Fast Cytoplasmic pH Regulation in Acid-Stressed Leaves

Induction of photosynthesis in leaves was prolonged, and steadystate photosynthesis was inhibited by very high CO₂ concentrationswhich cause cytoplasmic acidification. Prolonged exposure tohigh CO₂ relieved initially observed inhibition of photosynthesisat least partially. The sensitivity of carbon assimilation tohigh CO₂ was different in different plant species. Acidificationby CO₂ (or subsequent alkalization) was detected by measuringrapid CO₂-release from the tissue and by monitoring fluorescenceof pH-indicating dyes which had been fed to the leaves throughthe petiole. The results indicate that two different mechanismsoperate in leaves to achieve and maintain pH homeostasis. Rapidand efficient pH-adjustment is provided by proton/cation exchangeacross the tonoplast. Slower and less efficient regulation occursby formation or consumption of base. In the presence of highCO₂ concentrations, protons are pumped from the cytosol intoalready acidic vacuoles. In turn, vacuolar cations replace exportedprotons in the cytosol permitting bicarbonate accumulation andincreasing the pH of the acidified cytosol. Similarly effectiveand fast proton/cation exchange relieves acid-stress in thechloroplast stroma and permits photosynthesis to proceed withhigh quantum efficiency or high light-saturated rates in thepresence of CO₂ concentrations which would, in the absence offast cytoplasmic pH regulation, inhibit photosynthesis. By inference,proton/cation exchange must also occur across the mitochondrialboundary. After cytoplasmic pH adjustment in the presence ofhigh CO₂, removal of CO₂ results in transient cytoplasmic alkalizationand, subsequently, in the return of cytoplasmic pH values tolevels observed prior to acid-stress. In addition to fast pHregulation by rapid proton/cation exchange across biomembranes,slow base production (e.g. NH₃-formation) also contributes torelieving acid stress. Base produced in the presence of highCO₂ is rapidly consumed after removal of CO₂. Implications of the findings in regard to forest damage by potentiallyacidic air pollutants such as SO₂ are briefly discussed. (Received November 8, 1993; Accepted February 3, 1994)     

Inferential biases linked to unobservable states in complex occupancy models

Modeling of species distributions has undergone a shift from relying on equilibrium assumptions to recognizing transient system dynamics explicitly. This shift has necessitated more complex modeling techniques, but the performance of these dynamic models has not yet been assessed for systems where unobservable states exist. Our work is motivated by the impacts of the emerging infectious disease chytridiomycosis, a disease of amphibians that is associated with declines of many species worldwide. Using this host‐pathogen system as a general example, we first illustrate how misleading inferences can result from failing to incorporate pathogen dynamics into the modeling process, especially when the pathogen is difficult or impossible to survey in the absence of a host species. We found that traditional modeling techniques can underestimate the effect of a pathogen on host species occurrence and dynamics when the pathogen can only be detected in the host, and pathogen information is treated as a covariate. We propose a dynamic multistate modeling approach that is flexible enough to account for the detection structures that may be present in complex multistate systems, especially when the sampling design is limited by a species’ natural history or sampling technology. When multistate occupancy models are used and an unobservable state is present, parameter estimation can be influenced by model complexity, data sparseness, and the underlying dynamics of the system. We show that, even with large sample sizes, many models incorporating seasonal variation in vital rates may not generate reasonable estimates, indicating parameter redundancy. We found that certain types of missing data can greatly hinder inference, and we make study design recommendations to avoid these issues. Additionally, we advocate the use of time‐varying covariates to explain temporal trends in the data, and the development of sampling techniques that match the biology of the system to eliminate unobservable states when possible.     

High-Resolution Functional Profiling of the Norovirus Genome

Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex.     

Effect of metformin on insulin receptor binding and glycaemic control in type II diabetes.

To investigate the effect of metformin on insulin receptor binding and diabetic control, eight obese type II diabetic patients were studied before treatment, after one and four weeks of taking metformin (500 mg thrice daily), and four weeks after withdrawal of the drug. After one and four weeks of treatment the number of erythrocyte insulin receptors had increased by 116% and 184% respectively. This was due almost entirely to an increase in the number of low affinity binding sites. The number of receptors was still raised four weeks after metformin had been withdrawn. Diabetic control as assessed by urinary glucose, glycosylated haemoglobin (HbA1), and glucose tolerance values was significantly improved during metformin treatment, while plasma insulin concentrations were not altered. These results indicate that metformin produces a rapid and protracted increase in low affinity insulin receptors in type II diabetes, associated with greater insulin sensitivity and improved diabetic control.     

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-Arylproline analogs

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays. The impact of this systematic SAR on different drug properties is reported.     

Salmonella in Broiler Litter and Properties of Soil at Farm Location

Contamination of litter in a broiler grow-out house with Salmonella prior to placement of a new flock has been shown to be a precursor of the flock''s Salmonella contamination further down the production continuum. In the southern USA, broiler grow-out houses are primarily built on dirt pad foundations that are placed directly on top of the native soil surface. Broiler litter is placed directly on the dirt pad. Multiple grow-out flocks are reared on a single litter batch, and the litter is kept in the houses during downtime between flocks. The effects of environmental determinants on conditions in broiler litter, hence Salmonella ecology within it, has received limited attention. In a field study that included broiler farms in the states of Alabama, Mississippi and Texas we assessed Salmonella in broiler litter at the end of downtime between flocks, i.e. at the time of placement of a new flock for rearing. Here we utilized these results and the U.S. General Soil Map (STATSGO) data to test if properties of soil at farm location impacted the probability of Salmonella detection in the litter. The significance of soil properties as risk factors was tested in multilevel regression models after accounting for possible confounding differences among the farms, the participating broiler complexes and companies, and the farms'' geographical positioning. Significant associations were observed between infiltration and drainage capabilities of soil at farm location and probability of Salmonella detection in the litter.     

The influence of method-related partner violence on covert pill use and pill discontinuation among women living in La Paz, El Alto and Santa Cruz, Bolivia

Intimate partner violence is widespread worldwide. While assumed to impact women's ability to use contraceptive methods, few data are available to support this claim. In this study, eight focus group discussions were conducted to guide questionnaire development and to provide contextual information. Participants were women who were currently using the pill and women who had used the pill previously. In addition, 300 women were interviewed who initiated oral contraceptive pill use between December 1995 and April 1996. Participants were interviewed 3-6 months later to investigate the role intimate partner violence played in covert pill use and pill discontinuation. Special study procedures for asking women questions about violence were employed. Nineteen per cent of the women interviewed were using the pill covertly. The odds of covert pill use were four times higher in El Alto and La Paz than in Santa Cruz. Women who used the pill covertly were more likely to have experienced method-related partner violence (OR = 21.27) than women whose partners knew of their pill use. One-third of the women had discontinued pill use at the time of the interview. In the final multivariate analysis, having experienced side-effects (OR = 2.37) was a significant predictor of pill discontinuation and method-related partner violence was marginally predictive (OR = 1.91; 95% CI 1.0-3.66). While efforts are ongoing to incorporate men into family planning programmes, some male partners oppose, and in some situations violently oppose, contraceptive use. The needs of women with these types of partners must not be overlooked.     

Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.