Antigenic relationships among five reovirus-like (RVL) agents by complement fixation (CF) and development of new substitute CF antigens for the human RVL agent of infantile gastroenteritis.

The human reovirus-like (HRVL) agent, Nebraska calf diarrhea virus (NCDV), epizootic diarrhea of infant mice (EDIM) virus, simian agent (SA)-11, and the "O" (offal) agent were found to be similar, if not identical, in reciprocal complement fixation (CF) tests employing hyperimmune animal sera. In addition, in CF tests with paired sera from 35 diarrhea patients who shed the HRVL agent, 74% developed serologic evidence of infection with the HRVL antigen, 43% with NCDV, 51% with EDIM virus, 57% with SA-11, and 71% with the "O" agent. Thus, in addition to the NCDV, which had previously been described as a suitable substitute CF antigen for the HRVL agent, the SA-11, "O", and EDIM viruses may also be utilized as substitute antigens for the HRVL agent. However, the "O" agent appears to be the most efficient of the four substitute CF antigens and thus should be used preferentially when the HRVL agent is not available. The "O" agent was about as efficient as the HRVL agent and significantly more efficient than the NCDV for detecting seroresponses. The greatest efficiency for detecting infection with the HRVL agent resulted when sera were tested with both the HRVL and "O" agents as 31 (89%) of the patients developed serologic evidence of infection with one or both antigens. The finding of additional substitute CF antigens for the HRVL agent may have implications in the immunoprophylaxis against human disease.     

Effect of membrane potential on band 3 conformation in the human erythrocyte membrane detected by triplet state quenching experiments.

The triplet lifetime and absorption anisotropy decay of eosin-labeled band 3 was measured in resealed erythrocyte ghosts. Membrane potentials were generated by the addition of valinomycin in the presence of a K+ gradient. Neither negative nor positive membrane potentials had any detectable effect on the rotational diffusion of band 3 nor on the eosin triplet lifetime. The membrane potential did, however, affect quenching of the eosin triplet state by I- and TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Quenching was enhanced by a negative membrane potential (negative inside) and reduced by a positive membrane potential. In addition, it was found that a negative membrane potential enhanced the efficiency of eosin labeling of band 3 in intact erythrocytes. A positive membrane potential had the opposite effect. These results indicate that the eosin binding site on band 3 becomes more accessible to the extracellular aqueous phase in the presence of a negative membrane potential and less accessible in the presence of a positive membrane potential. Quenching by I- and TEMPO of the triplet state of eosin-labeled band 3 was further investigated as a function of pH. Quenching by TEMPO and its dependence on membrane potential were relatively insensitive to pH. In contrast, the rate of quenching by I- showed a marked decrease over the range pH 5.5-9.5. Moreover, the effect of a negative membrane potential on I- quenching also varied with pH. These results are discussed on the supposition that the eosin probe is located in the anion access channel of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between temperature and nutrients across levels of ecological organization

Temperature and nutrient availability play key roles in controlling the pathways and rates at which energy and materials move through ecosystems. These factors have also changed dramatically on Earth over the past century as human activities have intensified. Although significant effort has been devoted to understanding the role of temperature and nutrients in isolation, less is known about how these two factors interact to influence ecological processes. Recent advances in ecological stoichiometry and metabolic ecology provide a useful framework for making progress in this area, but conceptual synthesis and review are needed to help catalyze additional research. Here, we examine known and potential interactions between temperature and nutrients from a variety of physiological, community, and ecosystem perspectives. We first review patterns at the level of the individual, focusing on four traits – growth, respiration, body size, and elemental content – that should theoretically govern how temperature and nutrients interact to influence higher levels of biological organization. We next explore the interactive effects of temperature and nutrients on populations, communities, and food webs by synthesizing information related to community size spectra, biomass distributions, and elemental composition. We use metabolic theory to make predictions about how population‐level secondary production should respond to interactions between temperature and resource supply, setting up qualitative predictions about the flows of energy and materials through metazoan food webs. Last, we examine how temperature–nutrient interactions influence processes at the whole‐ecosystem level, focusing on apparent vs. intrinsic activation energies of ecosystem processes, how to represent temperature–nutrient interactions in ecosystem models, and patterns with respect to nutrient uptake and organic matter decomposition. We conclude that a better understanding of interactions between temperature and nutrients will be critical for developing realistic predictions about ecological responses to multiple, simultaneous drivers of global change, including climate warming and elevated nutrient supply.     

