External Loops at the C Terminus of Erwinia chrysanthemi Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.     

GERMINATION ECOPHYSIOLOGY OF HYDROPHYLLUM APPENDICULATUM,A MESIC FOREST BIENNIAL

In north central Kentucky, seeds of the mesic forest biennial Hydrophyllum appendiculatum Michx., are innately dormant at maturity in June. Under natural and simulated seasonal temperature changes, dormancy break occurred in two stages. Root dormancy was broken by high summer temperatures, and shoot dormancy was broken by low winter temperatures. Consequently, roots emerged from seeds during autumn, and cotyledons emerged the following spring. A 90-day warm (30/15 C) stratification treatment broke root dormancy, but the roots emerged only after transfer to lower temperatures. After the warm stratification treatment, roots emerged from 93, 73, 6 and 9% of the seeds incubated at 5, 15/6, 20/10 and 30/15 C (12/12 hr), respectively. Zero, 28, 56 and 84 days of cold (5 C) stratification of seeds with emerged roots resulted in 9, 21, 49 and 82% cotyledon emergence, respectively, at 20/10 C. Thus, H. appendiculatum exhibits a type of morpho-physiological dormancy known as epicotyl dormancy. Although many seeds germinate the first year, others remain dormant and germinate in successive years until the fourth season after ripening.     

Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

Evolutionary history of aphid-plant associations and their role in aphid diversification

Aphids are intimately linked with their host plants that constitute their only food resource and habitat, and thus impose considerable selective pressure on their evolution. It is therefore commonly assumed that host plants have greatly influenced the diversification of aphids. Here, we review what is known about the role of host plant association on aphid speciation by examining both macroevolutionary and population-level studies. Phylogenetic studies conducted at different taxonomic levels show that, as in many phytophagous insect groups, the radiation of angiosperms has probably favoured the major Tertiary diversification of aphids. These studies also highlight many aphid lineages constrained to sets of related host plants, suggesting strong evolutionary commitment in aphids’ host plant choice, but they fail to document cospeciation events between aphid and host lineages. Instead, phylogenies of several aphid genera reveal that divergence events are often accompanied by host shifts, and suggest, without constituting a formal demonstration, that aphid speciation could be a consequence of adaptation to new hosts. Experimental and field studies below the species level support reproductive isolation between host races as partly due to divergent selection by their host plants. Selected traits are mainly feeding performances and life cycle adaptations to plant phenology. Combined with behavioural preference for favourable host species, these divergent adaptations can induce pre- and post-zygotic barriers between host-specialized aphid populations. However, the hypothesis of host-driven speciation is seldom tested formally and must be weighed against overlooked explanations involving geographic isolation and non-ecological reproductive barriers in the process of speciation.     

Metabolic effect at six and twelve months of cyproterone acetate (2 mg) combined with ethinyl estradiol (35 micrograms) in 31 patients

The metabolic effects of cyproterone acetate (2 mg) combined with a new dose level of ethinyl estradiol (35 micrograms) were studied over a one-year period in 31 patients presenting moderate clinical hyperandrogenism. The following tests were performed before treatment, at 6 and 12 months, an oral glucose tolerance test (OGTT) with measurement of insulinemia, total cholesterol, HDL cholesterol and the LDL + VLDL/HDL ratio, A1 and B apoproteins. At six months and at one year of treatment, the weight and body mass index (kg/m2) were not modified. Glucose tolerance and insulinemia had not changed significantly at one year. Lipid test results showed an increase in triglycerides, as well as in total and HDL cholesterol levels. However, the LDL + VLDL/HDL and A1/B apoprotein ratios did not change during the study. We conclude from these results that the new combination does not have any adverse effects on glucose tolerance and has a predominantly estrogenic effect on lipid parameters, characterized by increases in total cholesterol, HDL cholesterol and triglycerides.     

