Epigenetics and fetal adaptation to perinatal events: diversity through fidelity.

Perinatal insults, including fetal undernutrition and hypoxia, are associated with an increased susceptibility to several adult-onset metabolic disorders. These include cardiovascular disease, insulin resistance, and obesity. However, the mechanisms driving the long-term phenotypic consequences have only recently begun to be elucidated. A primary mechanism accounting for perinatal adaptation is the epigenetic modification of chromatin. In this context, epigenetic modifications to chromatin are thought to arise in response to a perinatal insult in an effort to modulate gene expression and maximize fetal survival. In this symposium report, we discuss epigenetics as a mechanism by which perinatal adaptations can be made by the developing fetus. We examine the benefits of using multiple in vivo models to understand the interrelation of signals that come together and result in perinatal adaptation. Epigenetic effects on IGF-1 arising from a perinatal insult are discussed, as are the difficulties and challenges associated with this complex field. In conclusion, epigenetics provides a means of modulating gene transcription, thus allowing fetal adaptation to a broad variety of conditions.

A novel sensor array for field based ocular ultraviolet radiation measurements.

The intensification of terrestrial solar ultraviolet radiation (UVR) due to the diminution of the ozone layer has promoted a variety of research into establishing the impact of this elevated potential dose of UVR on biological tissues. Certain anterior ocular tissues have been found to be susceptible to damage by incident UVR and potentially blinding diseases such as pterygium are thought to be a direct result of absorbed UVR at the nasal limbus. There is a need for more accurate quantification and localisation of incident UVR at the anterior ocular surface. A novel solar blind photodiode sensor array system has been designed, constructed and tested for this purpose. Initial measurements to quantify the irradiance across the anterior ocular surface within the latitudes known as the 'pterygium belt' provide us with a set of core data for different head orientations and tilt angles and indicate the accuracy and stability of the system.

A universal molecular method for identifying underground plant parts to species.

As part of a large project to determine rooting depth and resource uptake on the Edwards Plateau of central Texas, we developed a DNA-based technique that allows the below-ground parts of all plants to be identified to the level of genus and usually to species. Identification is achieved by comparing DNA sequences of the internal transcribed spacer (ITS) region of the 18S-26S nuclear ribosomal DNA repeat, derived from below-ground plant material, with a reference ITS region database for plants at a site. The method works throughout plants because the plant ITS region can be PCR amplified using a set of universal primers. Congeneric species can usually be identified because the ITS region evolves relatively rapidly. In our study, all roots were easily identified to the level of genus; most congeneric species were identified solely by ITS sequence differences but some required a combination of ITS sequence data and above-ground surveys of species at a site. In addition to showing the feasibility and efficacy of our technique, we compare it with another DNA-based technique used to identify below-ground plant parts. Finally, we also describe a DNA extraction and purification technique that reliably provides high-quality DNA of sufficient quantity from roots so that PCR can be readily accomplished. Our technique should allow the below-ground parts of plants in any system to be identified and thereby open new possibilities for the study of below-ground plant communities.

What is the magnitude of blood pressure response to a programme of moderate intensity exercise? Randomised controlled trial among sedentary adults with unmedicated hypertension.

BACKGROUND: Current guidelines for the management of hypertension recommend regular, moderate intensity aerobic exercise such as brisk walking as a means of blood pressure reduction. However, there is a lack of consistent evidence regarding the magnitude of blood pressure response to such a prescription. In particular, no well designed studies have investigated the efficacy of a programme of exercise meeting current guidelines. AIM: To investigate the effect of a six-week programme of moderate intensity exercise on daytime ambulatory blood pressure (10.00 am to 10.00 pm) among unmedicated, sedentary adults aged 25 years to 63 years with office blood pressure of 150 mmHg to 180 mmHg systolic and/or 91 mmHg to 110 mmHg diastolic. METHOD: Randomised controlled trial of participants carrying out 30 minutes of moderate intensity exercise (brisk walking or equivalent) five days per week for six weeks compared with controls who maintained existing levels of physical activity. RESULTS: Compliance with the exercise programme was high. The reduction in mean daytime ambulatory blood pressure between baseline and six-week follow-up was greater in the intervention group than in the control group for both systolic and diastolic blood pressure. However, this net hypotensive effect was not statistically significant (systolic = -3.4 mmHg, 95% CI = -7.4 to 0.6; diastolic = -2.8 mmHg, 95% CI = -5.8 to 0.2). Adjusting for baseline differences in mean ambulatory blood pressure in an analysis of covariance led to a reduction in the estimated magnitude of the effect (systolic = -1.9 mmHg, 95% CI = -5.4 to 1.7, P = 0.31; diastolic = -2.2 mmHg, 95% CI = -4.9 to 0.5, P = 0.11). CONCLUSION: Despite high compliance with the exercise programme, the magnitude of the hypotensive effect of moderate intensity exercise was not as great as that found in studies of higher intensity exercise among hypertensives. Expectations of general practitioners and patients that a programme of moderate intensity exercise will lead to a clinically important reduction in the individual's blood pressure are unlikely to be realised.

