Exposure to traffic-related air pollution and changes in exhaled nitric oxide and DNA methylation in arginase and nitric oxide synthase in children with asthma.

BACKGROUND: Traffic-related air pollution (TRAP) has been associated with increased risk of airway inflammation in children with asthma. While epigenetic changes could potentially modulate TRAP-induced inflammatory responses, few studies have assessed the temporal pattern of exposure to TRAP, epigenetic changes and inflammation in children with asthma. Our goal was to test the time-lag patterns of personal exposure to TRAP, airway inflammation (measured as fractional exhaled nitric oxide, FeNO), and DNA methylation in the promoter regions of genes involved in nitric oxide synthesis among children with asthma. METHODS: We measured personal exposure to black carbon (BC) and FeNO for up to 30 days in a panel of children with asthma. We collected 90 buccal cell samples for DNA methylation analysis from 18 children (5 per child). Methylation in promoter regions of nitric oxide synthase (NOS1, NOS2A, NOS3) and arginase (ARG1, ARG2) was assessed by bisulfite pyrosequencing. Linear-mixed effect models were used to test the associations of BC at different lag periods, percent DNA methylation at each site and FeNO level. RESULTS: Exposure to BC was positively associated with FeNO, and negatively associated with DNA methylation in NOS3. We found strongest association between FeNO and BC at lag 0-6 h while strongest associations between methylation at positions 1 and 2 in NOS3 and BC were at lag 13-24 h and lag 0-24 h, respectively. The strengths of associations were attenuated at longer lag periods. No significant associations between exposure to TRAP and methylation levels in other NOS and ARG isoforms were observed. CONCLUSIONS: Exposure to TRAP was associated with higher levels of FeNO and lower levels of DNA methylation in the promoter regions of the NOS3 gene, indicating that DNA methylation of the NOS3 gene could be an important epigenetic mechanism in physiological responses to TRAP in children with asthma.

Lack of association between allelic status and myostatin content in lambs with the myostatin g+6723G>A allele.

Lambs with the myostatin (MSTN) g+6723G>A mutation have a greater muscle mass, which is believed to be associated with reduced myostatin protein abundance. This experiment was designed to determine if differences in allelic frequency of the MSTN g+6723G>A mutation affected abundance of myostatin protein from birth to 24 wk of age. A Poll Dorset cross White Suffolk ram (MSTN A/G) was mated to 35 White Suffolk cross Border Leicester cross Merino ewes (MSTN A/G, n=21, and MSTN G/G, n=14). The progeny of these matings delivered 44 lambs with MSTN A/A (n=9), MSTN A/G (n=21), and MSTN G/G (n=14) genotypes. At approximately 1, 4, and 12 wk of age, a biopsy sample was collected and a blood sample was taken to measure the abundance of myostatin protein in muscle and plasma. At approximately 24 wk of age, the wether lambs were slaughtered to determine carcass characteristics and muscle samples were taken from the bicep femoris. The abundance of mature myostatin protein in muscle from 1 wk old lambs was less (P=0.05) in MSTN A/A and MSTN A/G compared with MSTN G/G lambs. However, at 4 and 24 wk the MSTN A/A lambs had a greater (P=0.04) abundance of myostatin protein compared with the MSTN A/G and MSTN G/G lambs. The abundance of mature myostatin did not differ between genotypes in plasma but the myostatin protein did increase as the lambs aged. At slaughter the MSTN A/A wether lambs had greater dressing percentages (P=0.04), shortloin (P=0.01), topside (P<0.001), and round (P=0.01) weights but did not differ in final BW or HCW (P>0.05). The MSTN A/A lambs had more muscle fibers (P=0.02) in the cross-section of LM between the 12th and 13th rib. The MSTN A/A lambs also had greater lean (P=0.002), less fat (P=0.009), and reduced organ (heart, liver, spleen, and kidneys) mass as determined by computed tomography scanning than MSTN G/G lambs. The results of this study demonstrated that lambs homozygous for the MSTN g+6723G>A mutation have changes in carcass characteristics (dressing and total lean), organ weights, and muscle fiber number. This may be due to reduced myostatin protein early in utero, but after 4 wk of age there was no difference in the abundance of mature myostatin protein in muscle or plasma among MSTN A/A, MSTN A/G, and MSTN G/G genotypes.

Myostatin allelic status interacts with level of nutrition to affect growth, composition, and myofiber characteristics of lambs.

