Enterococci as indicators of Lake Michigan recreational water quality: comparison of two methodologies and their impacts on public health regulatory events

The frequency of poor-water-quality advisories issued in Milwaukee and Racine, Wisconsin, in the absence of identifiable sources of contamination brought into question the reliability of the present indicator organism, Escherichia coli. Enteroccoci have been suggested as an alternative to E. coli for freshwater monitoring due to their direct correlation to swimmer-associated gastroenteritis. The purpose of this research was threefold: (i) to explore enterococci as an alternative to E. coli for monitoring freshwater Lake Michigan beaches, (ii) to evaluate the impact of the two indicators on regulatory decisions, and (iii) to compare membrane filtration m-enterococcus agar with indoxyl-beta-D-glucoside to a chemical substrate technique (Enterolert) for the recovery of enterococci. Recreational water samples from Milwaukee (n = 305) and Racine (n = 153) were analyzed for the enumeration of E. coli and enterococci using IDEXX Colilert-18 and Enterolert. Correlation between the indicators was low (R(2) = 0.60 and 0.69). Based on U.S. Environmental Protection Agency bacterial indicator threshold levels of risk for full body immersion, using enterococci would have resulted in 56 additional unsafe-recreational-water-quality advisories compared to the total from using E. coli and the substrate-based methods. A comparison of the two enterococcal methods (n = 124) yielded similar results (R(2) = 0.62). This was further confounded by the frequent inability to verify enterococci from those wells producing fluorescence by the defined substrate test using conventional microbiological methods. These results suggest that further research is necessary regarding the use of defined substrate technology interchangeably with the U.S. Environmental Protection Agency-approved membrane filtration test for the detection of enterococci from fresh surface water.     

Membrane-anchored cytochrome c as an electron carrier in photosynthesis and respiration: past,present and future of an unexpected discovery

In the mid 1980s, it was observed that photosynthesis could still occur in the absence of the diffusible electron carrier cytochrome c ₂ in the purple non-sulfur facultative phototrophic bacterium Rhodobacter capsulatus. This serendipic finding led to the discovery of a novel class of membrane-anchored electron carrier cytochromes and their associated electron transfer pathways. Studies of cytochrome c _y of R. capsulatus (and its homologues in other species) have modified the previous dogma of electron transfer between photosynthetic and respiratory membrane protein complexes with a new paradigm, in which these proteins and their electron carriers can form `hard-wired' structural super-complexes. Here, we reminisce on the early days of this discovery, its impacts on our understanding of cellular energy transduction pathways and the physiological roles played by the electron carrier cytochromes c, and discuss the current knowledge and emerging future challenges of this field. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic engineering of Pichia pastoris to humanize N-glycosylation of proteins

The yeast Pichia pastoris is used extensively as the host cell for large-scale production of secreted recombinant proteins. Many proteins of pharmaceutical importance are N-glycosylated, and therefore require an expression host that yields N-linked oligosaccharides that are structurally and functionally identical to the human counterpart. The recent report by Choi et al. describes the use of combinatorial genetic libraries to alter the N-glycosylation pathway in P. pastoris to yield N-linked oligosaccharides with hybrid structures that are the same as the intermediates of mammalian-protein N-glycosylation. In view of recent progress in this area, the production of complex human glycans in yeasts is anticipated.     

Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.     

Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine biosynthetic machinery with the prohibitin complex of Saccharomyces cerevisiae

The majority of mitochondrial phosphatidylethanolamine (PtdEtn), a phospholipid essential for aerobic growth of yeast cells, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p) in the inner mitochondrial membrane (IMM). To identify components that become essential when the level of mitochondrial PtdEtn is decreased, we screened for mutants that are synthetically lethal with a temperature-sensitive (ts) allele of PSD1. This screen unveiled mutations in PHB1 and PHB2 encoding the two subunits of the prohibitin complex, which is located to the IMM and required for the stability of mitochondrially encoded proteins. Deletion of PHB1 and PHB2 resulted in an increase of mitochondrial PtdEtn at 30 degrees C. On glucose media, phb1Delta psd1Delta and phb2Delta psd1Delta double mutants were rescued only for a limited number of generations by exogenous ethanolamine, indicating that a decrease of the PtdEtn level is detrimental for prohibitin mutants. Similar to phb mutants, deletion of PSD1 destabilizes polypeptides encoded by the mitochondrial genome. In a phb1Delta phb2Delta psd1(ts) strain the destabilizing effect is dramatically enhanced. In addition, the mitochondrial genome is lost in this triple mutant, and nuclear-encoded proteins of the IMM are assembled at a very low rate. At the nonpermissive temperature mitochondria of phb1Delta phb2Delta psd1(ts) were fragmented and aggregated. In conclusion, destabilizing effects triggered by low levels of mitochondrial PtdEtn seem to account for synthetic lethality of psd1Delta with phb mutants.     

Platyamoeba nucleolilateralis n. sp. from the Chesapeake Bay Region

Members of the genus Platyamoeba are among the most common of the free-living brackish and marine amoebae; yet, to date only twelve species have been documented in the literature and only a limited number of habitats have been sampled globally. During the course of a systematic survey of salt-marsh amoebae along the east coast of the United States, a new species of Platyamoeba was discovered in sediment samples obtained from a salt marsh at Assateague Island, VA. The species can be distinguished from all other described species within the genus by the presence of a nucleus with a single parietal nucleolus and a floating form with long tapering pseudopods. Its shape varies from flabellate to spatulate as described for species of Platyamoeba and Vannella. The fine structure of the glycocalyx, however, is characteristic of Platyamoeba.     

Classification and comparison of biomechanical response strategies for accommodating landing impact

The purposes of this study were to (a) present a theoretical model to explain the methods by which individuals accommodate impact force in response to increases in an applied stressor, (b) use the model and a correlation procedure to classify a sample of individuals based on their observed response patterns, and (c) statistically evaluate the classification process. Ten participants performed landings from three heights while video and force platform data were being collected. Magnitudes of impact-force characteristics from ground reaction force and lower extremity joint moments were evaluated relative to changes in landing momentum. Correlation between impact force and landing momentum was used to classify participant responses into either a positive or negative biomechanical strategy, as defined by the model. Positive and negative groups were compared using the Mann-Whitney U test. Results indicated that all responses fit within the categories defined by the model. Some individuals preferred positive strategies while others preferred negative ones depending on the specific variable. Only one participant consistently exhibited the negative strategy for all variables. Positive and negative groups were determined to be statistically different, p < or = 0.05, for 61% of the comparisons, suggesting actual differences between groups. The proposed model appeared robust and accounted for all responses in the current experiment. The model should be evaluated further using landing and other impact activities; it should be refined and used to help researchers understand individual impact-response strategies in order to identify those who may be at risk for impact related injuries.     

Twenty years of therapy for HIV-1 infection

Antiretroviral therapy, where available, has transformed HIV-1 disease into a treatable and somewhat chronic infection. This article summarizes the accomplishments thus far and what lies ahead in our struggle to improve the treatment of, and possibly eliminate, HIV-1 infection.