In rats gestational iron deficiency does not change body fat or hepatic mitochondria in the aged offspring.

Mitochondrial dysfunction and resulting changes in adiposity have been observed in the offspring of animals fed a high fat (HF) diet. As iron is an important component of the mitochondria, we have studied the offspring of female rats fed complete (Con) or iron-deficient (FeD) rations for the duration of gestation to test for similar effects. The FeD offspring were ~12% smaller at weaning and remained so because of a persistent reduction in lean tissue mass. The offspring were fed a complete (stock) diet until 52 weeks of age after which some animals from each litter were fed a HF diet for a further 12 weeks. The HF diet increased body fat when compared with animals fed the stock diet, however, prenatal iron deficiency did not change the ratio of fat:lean in either the stock or HF diet groups. The HF diet caused triglyceride to accumulate in the liver, however, there was no effect of prenatal iron deficiency. The activity of the mitochondrial electron transport complexes was similar in all groups including those challenged with a HF diet. HF feeding increased the number of copies of mitochondrial DNA and the prevalence of the D-loop mutation, however, neither parameter was affected by prenatal iron deficiency. This study shows that the effects of prenatal iron deficiency differ from other models in that there is no persistent effect on hepatic mitochondria in aged animals exposed to an increased metabolic load.

The developmental origins of health and disease: current theories and epigenetic mechanisms.

The retrospective cohort studies of David Barker and colleagues during the late 1980s established the principle that the incidence of certain adult diseases such as stroke, type 2 diabetes and dyslipidaemia may be linked to in utero development. Later termed the "Developmental Origins of Health and Disease (DOHaD)" hypothesis, there have been several more recent attempts to explain this phenomenon. Although a general conceptual framework has been established to explain how mechanisms may have evolved to facilitate rapid adaptations to changing ecological conditions, it doesn't identify the actual mechanisms responsible for such effects. Extensive covalent modifications to DNA and related proteins occur from the earliest stages of mammalian development. These determine lineage-specific patterns of gene expression and so represent the most plausible mechanisms by which environmental factors can influence development during the life course. In providing a contemporary overview of chromatin modifications during early mammalian development, this review highlights both the complexity and our current lack of understanding of how epigenetic alterations may contribute to in utero programming. It concludes by providing some thoughts to future research endeavours where the emphasis should be on bettering our understanding of epigenesis and devising more thoughtful experimental approaches that focus on specific environmental factors in appropriate animal and cellular models.

DNA damaging agents increase gadd153 (CHOP-10) messenger RNA levels in bovine preimplantation embryos cultured in vitro.

DNA damage and other forms of stress are believed to be important factors in reducing the efficiency of in vitro embryo transfer techniques in farm animals. The expression of mRNAs from stress-responsive genes such as gadd153 (CHOP-10, ddit3) may provide a means of assessing the quality of embryos produced in vitro. Treatment of bovine granulosa cell cultures with the DNA-damaging agents, methyl methane-sulphonate (MMS) or sodium arsenite, induced the expression of an mRNA, which hybridized with the hamster gadd153 cDNA. Part of the corresponding bovine cDNA was amplified by nested polymerase chain reaction (PCR), cloned, and sequenced. Using a sensitive reverse transcriptase-PCR assay we have investigated the expression of gadd153 and beta-actin in blastocyst-stage bovine embryos treated with MMS or sodium arsenite. Both agents produced an increase in the ratio of gadd153 mRNA relative to beta-actin. These results show that there are changes in gene expression in blastocyst-stage bovine embryos in response to genotoxic stress, suggesting that an increase in gadd153 mRNA is a useful marker of DNA damage and metabolic stress in preimplantation embryos.

Maternal protein deficiency causes hypermethylation of DNA in the livers of rat fetuses.