The objective of this experiment was to determine if growth, carcass composition, and myofiber characteristics of lambs were affected by heterozygosity for a myostatin mutation (g+6723G>A) when offered differing allowances of feed administered with or without ractopamine. Heterozygote [MSTN A/G (n = 40)] and homozygote wildtype [MSTN G/G (n = 39)] castrate male lambs were individually fed ad libitum (HI; 1.8 × estimated ME(m)) or a restricted allowance (LO; 1.1 × estimated ME(m)) of a diet (191 g of CP/kg of DM and 12 MJ of ME/kg of DM), supplemented with (0.4 mg/kg of BW) or without the ß-adrenergic agonist ractopamine (RAC or NO RAC) for 47 d. The lambs were scanned by computed tomography at the beginning and completion of the feeding experiment to calculate composition of lean, fat, and bone in the carcass component of the body. The MSTN A/G HI intake lambs had significantly greater total daily carcass growth (P = 0.045) and loin eye depth (P = 0.022) and tended to have a greater daily growth of lean (P = 0.09) in the carcass, compared with MSTN G/G HI intake lambs. Conversely, MSTN A/G LO intake lambs tended to have less daily lean deposition (P = 0.09), significantly less total daily carcass growth (P = 0.045), and had a greater percentage of type IIX myofibers (P < 0.01) and total myofiber area (relative area) of type IIX myofibers (P = 0.013). The inclusion of RAC increased final BW (P = 0.03) and ADG (P = 0.02), percentage of type IIC (P < 0.001) and IIA (P = 0.012) myofibers, cross-sectional area of types I (P = 0.04) and IIAX (P = 0.04) fibers, and the relative area of type IIC (P = 0.003) and IIA (P = 0.01) myofibers in the LM. The experiment demonstrated that including RAC in the diet of lambs increased final BW and ADG, but not HCW, and increased proportion of type IIC and IIA myofibers and cross-sectional area of type I and IIAX myofibers. Our data suggest that RAC and the heterozygous myostatin mutation act together to increase growth of muscle on a high plane of nutrition. The experiment also demonstrated that poor nutritional background of lambs heterozygous for the myostatin mutation may negatively influence their growth rates and myofiber characteristics.

Randomized placebo-controlled trial of granulocyte-macrophage colony-stimulating-factor support for dose-intensive cyclophosphamide, etoposide, and cisplatin.

This is a double-blind randomized placebo-controlled trial to evaluate the efficacy and safety of granulocyte-macrophage colony-stimulating-factor (GM-CSF) after dose-intensive cyclophosphamide, etoposide, and cisplatin (DICEP). Fifty-six patients with lymphoma or breast carcinoma were randomized to receive GM-CSF 250 microg/m2 or placebo subcutaneously (SC) every 12 hr after each course of DICEP until recovery of absolute neutrophil count (ANC) of 1.5 x 10(9)/L. Each patient was to receive three courses of DICEP. There were 28 patients in each group. The median duration of ANC below 0.5 x 10(9)/L was 10 versus 12 days for Course 1 (P = 0.010), 10 versus 12 days for Course 2 (P = 0.248), and 16.5 versus 15 days for Course 3 (P = 0.126); platelet counts below 20 x 10(9)/L was 4 versus 4 days for Course 1 (P = 0.586), 8.5 versus 7 days for Course 2 (P = 0.013), and 23.5 versus 10.5 days for Course 3 (P = 0.104); hospitalization for patients readmitted with cytopenic fever were 4 versus 8 days for Course 1 (P = 0.035); 7 versus 6 days for Course 2 (P = 0.692); and 8 versus 12 days for Course 3 (P = 0.884) in the GM-CSF and placebo group, respectively. GM-CSF significantly shortens the duration of neutropenia and readmission only during the first course of DICEP. There was a delay in platelet recovery and an increase in transfusion requirement during subsequent courses in the GM-CSF group. The result cautions the routine use of lineage specific hematopoietic growth factors in supporting repeated cycles of dose-intensive chemotherapy.

Substrate and hormonal responses to exercise in women using oral contraceptives.

