Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.     

Suppression of Herpes Simplex Virus Infection by Phosphonoacetic Acid

Disodium phosphonoacetate when administered orally or topically to mice experimentally infected with herpes simplex virus was able to significantly reduce the mortality associated with the agent. In addition, this compound was able to reduce herpesvirus lesions on the corneas of infected rabbits.     

Spatial ecology of female bighorn sheep in a prairie landscape in Nebraska

Understanding the spatial ecology of small populations at the periphery of their range is important for identifying factors limiting population growth and developing sound management strategies. Bighorn sheep were reintroduced to Nebraska in 1981 and persist in a small population at the easternmost extent of the distribution of the species, where 1 of the 2 subpopulations is declining. Bighorn sheep space use and movement has been studied extensively in mountain and desert populations, but information is sparse from prairie populations in the Northern Great Plains. We investigated the spatial ecology of female bighorn sheep in Nebraska, USA, with global positioning system (GPS) telemetry. We tested the hypothesis that space use and movements would vary across seasons, years, and individuals but predicted that migration would involve relatively short distances in this translocated population (relative to native populations) occupying a fragmented landscape. Overall, females used smaller seasonal home ranges (3.3–7.8 km²) than most of those reported previously for the species and exhibited a high degree of variability in space use and movements across seasons, subpopulations, and individuals. Most females (92–100%) exhibited fidelity to their home ranges across seasons and years. Six females migrated between spatially distinct core lambing and winter ranges, although the distances (range = 7.9–8.7 km) and mean elevations (range = 31–41 m) between these core seasonal ranges were less than those reported for most native, migratory bighorn sheep populations. After accounting for variation in season, subpopulation, and years, home range size was positively associated with road density in both subpopulations (P < 0.001), suggesting that females incur greater energetic costs associated with greater space use in areas of higher fragmentation. Our results establish the basic spatial ecology of female bighorn sheep in Nebraska where their behavior appears to reflect the isolated nature of suitable habitat in this fragmented prairie landscape.     

Which population level environmental factors are associated with asthma,rhinoconjunctivitis and eczema? Review of the ecological analyses of ISAAC Phase One

The International Study of Asthma and Allergies in Childhood (ISAAC) Phase One showed large worldwide variations in the prevalence of symptoms of asthma, rhinoconjunctivitis and eczema, up to 10 to 20 fold between countries. Ecological analyses were undertaken with ISAAC Phase One data to explore factors that may have contributed to these variations, and are summarised and reviewed here.In ISAAC Phase One the prevalence of symptoms in the past 12 months of asthma, rhinoconjunctivitis and eczema were estimated from studies in 463,801 children aged 13 - 14 years in 155 centres in 56 countries, and in 257,800 children aged 6-7 years in 91 centres in 38 countries. Ecological analyses were undertaken between symptom prevalence and the following: Gross National Product per capita (GNP), food intake, immunisation rates, tuberculosis notifications, climatic factors, tobacco consumption, pollen, antibiotic sales, paracetamol sales, and outdoor air pollution.Symptom prevalence of all three conditions was positively associated with GNP, trans fatty acids, paracetamol, and women smoking, and inversely associated with food of plant origin, pollen, immunisations, tuberculosis notifications, air pollution, and men smoking. The magnitude of these associations was small, but consistent in direction between conditions. There were mixed associations of climate and antibiotic sales with symptom prevalence.The potential causality of these associations warrant further investigation. Factors which prevent the development of these conditions, or where there is an absence of a positive correlation at a population level may be as important from the policy viewpoint as a focus on the positive risk factors. Interventions based on small associations may have the potential for a large public health benefit.     

A composite enhancer element directing tissue-specific expression of mouse mammary tumor virus requires both ubiquitous and tissue-restricted factors

Mouse mammary tumor virus (MMTV) expression is restricted primarily to mammary epithelial cells. Sequences responsible for both the mammary-specific expression of MMTV and the activation of cellular oncogenes are located within two enhancer elements at the 5'-end of the long terminal repeat. Whereas the Ban2 enhancer (-1075 to -978) has been well characterized, the mammary-specific enhancer of MMTV from -956 to -862 has only recently been recognized as a key determinant of mammary-specific oncogene activation by MMTV. The present study identifies and characterizes three binding sites located within this element. Transient transfection of deletion and mutation constructs shows that all three sites contribute to the basal expression of MMTV in mammary cells. One of the binding activities (footprint I) is restricted to mammary cells, whereas the other two sites bind factors found in both mammary and nonmammary cells. The multimerized mammary-specific enhancer of MMTV on its own can enhance a minimal promoter in a mammary-specific fashion. However, the FpI binding site alone cannot mediate this effect. Thus, it is the binding of multiple factors in a combinatorial fashion that mediates the mammary-restricted expression of MMTV.     

Scaling of gill metabolic potential as a function of salinity in the euryhaline crab,Callinectes sapidus rathbun

The body-size scaling pattern of enzymes that are important in energy metabolism was examined in gills of the blue crab as a function of acclimation salinity. We hypothesized that the higher surface-area-to-volume ratio of small crabs would impose a greater metabolic cost for hyperosmoregulation, leading to an increase in the capacity for ATP production in gills. Postmetamorphic crabs spanning a 2,500-fold range in body mass were examined following a 7-d exposure to a salinity of 35, 17, or 5 ppt. The posterior gills, which are the principal site of osmoregulatory ion pumping, generally had higher activities than the anterior gills, which are primarily used for gas exchange, and this discrepancy was greatest in small crabs. A significant effect of salinity was found only for the enzyme citrate synthase, where the activity was highest at the lowest salinity. Although most enzymes scaled negatively with body mass, the activity was independent of size over a 250-fold size range that encompassed the body masses of juvenile crabs but decreased abruptly in the adult crabs. These data suggest that ion pumping associated with osmoregulation may represent a greater energetic challenge in smaller crabs, and this is reflected in the relatively higher metabolic potential of the posterior gills. However, acclimation to different salinity regimes does not lead to dramatic global changes in the capacity for energy metabolism.     

Myocardial gene reprogramming associated with a cardiac cross-resistant state induced by LPS preconditioning

Lipopolysaccharide (LPS) preconditioning induces cardiacresistance to subsequent LPS or ischemia. This study tested thehypothesis that resistance to LPS and resistance to ischemiaare two manifestations of cardiac cross-resistance which may involvereprogramming of cardiac gene expression. Rats were preconditioned witha single dose of LPS (0.5 mg/kg ip). Cardiac resistance to LPS wasexamined with a subsequent LPS challenge. Cardiac resistance toischemia was determined by subjecting hearts toischemia-reperfusion. Total RNA was extracted from myocardiumfor Northern analysis of mRNAs encoding protooncoproteins, antioxidantenzymes, and contractile protein isoforms. Rats preconditioned with LPS1-7 days earlier acquired cardiac resistance to endotoxemicdepression. This resistance temporally correlated with resistance toischemia. Pretreatment with cycloheximide (0.5 mg/kg ip)abolished resistance to both LPS and ischemia. LPSpreconditioning induced the expression of c-jun andc-fos mRNAs. LPS also transientlyincreased mRNAs encoding catalase and Mn-containing superoxidedismutase. The expression of both - and -myosin heavy chain mRNAswas upregulated, whereas the expression of cardiac -actin mRNA wassuppressed. We conclude that 1)LPS induces sustained cardiac resistance to both LPS and ischemia, 2) resistance toischemia and resistance to LPS seem to be two mechanisticallyindistinct components of cardiac cross-resistance, and3) the cardiac cross-resistance isassociated with reprogramming of myocardial gene expression.