Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation.

A virus that reproduces in a host without killing cells can easily establish a successful infection. Previously, we showed that dengue-2, a virus that threatens 40% of the world, induces autophagy, enabling dengue to reproduce in cells without triggering cell death. Autophagy further protects the virus-laden cells from further insults. In this study, we evaluate how it does so; we show that dengue upregulates host pathways that increase autophagy, namely endoplasmic reticulum (ER) stress and ataxia telangiectasia mutated (ATM) signaling followed by production of reactive oxygen species (ROS). Inhibition of ER stress or ATM signaling abrogates the dengue-conferred protection against other cell stressors. Direct inhibition of ER stress response in infected cells decreases autophagosome turnover, reduces ROS production and limits reproduction of dengue virus. Blocking ATM activation, which is an early response to infection, decreases transcription of ER stress response proteins, but ATM has limited impact on production of ROS and virus titers. Production of ROS determines only late-onset autophagy in infected cells and is not necessary for dengue-induced protection from stressors. Collectively, these results demonstrate that among the multiple autophagy-inducing pathways during infection, ER stress signaling is more important to viral replication and protection of cells than either ATM or ROS-mediated signaling. To limit virus production and survival of dengue-infected cells, one must address the earliest phase of autophagy, induced by ER stress.

Copper and cadmium: responses in Pseudomonas putida KT2440.

AIMS: To compare responses of a soil bacterium to Cu and Cd. METHODS AND RESULTS: In minimal medium, Cd caused a dose-dependent growth stasis of logarithmic phase cells of Pseudomonas putida, strain KT2440, whereas Cu did not compromise growth up to 10 mg l(-1). Proteomics showed changes in accumulation of both membrane and soluble proteins by 6 h of treatment; increased Krebs cycle enzymes were apparent. Transcript analysis showed Cd- and Cu-induced different genes. Cd-induced genes encoding the transcriptional regulator CzrR2; an outer membrane protein associated with lipopolysaccharide stability, H1; two oxidative stress protective proteins and the P-type ATPase, CadA2, associated with Cd(2+) efflux. The genes most responsive to Cu encoded the regulator CopR1 and the outer membrane resistance protein regulated by CopR1, CopB1; a putative porin, PorD and the Cu-binding protein, PacZ or CopZ, and CopA2. CONCLUSIONS: These findings support that a soil pseudomonad restricts internalization of the metals by using different sets of binding proteins and efflux pumps. Activation of mechanisms to protect against oxidative stress also was evident especially with Cd exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: The differential cellular responses to Cd and Cu suggest that risk assessment for Cd and Cu should be different.

Viral manipulation of cell death.

Elucidation of the mechanisms behind cell death has brought with it an appreciation for viral strategies that target these pathways as a means to promote viral propagation while avoiding or slowing the host immune response. Several redundant anti-viral pathways have evolved in eukaryotic cells that are designed to minimize the damage due to viral infection while quickly clearing the invading pathogen. Cell death is a commonly employed immune defense against viral infection, and many viruses potently induce or suppress cell death during infection. The past decade has seen an incredible increase in our understanding of how cell death assists in host immune response, as well as how viruses have evolved to hijack or disengage these systems. By targeting components of host cell death pathways, viruses have developed the ability to control host survival and death, ensuring efficient propagation while inactivating or avoiding the immune system consequences of infection. This review focuses on the most recent and important advances in our understanding of how a wide range of viruses manipulate the survival and death of their hosts.

Iron and arsenic release from aquifer solids in response to biostimulation.

Biostimulation has been used at various contaminated sites to promote the reductive dechlorination of trichloroethylene (TCE), but the addition of carbon and energy donor also stimulates bacteria that use Fe(III) as the terminal electron acceptor (TEA) in potential competition with dechlorination processes. Microcosm studies were conducted to determine the influence of various carbon donors on the extent of reductive dissolution of aquifer solids containing Fe(III) and arsenic. Glucose, a fermentable and respirable carbon donor, led to the production of 1500 mg Fe(II) kg(-1), or 24% of the total Fe in the aquifer sediment being reduced to Fe(II), whereas the same concentration of carbon as acetate resulted in only 300 mg Fe(II) kg(-1) being produced. The biogenic Fe(II) produced with acetate was exclusively associated with the solid phase whereas with fermentable carbon donors as whey and glucose, 22 and 54% of the Fe(II) was in solution. With fermentation, some of the metabolites appear to be electron shuttling chemicals and chelating agents that facilitate the reductive dissolution of even crystalline Fe(III) oxides. Without the presence of electron shuttling chemicals, only surficial Fe in direct contact with the bacteria was bioavailable, as illustrated when acetate was used. Regardless of carbon donor type and concentration, As concentrations in the water exceeded drinking water standards. The As dissolution appears to have been the result of the direct use of As as an electron acceptor by dissimilatory arsenic reducing bacteria. Our findings indicate that selection of the carbon and energy donor for biostimulation for remediation of chlorinated solvent impacted aquifers may greatly influence the extent of the reductive dissolution of iron minerals in direct competition with dechlorination processes. Biostimulation may also result in a significant release of As to the solution phase, contributing to further contamination of the aquifer.