Hormone and substrate responses to mild and heavy treadmill exercise were compared in women who used oral contraceptives (OC group; n = 7) and in normally menstruating women (control group; n = 8). Venous blood samples were obtained before exercise (-5 min), during exercise (15, 30, 45, and 60 min), and 30 min after exercise. All samples were analyzed for glucose, lactate, free fatty acids (FFA), glycerol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), human growth hormone (hGH), cortisol, insulin, estradiol (E2), and progesterone (P). Substrate patterns during exercise were not altered by the phase of the menstrual cycle or OC usage. However, in the OC group the FFA concentrations were consistently higher during mild exercise and the glucose concentrations were lower at rest and during exercise than in the control group (P less than 0.05). No differences in lactate or glycerol responses were observed between the groups (P greater than 0.05). The responses of insulin and hGH to exercise were not related to the OC use per se but rather to the steroid status, either endogenous or exogenous. Specifically, during the steroid phases (OC use phase and luteal phase) 1) insulin concentrations were not quite as markedly reduced (i.e., 12% higher when luteal phase and OC usage phase data were combined; P less than 0.05), and 2) hGH concentrations at rest and during light exercise were higher in the OC group during the OC use phase (P less than 0.05). LH patterns were not affected by exercise (P greater than 0.05), but a slight decrease was found in FSH (P less than 0.05). Increments in P and E2 were observed in the control group in both the follicular and luteal phase (P less than 0.05), but much greater increments in P occurred in the luteal phase than in the follicular phase (P less than 0.05). In contrast to the control group, no increments in P, E2, or cortisol occurred in the OC users during exercise (P greater than 0.05). Therefore the new observations in this study are that 1) insulin and growth hormone respond in a complex manner during exercise with either the phase of the menstrual cycle or the phases of OC use and disuse and 2) the steroid concentrations (P, E2, cortisol) are increased in the controls but not in the OC users during exercise. The latter point suggests that normal steroid increments are due to an increased rate of secretion rather than a decrease in the hepatic clearance of these steroids.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of anaesthesia on insulin-induced hypoglycemia in rabbits.

The aim of this study was to determine how anaesthetized rabbits survive much longer than awake rabbits after receiving an insulin overdose. Insulin appeared to act in both groups of rabbits because there was a prompt fall in circulating glucose, free fatty acids, and beta-hydroxybutyrate concentrations. Carbohydrate appeared to be the principal energy source for anaesthetized rabbits because their respiratory quotient approached unity. Although the fall in glycemia was similar in both groups of rabbits, the circulating lactate concentration rose only in the anaesthetized group. This rise in lactate in the initial 60 min after insulin was given could account for most of the fall in glycemia if the source of lactate was the glucose pool. The decline in hepatic glycogen was close to 100 mumol/g liver; this would account for about one-third of the total energy turnover and close to one-half of the measured glucose appearance in these anaesthetized rabbits. As judged from the rate of oxygen consumption, muscle glycogen seemed to supply two-thirds of the fuel to be oxidized in these rabbits. However, only one-third of the lactate released from muscle was first converted to glucose and the remainder was oxidized directly to CO2. Although insulin provided the metabolic setting for a rapid rate of glucose oxidation, this rate appeared to be diminished when the overall rate of oxygen consumption was lower during anaesthesia.

The structure of the hepatic insulin receptor and insulin binding.

Hepatocytes or hepatic plasma membranes were photoaffinity-labelled with radioiodinated N epsilon B29-monoazidobenzoyl-insulin. Analysis of the samples by SDS/polyacrylamide-gel electrophoresis and autoradiography revealed the insulin receptor as a predominant band of 450 kDa. When hepatic plasma membranes were first treated with clostridial collagenase and then photolabelled, the insulin receptor appeared as a predominant band of 360 kDa. This effect of collagenase treatment on the insulin receptor was due to Ca2+-dependent heat-labile proteinases contaminating the preparation of collagenase, and it could be mimicked by elastase. The decrease in size of the insulin receptor to 360 kDa resulted from the loss of a receptor component that was inaccessible to photolabelling. In contrast, the size of the insulin receptor of intact cells was not affected by collagenase treatment. This suggests that the site sensitive to proteolysis was located on the cytoplasmic side of the plasma membrane. In hepatic plasma membranes that were treated with collagenase or elastase, and contained the 360 kDa form of the insulin receptor, the binding affinity for insulin was increased by up to 2-fold. These findings support the concept that a component which is either a part of, or closely associated with, the insulin receptor may regulate its affinity for insulin.

Photoaffinity labelling of hepatic plasma membranes suggests two classes of hepatic insulin receptor.

Photoaffinity labelling of hepatic insulin receptors revealed specifically-labelled bands of 130, 90 and 40 kDa. Endogenous protease activity in hepatic plasma membranes, as well as contaminating proteases present in preparations of clostridial collagenase, degraded some of the 130-kDa insulin-binding subunit to a 115-kDa form. However, a large proportion of the 130-kDa subunits were resistant to degradation, suggesting the presence of two classes of insulin receptor in hepatic plasma membranes. In one class the 130-kDa subunit was sensitive to proteolysis, while in the other it was not. In contrast, the 130-kDa receptor subunits of adipose tissue were all resistant to such degradation. Scatchard analysis of control and collagenase-treated plasma membranes demonstrated that conversion of the 130-kDa subunit to a 115-kDa form did not affect the insulin-binding characteristics of the receptor. It was also apparent that insulin binds to a single class of high-affinity sites in hepatic plasma membranes.