Ecosystem rooting depth determined with caves and DNA.

Belowground vertical community composition and maximum rooting depth of the Edwards Plateau of central Texas were determined by using DNA sequence variation to identify roots from caves 5-65 m deep. Roots from caves were identified by comparing their DNA sequences for the internal transcribed spacer (ITS) region of the 18S-26S ribosomal DNA repeat against a reference ITS database developed for woody plants of the region. Sequencing the ITS provides, to our knowledge, the first universal method for identifying plant roots. At least six tree species in the system grew roots deeper than 5 m, but only the evergreen oak, Quercus fusiformis, was found below 10 m. The maximum rooting depth for the ecosystem was approximately 25 m. (18)O isotopic signatures for stem water of Q. fusiformis confirmed water uptake from 18 m underground. The availability of resources at depth, coupled with small surface pools of water and nutrients, may explain the occurrence of deep roots in this and other systems.

Sudden death of two horses associated with pulmonary aspergillosis.

The sudden death of two horses was attributed to the rapid and acute development of pulmonary aspergillosis. One horse was making excellent postoperative progress after a jejunal resection and anastomosis for intestinal adhesions. The other horse was being treated routinely for equine protozoal myeloencephalitis (EPM). Signs of fever and an increased respiratory rate were detected shortly before death in the first horse, but no premonitory clinical signs characteristic of pulmonary infection were detected in the horse being treated for EPM. Both horses developed rapidly debilitating, acute pulmonary mycosis and died unexpectedly.

HMG-CoA reductase guides migrating primordial germ cells.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is best known for catalysing a rate-limiting step in cholesterol biosynthesis, but it also participates in the production of a wide variety of other compounds. Some clinical benefits attributed to inhibitors of HMG-CoA reductase are now thought to be independent of any serum cholesterol-lowering effect. Here we describe a new cholesterol-independent role for HMG-CoA reductase, in regulating a developmental process: primordial germ cell migration. We show that in Drosophila this enzyme is highly expressed in the somatic gonad and that it is necessary for primordial germ cells to migrate to this tissue. Misexpression of HMG-CoA reductase is sufficient to attract primordial germ cells to tissues other than the gonadal mesoderm. We conclude that the regulated expression of HMG-CoA reductase has a critical developmental function in providing spatial information to guide migrating primordial germ cells.

Aorto-iliac thrombosis in a foal.

A six-day-old Missouri foxtrotter colt was examined because it had had diarrhoea since it was 24 hours old. A diagnosis of colitis, septicaemia, and disruption of the arterial blood flow to the pelvic limbs was made on the basis of clinical and laboratory findings. Despite intensive medical therapy, the foal died 13 hours after being examined. Postmortem examination revealed diffuse fibrinous enteritis with lymphoid necrosis, multifocal fibrinonecrotic typhlocolitis, disseminated intravascular coagulation, and a large occluding thrombus at the aortic termination. The results of bacteriological culturing supported the diagnosis of septicaemia leading to activation of the clotting cascade, disseminated intravascular coagulation, aorto-iliac thrombosis and infarction of the pelvic limbs.

Activation of extracellular matrix metalloproteinases in equine laminitis.