Evaluation of the immunochromatographic CORIS Giardia-Strip test for rapid diagnosis of Giardia lamblia.

The aim of this study was to evaluate the performance of the CORIS Giardia-Strip test (CORIS Bioconcept, Gembloux, Belgium) as a rapid initial method for the routine diagnosis of giardiasis. Compared to a commercial ELISA-coproantigen test (ProSpect Giardia-ELISA-microplate assay; Remel, Lenexa, KS, USA), the commercial strip test had a sensitivity of 58%, a specificity of 99%, a positive predictive value of 93% and a negative predictive value of 93% (n=158). These results are comparable to those obtained using microscopy of direct wet-mounted stool. Since the CORIS Giardia-Strip test is simpler to perform, it can replace direct wet-mounted stool microscopy for the rapid diagnosis of giardiasis; however, its sensitivity is inferior to that of other immunochromatographic antigen detection tests and fresh stool samples are required for its use. Nevertheless, the results suggest that a positive CORIS Giardia-Strip test outcome does not need confirmation, while samples with negative results should be re-examined using another, more sensitive, test.

Comparison of different PCR protocols for the detection and diagnosis of Plasmodium falciparum.

An assessment of differing PCR protocols for the diagnosis of Plasmodium falciparum infection was performed on samples from an area of holoendemic malaria transmission in western Burkina Faso. The PCR protocols had generally high sensitivities (>92%) and specificities (>69%), but the negative predictive values (NPV) were moderate and differed widely among the PCR protocols tested. These PCR protocols that amplified either the P. falciparum pfcrt gene or the small subunit ribosomal DNA were the most reliable diagnostic tools. However, the moderate NPV imply that more than one PCR protocol should be used for diagnosis in holoendemic areas.

Humic acid toxicity in biologically treated soil contaminated with polycyclic aromatic hydrocarbons and pentachlorophenol.

Contaminated soil from a land treatment unit at the Libby Groundwater Superfund Site in Libby, MT, was amended with 14C pyrene and incubated for 396 days to promote biodegradation and the formation of soil-associated bound residues. Humic and fulvic acids were extracted from the treated soil microcosms and analyzed for the presence of pyrene residues. Biologic activity promoted 14C association with the fulvic acid fraction, but humic acid-associated 14C did not increase with biologic activity. The Aboatox flash toxicity assay was used to assess the toxicity of humic and fulvic acid fractions. The fulvic acid gave no toxic response, but the humic acid showed significant toxicity. The observed toxicity was likely associated with pentachlorophenol, a known contaminant of the soil that was removed by solvent extraction of the humic acid and that correlated well with toxicity reduction.

Two- and three-factor solutions of the WAIS-III.

The third edition of the Wechsler Adult Intelligence Scale manual reports four-factor solutions for the WAIS-III, and subsequent research has validated four-factor solutions for a variety of samples. These four factors consistently correspond to the four Factor Indexes that are yielded by the WAIS-III. However, the WAIS-III still provides Verbal and Performance IQs, in addition to the Indexes, making it desirable to examine two-factor solutions as well. In addition, because the Wechsler literature includes much interpretation of three-factor solutions, these solutions were likewise examined. Principal factor analysis followed by Varimax and Oblimin rotations of two and three factors were performed on data for the total WAIS-III sample ages 16 to 89 years (N=2,450). The two-factor solutions were viewed as a construct validation of Wechsler's two separate IQs, although the Working Memory subtests tended to load higher on the Performance scale than on their intended scale (Verbal); three-factor solutions were interpreted within the context of Horn's expanded fluid-crystallized theory and research on working memory. Both the two- and three-factor Varimax-rotated solutions were related to similar factor analyses conducted previously for the Wechsler Adult Intelligence Scale-Revised and the Wechsler Intelligence Scale for Children-III. Coefficients of congruence between like-named factors consistently exceeded .90, and usually .98, across different Wechsler batteries.