Maternal protein deficiency during pregnancy is associated with changes in glucose tolerance and hypertension in the offspring of rats. In this study the growth of rat fetuses was examined when the dams were fed diets containing 18% casein, 9% casein or 8% casein supplemented with threonine. The extra threonine was added to reverse the decrease in circulating threonine concentrations that occurs when pregnant rats are fed protein-deficient diets. The fetuses of the group fed the low protein diet supplemented with threonine were significantly smaller than those of the control group and not significantly different from those fed low protein. Homogenates prepared from the livers of dams fed the diet containing 9% casein oxidized threonine at approximately twice the rate of homogenates prepared from dams fed the diet containing 18% casein. We conclude that circulating levels of threonine fall as a consequence of an increase in the activity of the pathway that metabolizes homocysteine produced by the transulfuration of methionine. Serum homocysteine was unaffected in the dams fed low protein diets compared with controls, but was significantly greater in dams fed the low protein diet supplemented with threonine. Elevated levels of homocysteine are associated with changes in the methylation of DNA. The endogenous methylation of DNA was greater than that of controls in the livers of fetuses from dams fed the 9% protein diets and increased further when the diet was supplemented with threonine. Our results suggest that changes in methionine metabolism increase homocysteine production, which leads to changes in DNA methylation in the fetus. An increase in maternal homocysteine may compromise fetal development, leading to the onset of glucose intolerance and hypertension in adult life.

Upregulation of CHOP-10 (gadd153) expression in the mouse blastocyst as a response to stress.

CHOP-10 (also known as gadd153 or Ddit3) is one of the genes overexpressed by mammalian cells exposed to cytotoxic agents or to nutrient stress. The response of this gene to stress was studied in the mouse blastocyst and in F9 embryonal carcinoma cells. When mouse blastocysts were exposed to the alkylating agent MMS, the metabolic inhibitor sodium arsenite or an inhibitor of protein glycosylation tunicamycin, levels of the CHOP-10 mRNA were increased by two- to threefold relative to the mRNA for beta-actin. There was no increase in gene expression when blastocysts were treated with the inhibitor of nucleotide synthesis PALA. These results show that the response of CHOP-10 is dependent on the type of stress applied to the embryo. When F9 embryonal carcinoma cells were treated with MMS or sodium arsenite, CHOP-10 expression was induced by fourfold within 4 hr of treatment. The induction following tunicamycin treatment was slower requiring at least 24 hr. The response to tunicamycin was greater in cells treated with retinoic acid to induce differentiation. The results suggest that there is a link between the extent of glycoprotein synthesis and the sensitivity of CHOP-10 to tunicamycin. The inhibitor PALA did not change CHOP-10 expression in the presence or absence of retinoic acid. In F9 cells an increase in the expression of CHOP-10 was followed by cell death due to apoptosis. The overexpression of CHOP-10 may be a marker for one of the pathways that lead to apoptosis in the blastocyst. These results suggest that there is more than one control system regulating growth arrest in the blastocyst and the fetal outcome may differ depending on the type of stress encountered in culture.

The effects of maternal protein restriction on the growth of the rat fetus and its amino acid supply.

Maternal protein deficiency causes fetal growth retardation which has been associated with the programming of adult disease. The growth of the rat fetus was examined when the mothers were fed on diets containing 180, 90 and 60 g protein/kg. The numbers of fetuses were similar in animals fed on the 180 and 90 g protein/kg diets but the number was significantly reduced in the animals fed on the 60 g protein/kg diet. The fetuses carried by the mothers fed on the 90 g protein/kg diet were 7.5% heavier than those of mothers fed on 180 g protein/kg diet on day 19 of gestation, but by day 21 the situation was reversed and the fetuses in the protein-deficient mothers were 14% smaller. Analysis of the free amino acids in the maternal serum showed that on day 19 the diets containing 90 and 60 g protein/kg led to threonine concentrations that were reduced to 46 and 20% of those found in animals fed on the control (180 g/kg) diet. The other essential amino acids were unchanged, except for a small decrease in the branched-chain amino acids in animals fed on the 60 g protein/kg diet. Both low-protein diets significantly increased the concentrations of glutamic acid+glutamine and glycine in the maternal serum. On day 21 the maternal serum threonine levels were still reduced by about one third in the group fed on the 90 g protein/kg diet. Dietary protein content had no effect on serum threonine concentrations in nonpregnant animals. Analysis of the total free amino acids in the fetuses on day 19 showed that feeding the mother on a low-protein diet did not change amino acid concentrations apart from a decrease in threonine concentrations to 45 and 26% of the control values at 90 and 60 g protein/ kg respectively. The results suggest that threonine is of particular importance to the protein-deficient mother and her fetuses. Possible mechanisms for the decrease in free threonine in both mother and fetuses and the consequences of the change in amino acid metabolism are discussed.

Expression of the growth arrest genes (GAS and GADD) changes during organogenesis in the rat fetus.

