 group: 45 mg/kg body weight per d). Liver histological analysis was performed by microscopy. Levels of hepatic carnitine palmitoyltransferase-Ia (CPT-Ia), acyl-coenzyme A oxidase (ACO), fatty acid synthase, glucose-6-phosphate dehydrogenase and malic enzyme were assessed by spectrophotometry, and acetyl-CoA carboxylase was assessed by radiometry. Commercial kits were used to determine serum TAG, NEFA, total HDL and non-HDL-cholesterol, glycerol, ketonic bodies, glucose, insulin, adiponectin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), hepatic TAG, thiobarbituric acid reactive substrates, GSH (GSSG) and superoxide dismutase. Resveratrol reduced liver weight and TAG content. It did not modify the activity of lipogenic enzymes but it did increase CPT-Ia and ACO activities. NEFA and ALP were reduced in both resveratrol-treated groups. AST/GOT was reduced only by the lowest dose. ALT/GPT, TAG and adiponectin remained unchanged. Resveratrol reduced liver oxidative stress. This study demonstrates that resveratrol can protect the liver from NAFLD by reducing fatty acid availability. Moreover, resveratrol also protects liver from oxidative stress.

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (<em>GPT</em>) of the inner plastid envelope membrane. In contrast to the wild type, <em>GPT</em>2, one of two functional <em>GPT</em> genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the <em>GPT</em> in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of <em>GPT</em> in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in <em>GPT</em>2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of At<em>GPT</em>1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of <em>GPT</em>2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of <em>GPT</em>2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.

Defects in the assembly of dolichol-linked oligosaccharide or its transfer to proteins result in severe, multi-system human diseases called Type I congenital disorders of glycosylation. We have identified a novel CDG type, CDG-Ij, resulting from deficiency in UDP-GlcNAc: dolichol phosphate N-acetyl-glucosamine-1 phosphate transferase (GPT) activity encoded by DPAGT1. The patient presents with severe hypotonia, medically intractable seizures, mental retardation, microcephaly, and exotropia. Metabolic labeling of cultured dermal fibroblasts from the patient with [2-(3)H]-mannose revealed lowered incorporation of radiolabel into full-length dolichol-linked oligosaccharides and glycoproteins. In vitro enzymatic analysis of microsomal fractions from the cultured cells indicated that oligosaccharyltransferase activity is normal, but the GPT activity is reduced to approximately 10% of normal levels while parents have heterozygous levels. The patient's paternal DPAGT1 allele contains a point mutation (660A>G) that replaces a highly conserved tyrosine with a cysteine (Y170C). The paternal allele cDNA produces a full-length protein with almost no activity when over-expressed in CHO cells. The maternal allele makes only about 12% normal mature mRNA, while the remainder shows a complex exon skipping pattern that shifts the reading frame encoding a truncated non-functional GPT protein. Thus, we conclude that the DPAGT1 gene defects are responsible for the CDG symptoms in this patient. Hum Mutat 22:144-150, 2003.