Small ubiquitin-like modifier modification of arrestin-3 regulates receptor trafficking

Nonvisual arrestins are regulated by direct post-translational modifications, such as phosphorylation, ubiquitination, and nitrosylation. However, whether arrestins are regulated by other post-translational modifications remains unknown. Here we show that nonvisual arrestins are modified by small ubiquitin-like modifier 1 (SUMO-1) upon activation of β(2)-adrenergic receptor (β(2)AR). Lysine residues 295 and 400 in arrestin-3 fall within canonical SUMO consensus sites, and mutagenic analysis reveals that Lys-400 represents the main SUMOylation site. Depletion of the SUMO E2 modifying enzyme Ubc9 blocks arrestin-3 SUMOylation and attenuates β(2)AR internalization, suggesting that arrestin SUMOylation mediates G protein-coupled receptor endocytosis. Consistent with this, expression of a SUMO-deficient arrestin mutant failed to promote β(2)AR internalization as compared with wild-type arrestin-3. Our data reveal an unprecedented role for SUMOylation in mediating GPCR endocytosis and provide novel mechanistic insight into arrestin function and regulation.     

Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils.

The effects of cGMP-dependent protein kinase (G-kinase), a major cellular receptor of cGMP, were investigated in activated human neutrophils. Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton and nucleus after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), and transiently co-localized with the intermediate filament protein, vimentin. During this time period, the most remarkable co-localization of G-kinase and vimentin was observed between 1-2.5 min stimulation with fMLP. At that time co-localization of G-kinase and vimentin was predominantly confined to filaments which extended from regions adjacent to the nucleus into the uropod. Distinctive localization for only G-kinase was observed at the microtubule organizing center and euchromatin of the nucleus. The filamentous staining pattern for G-kinase and vimentin was enhanced in the presence of 8-Br-cGMP. Coincident with co-localization of G-kinase and vimentin in adherent neutrophils was a transient increase in cGMP levels and an increase in the phosphorylation of vimentin in fMLP-stimulated cells. The increase in cGMP levels was dependent upon cell adherence, was enhanced by preincubating neutrophils with L-arginine (the precursor for nitric oxide synthesis), and attenuated with the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. Phosphorylation of vimentin in the fMLP-stimulated neutrophil was observed in the presence or absence of exogenous cGMP, although in the presence of low concentrations of 8-Br-cGMP a more rapid phosphorylation of vimentin was observed that correlated with the enhanced co-localization of G-kinase and vimentin. Phosphorylation of vimentin was not observed in non-activated cells treated with 8-Br-cGMP, suggesting that phosphorylation only occurs when G-kinase is co-localized with vimentin. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation during fMLP stimulation, while 8-Br-cGMP enhanced phosphorylation in fMLP-treated cells. This suggests that neither protein kinase C nor cAMP-dependent protein kinase catalyze the phosphorylation of vimentin in neutrophils activated by fMLP. These results indicate that vimentin and G-kinase are co-localized in neutrophils and that vimentin is phosphorylated by G-kinase in response to the co-localization of the two proteins. A model for the targeting of G-kinase and vimentin is presented which hypothesizes that the transient redistribution of G-kinase may regulate neutrophil activation.     

Malignant fibrous histiocytoma of the glans penis: a case report

BACKGROUND: Malignant fibrous histiocytoma has been regarded as the most common sarcoma of older adults. However, recent opinion regards pleomorphic malignant fibrous histiocytoma as an undifferentiated high grade pleomorphic sarcoma not otherwise classifiable utilizing current techniques available in surgical pathology. Notwithstanding controversy regarding its nomenclature, malignant fibrous histiocytoma involving the penis is exceedingly rare, with only 4 cases previously described, to our knowledge. CASE: An uncircumcised 73-year-old male presented with a painless, granular, partially necrotic lesion beneath the penile foreskin. There was no history of sexually transmitted disease, constitutional symptoms or dysuria. Examination of penile shaft, testicles, spermatic cord and inguinal lymph nodes were unremarkable. Biopsy revealed a markedly pleomorphic sarcoma. Subsequent, partial penectomy revealed the same lesion with an adjacent area of squamous cell carcinoma in situ. CONCLUSION: Malignant fibrous histiocytoma remains a diagnosis of exclusion. The investigation requires extensive tumor sampling in search of areas of differentiation and a complete battery of immunohistochemical markers. Therapeutically important entities in the differential diagnosis that must be ruled out include other poorly differentiated sarcomas, sarcomatoid squamous cell carcinoma and desmoplastic melanoma.     