Mammalian cells mount an active response to nutrient limitation by overexpressing the growth arrest specific (GAS) and the growth arrest and DNA damage (GADD) genes. During embryogenesis in rats, there are quantitative and temporal differences in GAS and GADD gene expression during the development of the placenta, heart and kidney. Genes associated with the inhibition of DNA synthesis (p53 and GAS1) were predominantly expressed during the early stages of development, whereas those genes associated with inhibition of protein synthesis [GADD153 (also known as CHOP-10 or Ddit3) and C/EBP-beta] were more highly expressed during the later stages. The GADD45 gene was expressed throughout development. There were distinct periods of GAS3 and GAS6 gene expression during the development of the placenta, heart and kidneys, which is consistent with the proposed roles of these genes in cell interactions. These results show that there is a change in the expression of genes associated with the negative regulation of growth as the fetus develops.

Effects of maternal vitamin A status on fetal heart and lung: changes in expression of key developmental genes.

Vitamin A is required during pregnancy for fetal lung development. These experiments monitored fetal lung morphology in normal and vitamin A-deficient rats. The expression of elastin and the growth arrest-specific gene 6 (gas6) in fetal and neonatal hearts and lungs was assessed by Northern blotting. In normal-fed rats, elastin and gas6 were expressed in the fetal lung and heart from day 19 of gestation up to day 2 postnatally. Maternal vitamin A deficiency altered fetal lung development. On day 20, the bronchial passageways were less developed and showed reduced staining for elastic fibers, and in the neonates, the relative air space and the size of the sacculi were reduced. In the fetal lung, the mRNAs for elastin and gas6 were reduced to 56 and 68% of the control values, respectively. In the fetal heart, the mRNA for elastin was reduced to 64% of the control value, whereas gas6 was increased twofold. In the neonate, there was no change in elastin expression in the lung or heart, but gas6 expression in the heart was increased twofold. These results suggest that, in the pregnant rat, vitamin A deficiency may retard fetal lung development or influence the differentiation of critical cell lines. The changes in elastin and gas6 expression may be used to identify the cell types affected.

Gastrointestinal tolerability of meloxicam and piroxicam: a double-blind placebo-controlled study.

AIMS: The aim of the study was to compare the effects of meloxicam and piroxicam on the gastroduodenal mucosa in healthy adults. METHODS: Forty-four healthy volunteers were given a 28 day course of either meloxicam 15 mg, piroxicam 20 mg or placebo. Damage to the oesophageal, gastric and duodenal mucosa was assessed, mucosal blood flow (MBF) measured at endoscopy and biopsies taken for prostaglandin content and microscopic assessment of damage before NSAID administration and during days 1, 7 and 28 of continued intake. RESULTS: Maximal macroscopic gastric mucosal damage (median grade+IQR) occurred within 24 h of piroxicam administration, the damage score increasing from 0 to 2.5 (0-3) (P=0.02) at day 1 before falling to 2.0 (0-2) at day 7 and 0 (0-1) at day 28 with resolution of damage observed in six out of the seven subjects who sustained acute injury. No significant macroscopic gastric damage occurred in either of the two other groups although some minor damage was observed in seven subjects taking placebo and five taking meloxicam. There was a trend towards piroxicam causing more acute gastric damage than meloxicam (P=0.06). Baseline antral, body and duodenal MBF were similar in all three groups. No significant changes occurred in any of the groups on any of the visits. There were also no changes in gastric mucosal prostaglandin content in any group. CONCLUSIONS: These observations suggest that meloxicam causes little acute damage to the upper gastrointestinal tract and piroxicam causes some acute gastric injury but such damage resolves in most subjects by 28 days.

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress.

The influence of age, gender, Helicobacter pylori and smoking on gastric mucosal adaptation to non-steroidal anti-inflammatory drugs.

