A maize thiamine auxotroph is defective in shoot meristem maintenance

Plant shoots undergo organogenesis throughout their life cycle via the perpetuation of stem cell pools called shoot apical meristems (SAMs). SAM maintenance requires the coordinated equilibrium between stem cell division and differentiation and is regulated by integrated networks of gene expression, hormonal signaling, and metabolite sensing. Here, we show that the maize (Zea mays) mutant bladekiller1-R (blk1-R) is defective in leaf blade development and meristem maintenance and exhibits a progressive reduction in SAM size that results in premature shoot abortion. Molecular markers for stem cell maintenance and organ initiation reveal that both of these meristematic functions are progressively compromised in blk1-R mutants, especially in the inflorescence and floral meristems. Positional cloning of blk1-R identified a predicted missense mutation in a highly conserved amino acid encoded by thiamine biosynthesis2 (thi2). Consistent with chromosome dosage studies suggesting that blk1-R is a null mutation, biochemical analyses confirm that the wild-type THI2 enzyme copurifies with a thiazole precursor to thiamine, whereas the mutant enzyme does not. Heterologous expression studies confirm that THI2 is targeted to chloroplasts. All blk1-R mutant phenotypes are rescued by exogenous thiamine supplementation, suggesting that blk1-R is a thiamine auxotroph. These results provide insight into the role of metabolic cofactors, such as thiamine, during the proliferation of stem and initial cell populations.     

Leukotrienes cause eosinophil emigration into conjunctival tissue

The ability of LTB4, LTC4, the 5S,6R and 5R,6S LTD4 stereoisomers, and LTE4 to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological and light microscopy techniques. LTD4 and LTE4 demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10 ng to 1000 ng), while there was only a minimal response to LTC4. LTB4 produced marked eosinophil infiltrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB4. The 5R,6S LTD4 stereoisomer did not evoke any leukocyte infiltration. The SRS-A antagonist, FPL 55712, abolished peptidoleukotriene-induced eosinophil emigration, and indomethacin pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10 micrograms to 1000 micrograms. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.     

Improving the design and analysis of high-throughput screening technology comparison experiments using statistical modeling

Contemporary small-molecule drug discovery frequently involves the screening of large compound files as a core activity. Subsequently cost, speed, and safety become critical issues. In order to meet this need, numerous technologies have been developed to allow mix and measure approaches, facilitate miniaturization, and to increase speed and to minimize the use of potentially hazardous reagents such as radioactive materials. However, despite the on-paper advantages of these new technologies, risks can remain undefined. For example, the question of whether the novel method will facilitate identification of active chemical series in a way that is comparable with conventional methods arises. In order to address this question, we have taken the approach of carrying out experiments to directly compare the output of high-throughput screens using a given novel approach and a traditional method. The concordance between the screening methods can then be determined via comparison of the numbers and structures of the active molecules identified. This article describes the approach taken in our laboratory to minimize variability in such experiments and shows data that exemplifies the general result of lower than expected concordance. Statistical modeling was subsequently used to facilitate this interpretation. The model used beta-distribution function to generate a real-activity frequency relationship with added normal random error and occasional outliers to represent assay variability. Hence, the effect of assay parameters such as the threshold, the number of real actives, and the number of outliers and the standard deviation could readily be explored. The model was found to describe the data reasonably and moreover was found to be of great utility when it came to planning further optimal experiments. A key conclusion from the model was that concordance between screening methods could appear poor even when one approach is compared with itself. This occurs simply because the result is a function of assay threshold, standard deviation and the true compound % activity. In response to this finding we have adopted alternative experimental designs that more reliably measure the concordance between screening methods.     

Ion-binding properties of calbindin D9k: a Monte Carlo simulation study

Monte Carlo simulations are used to calculate the binding constant of two Ca2+ ions to the protein bovine calbindin D9k. The change in binding constant with respect to mutation of charged amino acids, presence of various electrolytes, protein concentration, solution pH, and competitive binding of monovalent ions is investigated. Each of these factors may have a large influence on the binding constant. The simulations are performed in a dielectric continuum model, the so-called primitive model of electrolyte theory, with a fixed protein structure and a uniform dielectric permittivity. The calculated binding constants are in excellent agreement with experimental data and describe changes in the binding constant over six orders of magnitude.     

THE AUSTRALIAN SPECIES OF ORIUS WOLFF (HETEROPTERA: ANTHOCORIDAE)

Abstract
Triphleps australis China, 1926 is synonymised with Anthocoris tantillus Motschulsky, 1863 (both now in Orius Wolff) and additional taxonomic and distributional data are provided for O. tantillus and O. armatus Gross, 1954. O. heterorioides sp. n. and O. chadwicki sp. n. are described. The 4 species are keyed and their placements within the genus are discussed.     

Early effects of climate change: do they include changes in vector-borne disease?

The world's climate appears now to be changing at an unprecedented rate. Shifts in the distribution and behaviour of insect and bird species indicate that biological systems are already responding to this change. It is well established that climate is an important determinant of the spatial and temporal distribution of vectors and pathogens. In theory, a change in climate would be expected to cause changes in the geographical range, seasonality (intra-annual variability), and in the incidence rate (with or without changes in geographical or seasonal patterns). The detection and then attribution of such changes to climate change is an emerging task for scientists. We discuss the evidence required to attribute changes in disease and vectors to the early effects of anthropogenic climate change. The literature to date indicates that there is a lack of strong evidence of the impact of climate change on vector-borne diseases (i.e. malaria, dengue, leishmaniasis, tick-borne diseases). New approaches to monitoring, such as frequent and long-term sampling along transects to monitor the full latitudinal and altitudinal range of specific vector species, are necessary in order to provide convincing direct evidence of climate change effects. There is a need to reassess the appropriate levels of evidence, including dealing with the uncertainties attached to detecting the health impacts of global change.     

Mechanism of substrate hydrolysis by a thermophilic endoglucanase from Thermotoga maritima†

A cellulase from the thermophile, Thermotoga maritima, hydrolyzed oligosaccharide substrates by an exoglucanase mode of action but acted as an endoglucanase to rapidly reduce the viscosity of the soluble polysaccharides carboxymethylcellulose and barley -glucan. The V _max for hydrolysis of the substrate, p-nitrophenyl -d-cellobioside, was 42 mol min^–1 (mg protein)^–1, while that for barley -glucan was 637. The enzyme had little activity on crystalline cellulose.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.