Primary ciliary dyskinesia in mice lacking the novel ciliary protein Pcdp1

Primary ciliary dyskinesia (PCD) results from ciliary dysfunction and is commonly characterized by sinusitis, male infertility, hydrocephalus, and situs inversus. Mice homozygous for the nm1054 mutation develop phenotypes associated with PCD. On certain genetic backgrounds, homozygous mutants die perinatally from severe hydrocephalus, while mice on other backgrounds have an accumulation of mucus in the sinus cavity and male infertility. Mutant sperm lack mature flagella, while respiratory epithelial cilia are present but beat at a slower frequency than wild-type cilia. Transgenic rescue demonstrates that the PCD in nm1054 mutants results from the loss of a single gene encoding the novel primary ciliary dyskinesia protein 1 (Pcdp1). The Pcdp1 gene is expressed in spermatogenic cells and motile ciliated epithelial cells. Immunohistochemistry shows that Pcdp1 protein localizes to sperm flagella and the cilia of respiratory epithelial cells and brain ependymal cells in both mice and humans. This study demonstrates that Pcdp1 plays an important role in ciliary and flagellar biogenesis and motility, making the nm1054 mutant a useful model for studying the molecular genetics and pathogenesis of PCD.     

REPRODUCTIVE BIOLOGY OF ASCLEPIAS EXALTATA

An analysis of the relationships between plant size and survivorship and reproductive success was carried out by sampling four populations of the herbaceous perennial milkweed Asclepias exaltata in Virginia from 1980 to 1982. The annual survivorship rate (about 65%) is the lowest measured for any species of Asclepias. Survivorship was strongly size-dependent but showed no clear relationship with previous history of fruit production. Non-flowering plants were significantly smaller than flowering plants and showed very strong (r > 0.87) correlations between root dry weight and stem or leaf dry weight. Flowering plants were similar to nonflowering plants in root: shoot ratio (approximately 1:1) but differed in that root dry weight was not strongly correlated with stem or leaf dry weight. Components of inflorescence size were strongly correlated within a given level of comparison (e.g., stems per plant with flowers per plant) but less strongly correlated between levels (e.g., stems per plant with flowers per stem). Number of fruits per plant and percentage fruit-set were positively correlated with every component of inflorescence size. Although overall fruit-set was low (about 2%), fruits that were initiated had a high probability of surviving to maturity. There was no evidence of an early period of high fruit abortion: a relatively constant proportion of fruits aborted between each age class.     

Development and Application of a New Method for Specific and Sensitive Enumeration of Spores of Nonproteolytic Clostridium botulinum Types B,E, and F in Foods and Food Materials