Fate of pyrene in contaminated soil amended with alternate electron acceptors.

Creosote-contaminated soil samples from the Libby Ground Water Contamination Superfund Site in Libby, MT, were amended with the potential alternate electron acceptors (AEA) nitrate (KNO3), manganese oxide (MnO2), and amorphous iron oxyhydroxide (FeOOH) and incubated at low oxygen tensions (0-6% O2). The fate of 14C-pyrene was evaluated with respect to the different soil amendments. The fate of 14C from the radiolabeled pyrene with regard to mineralization and bound residue formation within soil humic fractions was not significantly different from controls for the iron and manganese amended soils. Nitrate amendments appeared to stimulate 14C-pyrene mineralization at a level of 170 mg NO3-N kg(-1), and inhibit mineralization at 340 mg NO3-N kg(-1). The stimulatory effect did not appear to be the result of nitrate serving as an electron acceptor. Although AEA amendments did not significantly affect the rate or extent of 14C-pyrene mineralization, results of oxygen-deprived incubations (purged with N2) indicate that AEA may be utilized by the microbial community in the unsaturated contaminated soil system.

Kinship analysis of Pacific salmon: insights into mating, homing, and timing of reproduction.

Multilocus microsatellite genotypes were used to infer kinship and relatedness in two species of Pacific salmon from three populations in Washington State. Even in the absence of direct genetic data from parents, clustering of individuals according to allele sharing and reconstruction of parental genotypes allowed resolution of full- and half-sib relationships among 135 chinook salmon (Oncorhynchus tshawytscha) sampled as preemergent juveniles from 14 redds in the Dungeness River. Inferred reproductive behaviors included single-pair matings, polyandry in which females mated with two to three males at a single redd, polygyny in which males mated with two females at different redds, use of two redds by a single female, and use of one redd site by two females. Greater average relatedness (rxy) in the upper reach of the Dungeness River implied within-reach homing of returning adults. In steelhead trout (O. mykiss), the frequency of related pairs (dyads) of mature individuals that migrated up Snow Creek less than a week apart was greater than expected for randomly chosen dyads, as was the frequency of steelhead dyads that were spawned on the same day in the Forks Creek hatchery. These results imply a heritable basis for upstream migration date and maturation date in steelhead trout.

Multiple inhibitor analysis of the brequinar and leflunomide binding sites on human dihydroorotate dehydrogenase.

Brequinar and the active metabolite of leflunomide, A77 1726, have been clearly shown to inhibit human dihydroorotate dehydrogenase (DHODH), but conflicting mechanisms for their inhibition have been reported. DHODH catalyses the conversion of dihydroorotate (DHO) to orotate concurrent with the reduction of ubiquinone. This study presents data that indicates brequinar is a competitive inhibitor versus ubiquinone; A77 1726 is noncompetitive versus ubiquinone and both are uncompetitive versus DHO. 2-Phenyl 5-quinolinecarboxylic acid (PQC), the core moiety of brequinar also shows competitive inhibition versus ubiquinone. Multiple inhibition experiments indicate that PQC (and thus brequinar) and A77 1726 have overlapping binding sites. Both PQC and A77 1726 are also mutually exclusive with barbituric acid (a competitive inhibitor versus DHO). In addition, we failed to observe brequinar binding to E.orotate by isothermal titration calorimetry (ITC). These results indicate that the E.DHO.inhibitor and E.orotate.inhibitor ternary complexes do not form. The absence of these complexes is consistent with the two-site ping-pong mechanism reported for DHODH. This kinetic data suggests that recent crystal structures of human DHODH complexed with orotate and A77 1726 or brequinar may not represent the relevant physiological binding sites for these inhibitors [Liu, S., Neidhardt, E. A., Grossman, T. H., Ocain, T., and Clardy J. (2000) Structure 8, 25-33].

Effect of weight reduction, obesity predisposition, and aerobic fitness on skeletal muscle mitochondrial function.

