Restriction of placental function alters heart development in the sheep fetus

Placental insufficiency, resulting in restriction of fetal substrate supply, is a major cause of intrauterine growth restriction (IUGR) and increased neonatal morbidity. Fetal adaptations to placental restriction maintain the growth of key organs, including the heart, but the impact of these adaptations on individual cardiomyocytes is unknown. Placental and hence fetal growth restriction was induced in fetal sheep by removing the majority of caruncles in the ewe before mating (placental restriction, PR). Vascular surgery was performed on 13 control and 11 PR fetuses at 110-125 days of gestation (term: 150 +/- 3 days). PR fetuses with a mean gestational Po(2) < 17 mmHg were defined as hypoxic. At postmortem (<135 or >135 days), fetal hearts were collected, and cardiomyocytes were isolated and fixed. Proliferating cardiomyocytes were counted by immunohistochemistry of Ki67 protein. Cardiomyocytes were stained with methylene blue to visualize the nuclei, and the proportion of mononucleated cells and length and width of cardiomyocytes were measured. PR resulted in chronic fetal hypoxia, IUGR, and elevated plasma cortisol concentrations. Although there was no difference in relative heart weights between control and PR fetuses, there was an increase in the proportion of mononucleated cardiomyocytes in PR fetuses. Whereas mononucleated and binucleated cardiomyocytes were smaller, the relative size of cardiomyocytes when expressed relative to heart weight was larger in PR compared with control fetuses. The increase in the relative proportion of mononucleated cardiomyocytes and the relative sparing of the growth of individual cardiomyocytes in the growth-restricted fetus are adaptations that may have long-term consequences for heart development in postnatal life.     

Thyroid Hormone Replacement Restores Circulating Enkephalin Concentrations in Hypophysectomized Fetal Sheep

Abstract: We have investigated the effects of fetal hypophysectomy (HX) with or without thyroxine (T₄) replacement on the plasma concentrations of free methionine-enkephalin (free Met-Enk), noradrenaline, and adrenaline in late gestation sheep fetus. Plasma adrenaline concentrations were significantly higher in intact fetal sheep (1.05 ± 0.12 pmol/L) between 125 and 140 days of gestation when compared with the HX + saline (0.64 ± 0.10 pmol/L) and HX + T₄ (0.61 ± 0.08 pmol/L) groups. During the first 15 days of the T₄ or saline infusion, the plasma concentrations of free Met-Enk were significantly higher in the HX + T₄ group (392 ± 40 pmol/L) than in the HX + saline group (299 ± 43 pmol/L). At this stage of gestation, however, circulating concentrations of free Met-Enk were significantly higher in intact fetal sheep (556 ± 51 pmol/L) than in either of the HX groups. Between 125 and 140 days of gestation, plasma free Met-Enk concentrations were similar and significantly higher in the intact and HX + T₄ groups than those measured in the HX + saline fetal sheep. We conclude that the decrease in circulating free Met-Enk concentrations after removal of the fetal pituitary is primarily a consequence of functional athyroidism.     

Differential Effects of Exposure to Maternal Obesity or Maternal Weight Loss during the Periconceptional Period in the Sheep on Insulin Signalling Molecules in Skeletal Muscle of the Offspring at 4 Months of Age

Exposure to maternal obesity before and/or throughout pregnancy may increase the risk of obesity and insulin resistance in the offspring in childhood and adult life, therefore, resulting in its transmission into subsequent generations. We have previously shown that exposure to maternal obesity around the time of conception alone resulted in increased adiposity in female lambs. Changes in the abundance of insulin signalling molecules in skeletal muscle and adipose tissue precede the development of insulin resistance and type 2 diabetes. It is not clear, however, whether exposure to maternal obesity results in insulin resistance in her offspring as a consequence of the impact of increased adiposity on skeletal muscle or as a consequence of the programming of specific changes in the abundance of insulin signalling molecules in this tissue. We have used an embryo transfer model in the sheep to investigate the effects of exposure to either maternal obesity or to weight loss in normal and obese mothers preceding and for one week after conception on the expression and abundance of insulin signalling molecules in muscle in the offspring. We found that exposure to maternal obesity resulted in lower muscle GLUT-4 and Ser 9 phospho-GSK3α and higher muscle GSK3α abundance in lambs when compared to lambs conceived in normally nourished ewes. Exposure to maternal weight loss in normal or obese mothers, however, resulted in lower muscle IRS1, PI3K, p110β, aPKCζ, Thr 642 phospho-AS160 and GLUT-4 abundance in the offspring. In conclusion, maternal obesity or weight loss around conception have each programmed specific changes on subsets of molecules in the insulin signalling, glucose transport and glycogen synthesis pathways in offspring. There is a need for a stronger evidence base to ensure that weight loss regimes in obese women seeking to become pregnant minimize the metabolic costs for the next generation.     