INTRODUCTION: Oral NSAIDs cause acute gastric injury that resolves, despite continued administration, by a process known as adaptation. Little is known about the factors that influence this process. METHODS: Sixty-two healthy volunteers were given a 28-day course of either etodolac 300 mg b.d. (13 subjects), naproxen 500 mg b.d. (23), enteric-coated diclofenac (10) or effervescent diclofenac 50 mg b.d. (16). All subjects were gastroscoped before and on days 1, 7 and 28 during drug administration, to assess gastric mucosal damage using a modified Lanza scale. Subjects were then divided into three categories: those who adapted completely, those who adapted incompletely and those who showed no adaptation. The proportion of subjects in each group was compared with respect to age, gender, smoking, the presence of Helicobacter pylori, and the NSAID prescribed. RESULTS: Fifty-nine subjects (median age 25.0 years, range 18-70) developed initial gastric injury to NSAIDs of whom 42 adapted completely, 13 adapted incompletely and four showed no evidence of adaptation. The mean age of subjects was lower in those who adapted (26.8 +/- 9.8 years) than those who adapted incompletely (32.5 +/- 10.3 years) and those who did not adapt (42.0 +/- 15.7 years, P = 0.01). There was no evidence of gender influencing adaptation. Of 17 H. pylori-positive subjects, a higher proportion had incomplete adaptation, with only nine subjects adapting completely (53% vs. 81%, P = 0.04). Sixteen subjects were smokers, of whom a greater proportion showed no evidence of adaptation (19% vs. 2%, P = 0.03). A smaller proportion of those who took naproxen (48%) adapted completely than those who took enteric-coated diclofenac (89%), effervescent diclofenac (75%) or etodolac (91%, P = 0.03). CONCLUSION: Some adaptation occurred in over 90% of subjects after 4 weeks dosing with an NSAID, but adaptation was less frequent in older subjects and in smokers. Complete adaptation occurred less frequently in H. pylori-positive subjects and in those who were given naproxen.

The growth arrest genes gas5, gas6, and CHOP-10 (gadd153) are expressed in the mouse preimplantation embryo.

The gas and gadd family of genes, known collectively as the growth arrest genes, are associated with the negative control of mammalian cell growth. The steady-state levels of their mRNAs are increased by three to fivefold when exponentially multiplying cells are exposed to a variety of stresses including inadequate nutrition or the removal of serum. Reverse transcription-polymerase chain reaction (RT-PCR) has been used to analyze growth arrest gene expression in the preimplantation mouse embryo. The gas5, gas6, and CHOP-10 (gadd153, Ddit3) genes were expressed from the eight-cell stage onward. The gas2 and gas3 genes associated with apoptosis were not expressed. Embryos were cultured in kSOM medium and a semiquantitative RT-PCR method was used to measure the relative gene expression using beta-actin mRNA as a reference. The ratio of gas5 to beta-actin mRNA was high at the eight-cell stage and fell three to fivefold during development. The decline in the gas5:beta-actin ratio corresponded to the activation of true cell growth (cytokinesis). The gas6:beta-actin ratio was low at the eight-cell stage and increased by twofold as the blastocyst formed. CHOP-10 was expressed at a constant level throughout development. Embryos that had developed in vivo were compared with the equivalent blastocyst-stage embryos cultured in kSOM medium. There were no significant differences in the ratio of CHOP-10, gas5, or gas6 mRNAs relative to beta-actin. These results suggest that these genes are expressed as part of normal early embryonic development. The potential roles of the growth arrest genes are discussed.

The use of percutaneous endoscopic gastrostomy (PEG) feeding tubes in patients with neurological disease.

Percutaneous endoscopic gastrostomy (PEG) is being used increasingly in the treatment of patients with neurogenic dysphagia to improve nutrition and prevent choking and aspiration pneumonia. PEG is used in a wide range of general medical conditions, but its role in clinical neurology is sometimes controversial. This paper reviews the place of PEG in the management of 32 patients with a variety of chronic and progressive neurological disorders. All the patients found it to be an effective and acceptable method of feeding that prevented weight loss, reduced chest infections, facilitated nursing care and improved their quality of life. PEG has an important role in neurological rehabilitation.

ob gene expression and secretion of leptin following differentiation of rat preadipocytes to adipocytes in primary culture.

Expression of the ob gene and production of leptin have been examined on differentiation of rat fibroblastic preadipocytes to adipocytes in primary culture. Preadipocytes were obtained from the inguinal fat pad of suckling rats, and following differentiation the cells contained lipid droplets and the mRNAs for both lipoprotein lipase and adipsin were detected by Northern blotting. ob mRNA was not, however, detected on Northern blots, but analysis by RT-PCR indicated that the ob gene was expressed, particularly after differentiation. Measurement of leptin in the culture medium by ELISA showed that the ob gene product was secreted by adipocytes from approximately 4 days after the induction of differentiation. Leptin production was sustained over a 2-week period with a peak at 8-10 days post-induction. Dexamethasone stimulated leptin production, while an inhibition was observed with the beta-adrenoceptor agonist isoprenaline. These results demonstrate that following the differentiation of fibroblastic preadipocytes to adipocytes in primary culture, leptin is secreted with the cells responding to stimuli which regulate production of the hormone.