We used (31)P magnetic resonance spectroscopy to measure maximal mitochondrial function in 12 obesity-prone women before and after diet-induced weight reduction and in 12 matched, never-obese, and 7 endurance-trained controls. Mitochondrial function was modeled after maximum-effort plantar flexion from the phosphocreatine recovery time constant (TC(PCr)), the ADP recovery time constant (TC(ADP)), and the rate of change in PCr during the first 14 s of recovery (OxPhos). Weight reduction was not associated with a significant change in mitochondrial function by TC(PCr), TC(ADP), or OxPhos. Mitochondrial function was not different between postobese and never-obese controls by TC(PCr) [35.1 +/- 2.5 (SE) vs. 34.6 +/- 2.5 s], TC(ADP) (22.9 +/- 1.8 vs. 21.2 +/- 1.8 s), or OxPhos (0.26 +/- 0. 03 vs. 0.25 +/- 0.03 mM ATP/s), postobese vs. never-obese, respectively. However, TC(ADP) was significantly faster (14.5 +/- 2. 3 s), and OxPhos was significantly higher (0.38 +/- 0.04 mM ATP/s) in the endurance-trained group. These results suggest that maximal mitochondrial function is not impaired in normal-weight obesity-prone women relative to their never-obese counterparts but is increased in endurance-trained women.

Communication forms and functions of children and adults with severe mental retardation in community and institutional settings.

The forms and functions of expressive communication produced by 84 individuals with severe mental retardation were assessed, using a structured communication sampling procedure. Symbolic communication acts were produced by 39 participants, and 27 of these symbolic communicators produced one or more multiword/multisymbol utterances. Of the remaining participants, 38 produced intentional but nonsymbolic communication acts; 7 were not observed to produce any intentional communication. For all participants who produced intentional communication, there were significantly more imperative than declarative communication acts. Significant differences in the frequencies and functions of communication acts produced by these participants were associated with differences in their communication levels (contact gesture, distal gesture, or symbolic), age (child vs. adult), and residential status (community home vs. large facility).

Expression and characterization of E. coli-produced soluble, functional human dihydroorotate dehydrogenase: a potential target for immunosuppression.

Human dihydroorotate dehydrogenase (huDHODH) is essential for de novo biosynthesis of pyrimidines and the target of two immunosuppressive drugs, brequinar and the leflunomide metabolite A77-1726 (Chen et al., 1992; Davis et al., 1996). Using a T7 RNA polymerase expression system, we produced huDHODH as a fusion protein containing an amino-terminal decahistidine tag. Escherichia coli growth and expression conditions were optimized to enhance huDHODH solubility and to permit purification of the enzyme in the absence of detergent. Soluble huDHODH, purified by a simple two-step procedure, was catalytically active, monomeric, and contained a flavin mononucleotide (FMN) cofactor in a 1:1 FMN/protein molar ratio. Kinetic analysis showed that huDHODH uses a two site ping-pong mechanism, where DHO is oxidized at one site and the second substrate, ubiquinone, is reduced at the other. This result is consistent with the mechanism proposed for bovine liver DHODH (Hines and Johnston, 1989).

A front end to a theory of picture recognition.

We describe the results of four picture-recognition memory experiments in which we systematically manipulated four variables: stimulus duration, stimulus contrast, the duration of a blank gap between successive presentations of the same stimulus, and the presence or absence of a noise mask that immediately followed stimulus offset. The patterns of obtained data confirmed a simple extension of a theory previously used to account for digit recall data. This theory consists of a low-pass linear-filter front end that generates a sensory response from the physical stimulus, followed by an information-sampling process whose instantaneous sampling rate is based in part on the sensory response magnitude. The data confirm both qualitative and quantitative theoretical predictions, some of which were previously untestable in digit recall tasks because of ceiling effects that were not present in our picture-recognition tasks. We describe the role of our theory within the broader family of picture-memory theories, and we briefly discuss our theory's unification of two salient facets of visual behavior: information acquisition on the one hand, and phenomenological appearance on the other hand.

Marine population structure in an anadromous fish: life-history influences patterns of mitochondrial DNA variation in the eulachon, Thaleichthys pacificus.

Due to the apparent decline in size of a number of populations, eulachon, Thaleichthys pacificus, have recently become the focus of a conservation movement in the northeast Pacific. Little is known of the marine life-history phase of this anadromous fish, and although it has been suggested that eulachon spawning in different rivers may form distinct populations, nothing is known of their population structure. Molecular genetic data were used to investigate population structure and possible management schemes. Mitochondrial DNA genotypes, determined through restriction fragment length polymorphisms (RFLP) analysis, were resolved in fish from several rivers throughout the geographical range of eulachon. Our data support the idea that extant eulachon populations result from postglacial dispersal from a single Wisconsinan glacial refuge. Further, while three of the 37 haplotypes recovered account for approximately 79% of the samples, many private haplotypes were observed, suggesting possible regional population structure. While a great deal of genetic variation was observed (37 haplotypes in 315 samples), an AMOVA showed that > 97% of the total variation was detected within populations. As yet, it is unclear whether genetically distinct populations of eulachon exist, or if these fish may be treated as one or a few large populations. Results were tested against predictions made from hypotheses concerning the origin and persistence of subdivided populations in marine species, and seem to be more consistent with the Member-Vagrant hypothesis than isolation by distance. Eulachon present an interesting situation that illustrates the difficulties involved in defining management units in organisms with high levels of gene flow.