Characterization of oxylipins and dioxygenase genes in the asexual fungus Aspergillus niger

Background  

Aspergillus niger is an ascomycetous fungus that is known to reproduce through asexual spores, only. Interestingly, recent genome analysis of A. niger has revealed the presence of a full complement of functional genes related to sexual reproduction [1]. An example of such genes are the dioxygenase genes which in Aspergillus nidulans, have been shown to be connected to oxylipin production and regulation of both sexual and asexual sporulation [2–4]. Nevertheless, the presence of sex related genes alone does not confirm sexual sporulation in A. niger.     

Systematic Reduction of a Stochastic Signalling Cascade Model

Biochemical systems involve chemical reactions occurring in low-number regimes, wherein fluctuations are not negligible and thus stochastic models are required to capture the system behaviour. The resulting models are often quite large and complex, involving many reactions and species. For clarity and computational tractability, it is important to be able to simplify these systems to equivalent ones involving fewer elements. While many model simplification approaches have been developed for deterministic systems, there has been limited work on applying these approaches to stochastic modelling. Here, we propose a method that reduces the complexity of stochastic biochemical network models, and apply this method to the reduction of a mammalian signalling cascade. Our results indicate that the simplified model gives an accurate representation for not only the average number of all species, but also for the associated fluctuations and statistical parameters.     

Distribution of living polycystine radiolarians in the Gulf of Mexico and Caribbean Sea,and comparison with the sedimentary record

Plankton tows and bottom samples from the Gulf of Mexico and Caribbean Sea were examined to study the distribution, ecology, and preservation of modern radiolarians. Abundances of radiolarians from this region, an area of low primary productivity, are low. Diversities are variable in the surface waters; diversity indices decrease with increasing water depth, indicating that fewer niches are occupied by radiolarians in deep water. Living radiolarians are confined to specific water masses, some species ranging between two masses, and can be used as indicators of these water masses in ecologic or paleo-oceanographic studies.The record of radiolarian tests present in the sediment is dissimilar to the record of living radiolarians in the water column, but is similar to the record of empty tests falling through the water column. This indicates that a considerable length of time is involved in the settling of radiolarian tests. It also confirms the validity of ecologic interpretations based on a study of sediments only.     

Physiological and immunological changes in the brain associated with lethal eastern equine encephalitis virus in macaques

Aerosol exposure to eastern equine encephalitis virus (EEEV) can trigger a lethal viral encephalitis in cynomolgus macaques which resembles severe human disease. Biomarkers indicative of central nervous system (CNS) infection by the virus and lethal outcome of disease would be useful in evaluating potential medical countermeasures, especially for therapeutic compounds. To meet requirements of the Animal Rule, a better understanding of the pathophysiology of EEEV-mediated disease in cynomolgus macaques is needed. In this study, macaques given a lethal dose of clone-derived EEEV strain V105 developed a fever between 2–3 days post infection (dpi) and succumbed to the disease by 6 dpi. At the peak of the febrile phase, there was a significant increase in the delta electroencephalography (EEG) power band associated with deep sleep as well as a sharp rise in intracranial pressure (ICP). Viremia peaked early after infection and was largely absent by the onset of fever. Granulocytosis and elevated plasma levels of IP-10 were found early after infection. At necropsy, there was a one hundred- to one thousand-fold increase in expression of traumatic brain injury genes (LIF, MMP-9) as well as inflammatory cytokines and chemokines (IFN-γ, IP-10, MCP-1, IL-8, IL-6) in the brain tissues. Phenotypic analysis of leukocytes entering the brain identified cells as primarily lymphoid (T, B, NK cells) with lower levels of infiltrating macrophages and activated microglia. Massive amounts of infectious virus were found in the brains of lethally-infected macaques. While no infectious virus was found in surviving macaques, quantitative PCR did find evidence of viral genomes in the brains of several survivors. These data are consistent with an overwhelming viral infection in the CNS coupled with a tremendous inflammatory response to the infection that may contribute to the disease outcome. Physiological monitoring of EEG and ICP represent novel methods for assessing efficacy of vaccines or therapeutics in the cynomolgus macaque model of EEEV encephalitis.     

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

AIMS: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. METHODS AND RESULTS: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 10(10)R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. CONCLUSIONS: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. SIGNIFICANCE AND IMPACT OF THE STUDY: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.