Identifying novel targets in renal cell carcinoma: Design and synthesis of affinity chromatography reagents

Two novel scaffolds, 4-pyridylanilinothiazoles (PAT) and 3-pyridylphenylsulfonyl benzamides (PPB), previously identified as selective cytotoxins for von Hippel–Lindau-deficient Renal Carcinoma cells, were used as templates to prepare affinity chromatography reagents to aid the identification of the molecular targets of these two classes. Structure–activity data and computational models were used to predict possible points of attachment for linker chains. In the PAT class, Click coupling of long chain azides with 2- and 3-pyridylanilinothiazoleacetylenes gave triazole-linked pyridylanilinothiazoles which did not retain the VHL-dependent selectivity of parent analogues. For the PPB class, Sonagashira coupling of 4-iodo-(3-pyridylphenylsulfonyl)benzamide with a propargyl hexaethylene glycol carbamate gave an acetylene which was reduced to the corresponding alkyl 3-pyridylphenylsulfonylbenzamide. This reagent retained the VHL-dependent selectivity of the parent analogues and was successfully utilized as an affinity reagent.     

Structure–activity relationships (SAR) and structure–kinetic relationships (SKR) of bicyclic heteroaromatic acetic acids as potent CRTh2 antagonists III: The role of a hydrogen-bond acceptor in long receptor residence times

The correct positioning and orientation of an hydrogen bond acceptor (HBA) in the tail portion of the biaryl series of CRTh2 antagonists is a requirement for long receptor residence time. The HBA in combination with a small steric substituent in the core section (R^core ≠ H) gives access to compounds with dissociation half-lives of ⩾24 h.     

A role for Rev in the association of HIV-1 gag mRNA with cytoskeletal β-actin and viral protein expression

Human immunodeficiency virus type-1 (HIV-1) Rev acts by inducing the specific nucleocytoplasmic transport of a class of incompletely spliced RNAs that encodes the viral structural proteins. The transfection of HeLA cells with a rev-defective HIV-1 expression plasmid, however, resulted in the export of overexpressed, intron-containing species of viral RNAs, possibly through a default process of nuclear retention. Thus, this system enabled us to directly compare Rev⁺ and Rev⁻ cells as to the usage of RRE-containing mRNAs by the cellular translational machinery. Biochemical examination of the transfected cells revealed that although significant levels of gag and env mRNAs were detected in both the presence and absence of Rev, efficient production of viral proteins was strictly dependent on the presence of Rev. A fluoroscence in situ hybridisation assay confirmed these findings and provided further evidence that even in the presence of Rev, not all of the viral mRNA was equally translated. At the early phase of RNA export in Rev⁺ cells, gag mRNA was observed throughout both the cytoplasm and nucleoplasm as uniform fine stippling. In addition, the mRNA formed clusters mainly in the perinuclear region, which were not observed in Rev⁻ cells. In the presence of Rev, expression of the gag protein was limited to these perinuclear sites where the mRNA accumulated. Subsequent staining of the cytoskeletal proteins demonstrated that in Rev⁺ cells gag mRNA is colocalized with β-actin in the sites where the RNA formed clusters. In the absence of Rev, in contrast, the gag mRNA failed to associate with the cytoskeletal proteins. These results suggest that in addition to promoting the emergence of intron-containing RNA from the nucleus, Rev plays an important role in the compartmentation of translation by directing RRE-containing mRNAs to the β-actin to form the perinuclear clusters at which the synthesis of viral structural proteins begins.     

Cub growth and maternal care in cheetahs

Using cub growth as an index, I examine the influence of maternalnutrition, litter size, and cub sex on maternal care in cheetahs(Acinonyx jubatus) and compare cub and litter growth rates withthose of other large feilds. Seventy-nine free-living cheetahcubs in 21 litters from 15 mothers were weighed at least oncebetween 6 and 48 days of age. Eleven litters were weighed atthe beginning and end of a 5-day observation of their mothers.The mean cub growth rate varied significantly between litters,due primarily to differences in maternal food intake. Growthdeclined sharply when maternal food intake was less than 1.5kg/ day, but did not increase with greater levels of food intake.Lower limits of growth rates may therefore have been set bythe mother's food intake, whereas upper limits may be set bythe intrinsic physiological ability of cubs to grow. Althoughmale cubs were heavier than female cubs in the same litter whenfirst weighed, major differences in growth rate between thesexes were not apparent at this stage. Both cheetah cubs andlitters grow fast relative to other large felids, and I arguethat this may be an adaptation to the high rate of cheetah juvenilemortality from predation.     

