Toward an optimal sampling protocol for Hemiptera on understorey plants

There are no standardised sampling protocols for inventorying Hemiptera from understorey or canopy plants. This paper proposes an optimal protocol for the understorey, after evaluating the efficiency of seven methods to maximise the richness of Hemiptera collected from plants with minimal field and laboratory time. The methods evaluated were beating, chemical knockdown, sweeping, branch clipping, hand collecting, vacuum sampling and sticky trapping. These techniques were tested at two spatial scales: 1 ha sites and individual plants. In addition, because efficiency may differ with vegetation structure, sampling of sites was conducted in three disparate understorey habitats, and sampling of individual plants was conducted across 33 plant species. No single method sampled the majority of hemipteran species in the understorey. Chemical knockdown, vacuum sampling and beating yielded speciose samples (61, 61 and 30 species, respectively, representing 53, 53 and 26% of total species collected). The four remaining methods provided species-poor samples (<18 species or <16% of total species collected). These methods also had biases towards particular taxa (e.g., branch clipping and hand collecting targeted sessile Hemiptera, and sticky trapping were dominated by five species of Psyllidae). The most time-efficient methods were beating, sweeping and hand collecting (200 minutes of field and laboratory time yielded >7 species for each technique). By comparison, vacuum sampling, sticky trapping, branch clipping and chemical knockdown yielded <5 species for the same period. Chemical knockdown had further disadvantages; high financial cost and potential spray drift. The most effective methods for a standardised sampling protocol to inventory Hemiptera from the understorey are beating and vacuum sampling. If used in combination, these methods optimise the catch of understorey hemipteran species, as their samples have high complementarity.     

Regulatory mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae. I. Kinetic studies

Homocitrate synthase (HCS) catalyzes one of the regulated steps of the alpha-aminoadipate pathway for lysine biosynthesis in fungi. The kinetic mechanism of regulation of HCS from Saccharomyces cerevisiae by Na+ and the feedback inhibitor lysine was studied by measuring the initial rate in the absence and presence of the effectors. The data suggest that Na+ is an activator at low concentrations and an inhibitor at high concentrations and that these effects occur as a result of the monovalent ion binding to two different sites in the free enzyme. Inhibition and activation by Na+ can occur simultaneously, with the net rate of the enzyme determined by Na+/K(iNa+) and Na+/K(act), where K(iNa+) and K(act) are the inhibition and activation constants, respectively. The inhibition by Na+ was eliminated at high concentrations of acetyl-CoA, the second substrate bound, but the activation remained. Fluorescence binding studies indicated that lysine bound with high affinity to its binding site as an inhibitor. The inhibition by lysine was competitive versus alpha-ketoglutarate and linear in the physiological range of lysine concentrations up to 5 mm. The effects of Na+ and lysine were independent of one another. A model is developed for regulation of HCS that takes into account all of the effects discussed above.     

Detection and quantification of Legionella pneumophila in water samples using competitive PCR

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.     

Chimeric proteins suggest that the catalytic and/or C-terminal domains give CesA1 and CesA3 access to their specific sites in the cellulose synthase of primary walls

CesA1 and CesA3 are thought to occupy noninterchangeable sites in the cellulose synthase making primary wall cellulose in Arabidopsis (Arabidopsis thaliana L. Heynh). With domain swaps and deletions, we show that sites C terminal to transmembrane domain 2 give CesAs access to their individual sites and, from dominance and recessive behavior, deduce that certain CesA alleles exclude others from accessing each site. Constructs that swapped or deleted N-terminal domains were stably transformed into the wild type and into the temperature-sensitive mutants rsw1 (Ala-549Val in CesA1) and rsw5 (Pro-1056Ser in CesA3). Dominant-positive behavior was assayed as root elongation at the restrictive temperature and dominant-negative effects were observed at the permissive temperature. A protein with the catalytic and C-terminal domains of CesA1 and the N-terminal domain of CesA3 promoted growth only in rsw1 consistent with it accessing the CesA1 site even though it contained the CesA3 N-terminal domain. A protein having the CesA3 catalytic and C-terminal domains linked to the CesA1 N-terminal domain dramatically affected growth, but only in the CesA3 mutant. This is consistent with the operation of the same access rule taking this chimeric protein to the CesA3 site. In this case, however, the transgene behaved as a genotype-specific dominant negative, causing a 60% death rate in rsw5, but giving no visible phenotype in wild type or rsw1. We therefore hypothesize that possession of CesA3(WT) protects Columbia and rsw1 from the lethal effects of this chimeric protein, whereas the mutant protein (CesA3(rsw5)) does not.     

NALL-1, an acute lymphoblastic leukemia cell line with peroxidase activity

Summary All cells examined from the non-B, non-T acute lymphoblastic leukemia cell line, NALL-1, stained positive both for terminal deoxynucleotidyl transferase and for common ALL antigen. In addition, peroxidase activity was detected by light microscopy in 55 to 75% of cells and peroxidase-positive granules were detected ultrastructurally in >80% of cells. Peroxidase activity in NALL-1 may result from derepression of peroxidase genes or clonal proliferation of a biphenotypic precursor cell.     

Bacterial responses to background organic pollutants in the northeast subarctic Pacific Ocean

Thousands of man-made synthetic chemicals are released to oceans and compose the anthropogenic dissolved organic carbon (ADOC). Little is known about the effects of this chronic pollution on marine microbiome activities. In this study, we measured the pollution level at three sites in the Northeast Subarctic Pacific Ocean (NESAP) and investigated how mixtures of three model families of ADOC at different environmentally relevant concentrations affected naturally occurring marine bacterioplankton communities' structure and metabolic functioning. The offshore northernmost site (North) had the lowest concentrations of hydrocarbons, as well as organophosphate ester plasticizers, contrasting with the two other continental shelf sites, the southern coastal site (South) being the most contaminated. At North, ADOC stimulated bacterial growth and promoted an increase in the contribution of some Gammaproteobacteria groups (e.g. Alteromonadales) to the 16 rRNA pool. These groups are described as fast responders after oil spills. In contrast, minor changes in South microbiome activities were observed. Gene expression profiles at Central showed the coexistence of ADOC degradation and stress-response strategies to cope with ADOC toxicities. These results show that marine microbial communities at three distinct domains in NESAP are influenced by background concentrations of ADOC, expanding previous assessments for polar and temperate waters.     

Flocculation in ale brewing strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: re-evaluation of the role of cell surface charge and hydrophobicity

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.     

Structural elucidation of the O-antigenic polysaccharide from enterohemorrhagic (EHEC) Escherichia coli O48:H21

The structure of the antigenic O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) produced by the enterohemorrhagic strain of Escherichia coli O48:H21 (EHEC) has been elucidated. The O-PS obtained by mild acid hydrolysis of the LPS had [alpha]D +95 (water) and was composed of L-rhamnose (L-Rha), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN), and D-galacturonic acid (D-GalA) (1:1:1:1:1). From the results of methylation analysis, mass spectrometry, 2D NMR, and DOC-PAGE, the O-PS was shown to be a high molecular mass polymer of a repeating pentasaccharide unit having the structure: [structure: see text]. The D-Gal pA non-reducing end groups in the O-PS were partially O-acetylated (approximately 30%) at the O-2 and O-3 positions and the degree of acetylation was variable from batch to batch cell production.