Hyperinsulinemia and in vivo very-low-density lipoprotein-triglyceride kinetics.

The effect of hyperinsulinemia (2 wk of twice daily NPH insulin) on the kinetics of very-low-density lipoprotein (VLDL)-triglyceride (TG) was studied in rats. To avoid profound hypoglycemia the rats were allowed sucrose ad libitum. Two control groups were needed: chow only and ad libitum sucrose-supplemented (high-CHO). The insulin-treated rats had 15 times higher IRI and 50% lower plasma glucose levels than either control group. Their TG production exceeded and their TG concentrations were less than those of either control group. This indicated that their TG removal was increased even more than their TG production. This increase in TG production occurred despite lower plasma free fatty acid (FFA) levels, suggesting that a greater proportion of TG fatty acids came from a source other than FFA. Compared with chow controls, high-CHO controls had the same peripheral IRI, a slight increase in TG production, and an increase in TG concentration. The differences between the effects of CHO supplementation alone or together with injected insulin may relate to the IRI and/or the route of access of insulin (peripheral vs. portal). The present studies indicate that hyperinsulinemia, either directly or indirectly, accelerates triglyceride turnover.

Effects of menstrual cycle on metabolic responses to exercise.

Selected substrate and hormonal responses to exercise were compared in two phases of the menstrual cycle. Exercise-induced changes in substrate [glucose, lactate, free fatty acids (FFA), glycerol] and hormonal patterns [luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin, progesterone (P), growth hormone (GH), cortisol] were compared in the follicular and luteal phases of the menstrual cycle in 24-h-fasted (n = 5), glucose-loaded (n = 6; 1.50 g/kg, 20% solution), and control subjects (n = 8). A treadmill walk was maintained for 60 min (30 min, 40% VO2 max; 30 min, 80% VO2 max). Blood samples were obtained 5 min before, 15, 30, 45, and 60 min during, and 30 min after exercise. In the glucose group a blood sample was also taken 20 min before exercise, and glucose was ingested 15 min before exercise. Within each nutritional group the metabolic and endocrine responses to exercise were similar in the two phases for glucose, lactate, glycerol, LH, FSH, and cortisol (P greater than 0.05). In the glucose group the FFA response was lower in the luteal phase (P less than 0.05). In the fasted subjects insulin and GH responses were elevated in the luteal phase (P less than 0.05). P responses in the control and glucose groups were markedly greater in the luteal phase (P less than 0.05). In the fasted subjects no alteration in P occurred in either phase (P less than 0.05), and the LH concentration was lower in these subjects relative to the control groups (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Anomalous pulmonary venous return with intact atrial septum: diagnosis and pathophysiology.

Twenty-one patients with partial anomalous pulmonary venous drainage with intact atrial septum have been studied. These include 13 patients not previously reported from our laboratories and eight patients with complete hemodynamics reported by others. Methods for identification of this abnormality and for identification of an intact atrial septum are described, including differential indicator dilution curves, catheter probing of the atrial septum and pulmonary angiography. Blood flow through anomalously draining lobes of the lung is usually higher than through normally drainage lobes attributable to the higher pressure differences across the anomalous lung, right atrial pressure being uniformly lower than left atrial pressure. The pulmonary vascular resistance when "standardized" to the flow of blood normally present in different portions of the lung indicated that no significant differences existed between normally and anomalously draining lobes. Six patients had coexisting rheumatic mitral stenosis and one had congenital mitral stenosis. Its influence on the hemodynamic changes produced by PAPVD is discussed.

Genetic basis of heterosis for yield in the autotetraploid potato.

A model of overdominant gene action to explain heterosis for yield in the autotetraploid potato has been presented. Loci with multiple allcles and a maximum heterotic value for quadrigenic genotypic structures have been postulated. Various experimental results have been analyzed on the basis of such a model in contrast with a dominance situation. The analysis suggests a close positive correlation between heterozygosity and yield. The implication of the proposed overdominant model to potato breeding would be that substantial genetic advance in yield should be made upon increasing the genetic diversity of the parental clones. However, the alien sources of germplasm should undergo some previous selection for adaptation. A proper balance between heterozygosity and adaptation, mainly to photoperiod, should maximize the heterosis for yield.