The highly potent botulinum neurotoxins are responsible for botulism, a severe neuroparalytic disease. Strains of nonproteolytic Clostridium botulinum form neurotoxins of types B, E, and F and are the main hazard associated with minimally heated refrigerated foods. Recent developments in quantitative microbiological risk assessment (QMRA) and food safety objectives (FSO) have made food safety more quantitative and include, as inputs, probability distributions for the contamination of food materials and foods. A new method that combines a selective enrichment culture with multiplex PCR has been developed and validated to enumerate specifically the spores of nonproteolytic C. botulinum. Key features of this new method include the following: (i) it is specific for nonproteolytic C. botulinum (and does not detect proteolytic C. botulinum), (ii) the detection limit has been determined for each food tested (using carefully structured control samples), and (iii) a low detection limit has been achieved by the use of selective enrichment and large test samples. The method has been used to enumerate spores of nonproteolytic C. botulinum in 637 samples of 19 food materials included in pasta-based minimally heated refrigerated foods and in 7 complete foods. A total of 32 samples (5 egg pastas and 27 scallops) contained spores of nonproteolytic C. botulinum type B or F. The majority of samples contained <100 spores/kg, but one sample of scallops contained 444 spores/kg. Nonproteolytic C. botulinum type E was not detected. Importantly, for QMRA and FSO, the construction of probability distributions will enable the frequency of packs containing particular levels of contamination to be determined.Food-borne botulism is a severe and deadly intoxication caused by the consumption of food containing as little as 30 to 100 ng of preformed botulinum neurotoxin (45). More than 2,500 cases of botulism were reported in Europe in 1999 and 2000, with the majority of cases in the east of the continent (44). Currently, 25 to 50 food-borne botulism cases are diagnosed annually in the United States (27). There are seven distinct botulinum neurotoxins (types A to G) and a number of subtypes (6, 26, 45). In view of the potency of the botulinum neurotoxin and the severity of botulism, four phylogenetically distinct bacteria are grouped together as the Clostridium botulinum species, solely on the basis of their ability to form botulinum neurotoxin. The divergence between these four distinct bacteria is strong enough to merit their classification as distinct species and in some cases is significantly greater than that between bacteria belonging to different genera, e.g., Bacillus subtilis and Staphylococcus aureus (7). Two of these bacteria (proteolytic C. botulinum and nonproteolytic C. botulinum) are responsible for the majority of cases of food-borne botulism. Strains of proteolytic C. botulinum produce neurotoxins of type A, B, or F, form spores of high heat resistance, and have a minimum growth temperature of approximately 12°C (39). Strains of nonproteolytic C. botulinum produce neurotoxins of type B, E, or F, form spores of moderate heat resistance, and are able to grow and form toxin at 3°C (18, 48) and are recognized as the major hazard associated with minimally heated refrigerated foods (4, 37, 43, 44, 48). These new foods meet consumer demand for high-quality, convenient foods that are low in preservatives, and sales are presently increasing by about 10% per annum in many countries (3, 47).Quantitative microbiological risk assessment (QMRA) is now established as an important microbiology food safety tool (42). Process risk models have been used to assess the safety of specific foods with respect to nonproteolytic C. botulinum and the food-borne botulism hazard (e.g., 2, 41). These process risk models benefit from high-quality information, including that on the incidence of spores of nonproteolytic C. botulinum spores in food materials. The implementation of food safety objectives (FSO) also benefits from the availability of high-quality information on the microbial contamination of foods and food materials (24). This information is most effective in the form of probability distributions rather than as average spore concentrations or other statistics.The difficulty with enumerating nonproteolytic C. botulinum in foods is that there is no effective selective culture medium available. Surveys of the extent of contamination of foods and food materials have used a nonselective enrichment followed by either testing for neurotoxin using a mouse test or enzyme-linked immunosorbent assay (ELISA) or testing for the presence of neurotoxin genes using a PCR test (3, 10, 13, 35, 38, 39). This approach, however, is not optimized for nonproteolytic C. botulinum or proteolytic C. botulinum (therefore potentially failing to recover all spores of either organism) and may also not distinguish nonproteolytic C. botulinum from proteolytic C. botulinum. Heating at 80°C for 10 min followed by incubation at 35°C (54) may be reasonably selective for proteolytic C. botulinum, but there is no similar approach for nonproteolytic C. botulinum, although incubation at 28°C (54) may offer an element of selection. It is necessary, therefore, to develop a method to enumerate spores of nonproteolytic C. botulinum in food materials that is robust and optimized, as well as sensitive and specific for this particular pathogen (and does not also detect proteolytic C. botulinum). When enumerating bacteria in foods, it is essential to demonstrate the efficiency of the method by verifying that small concentrations (in the present study, spores of nonproteolytic C. botulinum) can be detected following addition to test samples.This paper describes the development, validation, and application of a new method to enumerate spores of nonproteolytic C. botulinum in foods and in food materials. This method has been designed to generate data for the construction of probability distributions that can be used in QMRA and FSO settings. Most of the effort has been dedicated to the development and evaluation of the enrichment procedure rather than the PCR test, as the PCR test has received much attention from others (e.g., 3, 10, 16, 36, 38). A low-temperature selective-enrichment procedure is described that has been optimized specifically for nonproteolytic C. botulinum over proteolytic C. botulinum and other bacteria. In order to detect low concentrations of spores, large quantities (200 g) of food materials and foods have been tested. Specific detection of neurotoxin genes is achieved by the use of an established multiplex PCR (36), with an internal amplification control now included (25). By the use of a set of control samples inoculated with defined concentrations of spores of nonproteolytic C. botulinum, the detection limit has been estimated for each food material and food tested. The method has been used in an extensive survey of raw materials intended for use in pasta ready meals, as well as the final meals themselves. The implications for risk assessment and risk management of chilled foods are discussed.