Samples of connective tissue obtained from the hoof of six laminitic and eight non-laminitic adult horses were analysed zymographically to investigate whether connective tissue matrix metalloproteinases are activated or induced during laminitis. The activity or matrix metalloproteinases was substantially greater in the tissues from the laminitic horses than in the tissues from the non-laminitic horses. A comparison of the collagenolytic activity in the laminitic and control tissues showed that collagenolytic activities corresponding to the 92 kDa (P < 0.001), 72 kDa (P < 0.01) and 66 kDa (P < 0.01) bands were induced in the laminitic tissues.

Gonadal mesoderm and fat body initially follow a common developmental path in Drosophila.

During gastrulation, the Drosophila mesoderm invaginates and forms a single cell layer in close juxtaposition to the overlying ectoderm. Subsequently, particular cell types within the mesoderm are specified along the anteroposterior and dorsoventral axes. The exact developmental pathways that guide the specification of different cell types within the mesoderm are not well understood. We have analyzed the developmental relationship between two mesodermal tissues in the Drosophila embryo, the gonadal mesoderm and the fat body. Both tissues arise from lateral mesoderm within the eve domain. Whereas in the eve domain of parasegments 10-12 gonadal mesoderm develops from dorsolateral mesoderm and fat body from ventrolateral mesoderm, in parasegments 4-9 only fat body is specified. Our results demonstrate that the cell fate decision between gonadal mesoderm and fat body identity within dorsolateral mesoderm along the anteroposterior axis is determined by the combined actions of genes including abdA, AbdB and srp; while srp promotes fat body development, abdA allows gonadal mesoderm to develop by repressing srp function. Furthermore, we present evidence from genetic analysis suggesting that, before stage 10 of embryogenesis, gonadal mesoderm and the fat body have not yet been specified as different cell types, but exist as a common pool of precursor cells requiring the functions of the tin, zfh-1 and cli genes for their development.

zfh-1 is required for germ cell migration and gonadal mesoderm development in Drosophila.

In Drosophila as well as many vertebrate systems, germ cells form extraembryonically and migrate into the embryo before navigating toward gonadal mesodermal cells. How the gonadal mesoderm attracts migratory germ cells is not understood in any system. We have taken a genetic approach to identify genes required for germ cell migration in Drosophila. Here we describe the role of zfh-1 in germ cell migration to the gonadal mesoderm. In zfh-1 mutant embryos, the initial association of germ cells and gonadal mesoderm is blocked. Loss of zfh-1 activity disrupts the development of two distinct mesodermal populations: the caudal visceral mesoderm and the gonadal mesoderm. We demonstrate that the caudal visceral mesoderm facilitates the migration of germ cells from the endoderm to the mesoderm. Zfh-1 is also expressed in the gonadal mesoderm throughout the development of this tissue. Ectopic expression of Zfh-1 is sufficient to induce additional gonadal mesodermal cells and to alter the temporal course of gene expression within these cells. Finally, through analysis of a tinman zfh-1 double mutant, we show that zfh-1 acts in conjunction with tinman, another homeodomain protein, in the specification of lateral mesodermal derivatives, including the gonadal mesoderm.

Identification of genes controlling germ cell migration and embryonic gonad formation in Drosophila.

Gonadogenesis in the Drosophila embryo is a complex process involving numerous cellular migratory steps and cell-cell interactions. The mechanisms guiding germ cells to move through, recognize and adhere to specific cell types are poorly understood. In order to identify genes that are required for these processes, we have conducted an extensive mutagenesis of the third chromosome and screened for mutations disrupting germ cell migration at any point in embryonic development. Phenotypic analysis of these mutants demonstrates that germ cell migration can be broken down into discrete developmental steps, with each step requiring a specific set of genes. Many of these genes are involved in the development of gonadal mesoderm, the tissue that associates with germ cells to form the embryonic gonad. Moreover, mutations that we isolated affecting embryonic patterning as well as germ cell migration suggest that the origin of gonadal mesoderm lies within the eve domain of the developing mesoderm.

Management of headshaking in three horses by treatment for protozoal myeloencephalitis.