An investigation into the relationship between interests and intelligence.

This article relates the six General Occupational Themes (GOTs) and the 23 Basic Interest Scales (BISs) yielded by the Strong Interest Inventory (SII) to age, gender, and performance on the KAIT. The sample included 936 males and females aged 16 to 65 years. MANOVAs and MANCOVAs (covarying education) were conducted, followed by univariate ANOVAs and ANCOVAs. IQ level on KAIT was significantly related to the Investigative and Realistic themes and to numerous interest scales, most notably Writing, Nature, Teaching, Mathematics, and Art. The discrepancy between fluid and crystallized intelligence on the KAIT related significantly to a few variables, but relationships were generally small in magnitude. Significant relationships with age were few; gender related significantly to most variables, consistent with previous research. These findings were interpreted in the context of previous research on the Strong, and on the integration of interests and intellect.

Black-white differences on the Strong Interest Inventory General Occupational Themes and Basic Interest Scales at ages 16 to 65.

Black-white differences on the Strong Interest Inventory were examined for a heterogeneous sample of 756 Whites and 85 Blacks aged 16 to 65 years. Multivariate and univariate analyses of variance and covariance were conducted. Race, gender, and IQ were independent variables; the six Holland General Occupational Themes and the 23 Basic Interest Scales were dependent variables; and educational attainment was the covariate. All interactions were nonsignificant, but race was consistently a significant main effect. In general, Whites scored higher than Blacks on Realistic and Investigative themes and scales, and Blacks scored higher in the Social, Enterprising, and Conventional areas. These findings were viewed in the context of counselors' and psychologists' roles in interpreting interest pattern of Black individuals.

De novo heme proteins from designed combinatorial libraries.

We previously reported the design of a library of de novo amino acid sequences targeted to fold into four-helix bundles. The design of these sequences was based on a "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains is varied extensively (Kamtekar S, Schiffer JM, Xiong H, Babik JM, Hecht MH, 1993, Science 262:1680-1685). Because of this variability, the resulting collection of amino acid sequences may include de novo proteins capable of binding biologically important cofactors. To probe for such binding, the de novo sequences were screened for their ability to bind the heme cofactor. Among an initial collection of 30 binary code sequences, 15 are shown to bind heme and form bright red complexes. Characterization of several of these de novo heme proteins demonstrated that their absorption spectra and resonance Raman spectra resemble those of natural cytochromes. Because the design of these sequences is based on global features of polar/ nonpolar patterning, the finding that half of them bind heme highlights the power of the binary code strategy, and demonstrates that isolating de novo heme proteins does not require explicit design of the cofactor binding site. Because bound heme plays a key role in the functions of many natural proteins, these results suggest that binary code sequences may serve as initial prototypes for the development of large collections of functionally active de novo proteins.

Profiles of Hispanic adolescents and adults on the Holland Themes and Basic Interest Scales of the Strong Interest Inventory.

The Strong Interest Inventory and its predecessors have been the subject of decades of research. Many studies have used Holland's Six General Occupational Themes, and some have explored the 23 Basic Interest Scales. The literature addresses IQ in vocational counseling and includes studies relating Holland's themes and the 23 interest scales. The subjects included a nationwide sample of 894 13- to 65-yr.-olds tested during the standardization of the Kaufman Adolescent and Adult Intelligence Test. Multivariate analyses of variance were conducted with age, gender, and IQ on the intelligence test as independent variables, and the six Holland themes and 23 interest scales as dependent variables. IQ was related to Investigative and Artistic themes. Individuals with high IQs scored nearly one standard deviation higher than individuals with low IQs on each of these scales. A number of Basic Interest Scales correlated significantly with IQs, most of which were associated with either the Investigative or Artistic themes. Correlations of IQ with Holland Themes and scores on Basic Interest Scales were observed for both genders. The information gained from an individually administered intelligence test can enhance interpretation of the Holland themes and interest scales for vocational counseling.