The public health implications of maternal care trade-offs

The socioeconomic and ethnic characteristics of parents are some of the most important correlates of adverse health outcomes in childhood. However, the relationships between ethnic, economic, and behavioral factors and the health outcomes responsible for this pervasive finding have not been specified in child health epidemiology. The general objective of this paper is to propose a theoretical approach to the study of maternal behaviors and child health in diverse ethnic and socioeconomic environments. The specific aims are: (a) to describe a causal pathway between the utility that women obtain through work outside the home and through child care and disease hazard rates in childhood using an optimization model; (b) to specify the influence of ethnic and socioeconomic factors on model constraints; (c) to use the model as a tool to learn about how different combinations of maternal wage labor and child care time might influence child health outcomes in diverse social contexts; (d) to identify parameters that will require measurement in future research; (e) to discuss research strategies that will enable us to obtain these measurements; and (f) to discuss the implications of the model for biostatistical modeling and public health intervention. Optimization models are powerful heuristic tools for understanding how ethnic, environmental, family, and personal characteristics can place important constraints on both the quality and quantity of care that women can provide to their children. They provide a quantitative appreciation for the difficult trade-offs that most women face between working in order to purchase basic goods that children cannot do without (e.g., food, clothing, shelter, health insurance), and increasing offspring well-being through child care (e.g., training in social skills, affection, protection from environmental hazards, help with homework). The research was funded by a Faculty Scholars Award from the William T. Grant Foundation to A. Magdalena Hurtado. A. Magdalena Hurtado, Ph.D., is an associate professor in the Department of Anthropology, University of New Mexico. Her research interests include the origins of the sexual division of labor, epidemiology of indigenous peoples, disease susceptibility, the development and intergenerational transmission of antigens and immune defense, immune function and allergic sensitization, and trauma. She also works on public health interventions, biological capital and poverty, and land tenure and human rights in native communities of South America. Carol Lambourne, M.Sc., is a doctoral candidate in the Department of Anthropology, University of New Mexico. Her research interests are evolutionary models of child and adolescent development, life history theory, family composition and investment patterns, pubertal timing and psychosexual maturation, juvenile stress, and infanticide. Kim Hill, Ph.D., is a professor in the Department of Anthropology, University of New Mexico. His research interests are modern hunter-gatherers, including extensive fieldwork in lowland South America. Current topics of interest include human evolution, economic strategies, life history theory, the evolution of cooperation, and the emergence of social norms enforced by punishment. He is also involved in economic development, health and education projects with lowland South American native populations. Karen Kessler received her M.S. in Anthropology from the University of New Mexico in 1996. Her research interests are the application of mathematical modeling to the prevention of diabetes and other causes of morbidity and mortality in historical populations.     

An empirical analysis of the factors contributing to 20-year decrease in soil pH in an old-field plantation of loblolly pine

The pH of weak-acid solutions is controlled by acid concentration (HA + A^–), the degree of acid dissociation (A^–/HA), and the strength of the acids present (pK_a). We developed an empirical approach that allows the relative importance of each of these factors to be estimated for soils. This empirical model was applied to soils collected from an old-field plantation of loblolly pine (Pinus taeda L.) at 5 and 25 years of age. During this period, soil pH dropped by 0.3 to 0.8 units, and extractable calcium, magnesium and potassium declined by 20 to 80%. The empirical model indicates that the decline in pH resulted largely from the reduction in base saturation of the exchange complex. However, the average acid strength of the exchange complex decreased during the 20 years, preventing a greater decline of perhaps 0.1 to 0.2 units in the observed pH. The rate of decrease in the acid neutralizing capacity to pH 3.5 was about 1.3 kmol_c/ha annually, while the increase in base neutralizing capacity was about 2.7 and 1.6 kmol_c/ha annually to pH 5.5 and 8.2, respectively. Extractable alkali and alkaline earth cations declined by about 2.2 kmol_c/ha annually, matched by the rate of increase in aluminium. These changes demonstrated the dynamic nature of poorly buffered soils, and indicated that changes in soil acidity may be expected over a period of decades (especially following changes in land-use).