Unlike the incidence of equine protozoal myeloencephalitis (EPM), which appears to be increasing, headshaking is an uncommon problem for horses in Missouri and the adjacent states. Equine protozoal myeloencephalitis was incriminated in three horses examined for the treatment of headshaking on the basis of a neurological examination, an analysis of cerebrospinal fluid and their response to treatment. The headshaking and stereotypical behaviour associated with EPM was successfully treated with potentiated sulphonamides and pyrimethamine.

Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken.

Two unique cDNA clones containing chicken slow myosin heavy chain (MyHC) inserts have been isolated from an expression library. Immunochemical analyses of the expressed proteins using different slow MyHC specific monoclonal antibodies were consistent with the two clones encoding slow MyHC 1 (SM1) and slow MyHC 2 (SM2) protein sequences. Northern blot analyses showed that the clones hybridized with 6-kb mRNAs that are differentially expressed in developing and adult slow muscles, further supporting the conclusion that these two clones represent SM1 and SM2 cDNAs. Sequence analyses show that both clones encode the highly conserved light meromyosin portion of the sarcomeric myosin rod and are 78-81% homologous to a mammalian slow/cardiac beta-MyHC cDNA. Hybridization using PCR generated probes specific for SM1 and SM2 sequences demonstrated that the genes encoding these two slow MyHCs colocalized to an 80-kb BssHII genomic fragment. We further show that a probe specific to a third slow MyHC gene also hybridized with the same 80-kb genomic fragment. We conclude that in the chicken genome there is a slow MyHC locus containing at least three distinct slow MyHC genes.

Expression of fast myosin heavy chain transcripts in developing and dystrophic chicken skeletal muscle.

The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dotblot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation.

Selective stability-indicating high-performance liquid chromatographic assay for recombinant human regular insulin.

This report presents a selective HPLC assay capable of separating recombinant human regular insulin from insulin decomposition and transformation products. The assay utilizes an isocratic delivery of mobile phase, a C18 peptide column, UV detection and is performed at ambient temperature. The standard curve ranges from 0.2 to 2.5 U/ml. The inter-day and intra-day variabilities are less than 7 and 5%, respectively, at the concentrations studied. The accuracy and precision are within 5% over the range of the standard curve.

Psychometric properties of the Social Problem Solving Inventory (SPSI) with normal and emotionally disturbed adolescents.

The factor structure of the Social Problem Solving Inventory (SPSI; D'Zurilla & Nezu, 1990) was evaluated with a sample of 708 normal adolescents. Confirmatory factor analysis supported the empirically derived five-factor model reported by D'Zurilla and Maydeu-Olivares (1994) using an adult sample, but not the theoretically derived seven-factor structure of the original SPSI. The psychometric properties of the original and revised inventories are reported for normal adolescents and psychiatrically hospitalized adolescents (n = 63). Internal consistency and reliability estimates were adequate. Support for the validity of the revised SPSI was demonstrated by examining the relationship between social skills, depression, and social problem solving; in addition, differences between normal and inpatient adolescent samples were examined. The findings are discussed in terms of the utility of the inventories with adolescents.

Mycobacterium kansasii among patients infected with human immunodeficiency virus in Kansas City. Kansas City AIDS Research Consortium.

Previous reports of infection due to Mycobacterium kansasii among patients infected with human immunodeficiency virus (HIV) have conflicted with regard to the significance of the isolate; the clinical, radiographic, and laboratory features of the disease; and the response to therapy. To clarify the spectrum of M. kansasii infection in this population, we conducted a retrospective study of 35 patients. Twenty-eight of these patients were believed to have disease due to M. kansasii, while the remaining seven patients were probably colonized with the organism. All but two patients presented with advanced HIV infection; the median CD4 cell count was 12/microL. Most patients with pulmonary disease presented with fever, cough, and dyspnea, but only eight of these 22 patients had radiographic findings of either pulmonary cavitation or predominantly upper-lobe disease. Ten patients had M. kansasii isolated from blood or bone marrow. The majority of patients with pulmonary or disseminated disease responded to therapy. However, 11 patients died either before mycobacterial infection was diagnosed or early in the course of treatment, and two had a relapse of infection during therapy.