An increased number of circulating γ/δ TCR + T cells in patients with chronic viral hepatitis

Abstract There is evidence that γ/δ TCR + T cells are specialized in recognizing different antigens, but their immunologic role as a second TCR is still unclear. The aim of this study was to investigate the percentage and absolute numbers of circulating γ/δ TCR + T cells in patients with chronic viral hepatitis (CVH) and to compare with HBsAg⁺, HCV healthy carriers and healthy subjects. Forty nine patients with CVH-24 with chronic active (CAH) and 25 with chronic persistent hepatitis (CPH)-, 21 HBsAg⁺, 20 HCV asymptomatic carriers and 20 healthy subjects were enrolled in the study. Lymphocyte subsets were determined after incubation with monoclonal antibodies to T total (CD5) and T γ/δ cells (γ/δ-1) using immunofluorescence microscopy. An increased number of circulating γ/δ TCR + T cells was found in patients with CVH in comparison with asymptomatic carriers and normal controls: this increase was more profound in patients with CAH, compared to CPH patients. These results indicate a correlation between circulating γ/δ TCR + T cells in CVH patients and activity and chronicity of the disease.     

Hypersensitive response of wheat to the Hessian fly

Hessian flyMayetiola destructor (Say) larvae are able to obtain food from their host plant without inflicting mechanical damage to the plant surface, apparently by secreting substances which elicit release of nutrients from plant cells surrounding the feeding site. Cells of fully susceptible plants retain their normal appearances, while in resistant plants extensive areas of cellular collapse occur. These responses indicate that hypersensitivity is the basis of wheat's resistance to the Hessian fly. The fly's feeding mechanism more closely resembles that of a pathogen than of a phytophagous insect; correspondingly, both the genetic relationship and resistance mechanism of the host plant to the parasite are of the sorts commonly associated with bacterial and fungal pathogens.     

Effects of Peanut Genotypes on Meloidogyne Species Interactions

A 3-year microplot study was conducted to characterize the interaction between Meloidogyne arenaria race 1 (MA1) and M. hapla (MH), as affected by the five peanut genotypes: Florigiant, NC 7, NC 6, NC Ac 18416, and NC Ac 18016. The interactive effects on infection (total parasitic forms per root unit) and reproduction potentials of each nematode species and crop damage were determined. As a single population, MA1 had greater infection capacity and caused more crop damage than did MH, but both species had similar reproduction potentials. In mixed infestations, MA1 was more competitive than MH, as reflected by incidence of infection. Infection and reproduction potentials, and crop-damage capabilities of the mixed populations were similar to those of MA1 alone. All peanut genotypes were susceptible to infection by both nematodes. NC 6 was less susceptible to damage by MA1 and the mixed populations than other genotypes. A nematode treatment x genotype interaction was detected for root infection and crop damage, but not for population density or reproduction. With high preplant nematode levels (Pi), the populations reached their peak by midseason, whereas those with low Pi peaked after midseason. Crop damage in the second and third years was correlated with Pi level.     

The Ca2+ Pump of the Plasma Membrane of Arabidopsis thaliana: Characteristics and Sensitivity to Fluorescein Derivatives

The transport and hydrolytic activities of the plasma membrane (PM) Ca²⁺ pump were characterized in a PM fraction purified from seedlings of Arabidopsis thaliana by the aqueous two-phase partitioning technique. Ca²⁺ uptake could be energized by ATP and by ITP (at about 70% the rate sustained by ATP). This characteristic was used to measure the hydrolytic activity of the enzyme as Ca²⁺-dependent ITPase activity. The PM Ca²⁺ pump displayed a broad pH optimum around pH 7.2, was drastically inhibited by erythrosin B (EB), and was half-saturated by 60 μM ITP. It was stimulated by CaM, specially at low, non-saturating Ca²⁺ concentrations. All of these characteristics closely resemble those of the PM Ca²⁺ pump in other plant materials. Analysis of the effects of EB and other fluorescein derivatives (eosin Y and rose bengal) showed that: i) EB behaved as a competitive inhibitor with respect to ITP; ii) the PM Ca²⁺ pump was drastically inhibited by concentrations of fluorescein derivatives (submicromolar), much lower than those required to inhibit the PM H⁺-ATPase; iii) the different fluorescein derivatives were diversely efficient in inhibiting the activities of the Ca²⁺ pump and of the H⁺-ATPase of the PM (eosin Y was about 10000-fold, EB 1000-fold and rose bengal only 50-fold more active on the Ca²⁺ pump than on the H⁺-ATPase); and iv) the effectiveness of EB in inhibiting the Ca²⁺ pump was strongly affected by the protein concentration in the assay medium.