CYP3A4-V and prostate cancer in African Americans: causal or confounding association because of population stratification?

CYP3A4-V, an A to G promoter variant associated with prostate cancer in African Americans, exhibits large differences in allele frequency between populations. Given that the African American population is genetically heterogeneous because of its African ancestry and subsequent admixture with European Americans, case-control studies with African Americans are highly susceptible to spurious associations. To test for association with prostate cancer, we genotyped CYP3A4-V in 1376 (2 N) chromosomes from prostate cancer patients and age- and ethnicity-matched controls representing African Americans, Nigerians, and European Americans. To detect population stratification among the African American samples, 10 unlinked genetic markers were genotyped. To correct for the stratification, the uncorrected association statistic was divided by the average of association statistics across the 10 unlinked markers. Sharp differences in CYP3A4-V frequencies were observed between Nigerian and European American controls (0.87 and 0.10, respectively; P<0.0001). African Americans were intermediate (0.66). An association uncorrected for stratification was observed between CYP3A4-V and prostate cancer in African Americans (P=0.007). A nominal association was also observed among European Americans (P=0.02) but not Nigerians. In addition, the unlinked genetic marker test provided strong evidence of population stratification among African Americans. Because of the high level of stratification, the corrected P-value was not significant (P=0.25). Follow-up studies on a larger dataset will be needed to confirm whether the association is indeed spurious; however, these results reveal the potential for confounding of association studies by using African Americans and the need for study designs that take into account substructure caused by differences in ancestral proportions between cases and controls.     

Proteolytic activity against the light-harvesting complex and the D1/D2 core proteins of Photosystem II in close association to the light-harvesting complex II trimer

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 degrees C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing "activity gels". Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being "self-digested", also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 degrees C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg(2+), and inhibited by Zn(2+), Cd(2+), EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing "activity gels" or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.     

Triazines facilitate neurotransmitter release of synaptic terminals located in hearts of frog (Rana ridibunda) and honeybee (Apis mellifera) and in the ventral nerve cord of a beetle (Tenebrio molitor)

Three triazine herbicides, atrazine, simazine and metribuzine, and some of their major metabolites (cyanuric acid and 6-azauracil) were investigated for their action on synaptic terminals using three different isolated tissue preparations from the atria of the frog, Rana ridibunda, the heart of the honeybee, Apis mellifera macedonica, and the ventral nerve cord of the beetle, Tenebrio molitor. The results indicate that triazines facilitate the release of neurotransmitters from nerve terminals, as already reported for the mammalian central nervous system. The no observed effect concentration, the maximum concentration of the herbicide diluted in the saline that has no effect on the physiological properties of the isolated tissue, was estimated for each individual preparation. According to their relative potency, the three triazines tested can be ranked as follows: atrazine (cyanuric acid), simazine>metribuzine (6-azauracil). The action of these compounds on the cholinergic (amphibians, insects), adrenergic (amphibian) and octopaminergic (insects) synaptic terminals is discussed.     

The simultaneous determination of 1-aminocyclopropane-1-carboxylic acid and cyclopropane-1,1-dicarboxylic acid in Lycopersicum esculentum by high-performance liquid chromatography--electrospray tandem mass spectrometry

Varying concentrations of cyclopropane-1,1-dicarboxylic acid (CDA), an inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase, added to the solid culture medium of tomato nodal shoot segments resulted in a reduction in the level of endogenous ethylene according to the concentration of inhibitor applied. Following treatment with inhibitor, plants were homogenised and the concentrations of CDA and of 1-aminocyclopropane-1-carboxylic acid (ACC) were measured simultaneously in the resulting juice using an HPLC-ESI/MS-MS method. The levels of CDA and ACC measured in the plant tissues were associated with the concentration of inhibitor added to the solid medium. The HPLC-ESI/MS-MS method described produced limits of detection of 0.8 pmol for ACC and of 4 pmol for CDA.     

Hybridization and karyotype variability of three endemic Fritillaria L. (Liliaceae) in Argolis Peninsula (Greece)

Abstract

The Argolis Peninsula covers the north-eastern part of Peloponnisos and is surrounded by the Gulf of Argosaronic. The area hosts three species of the genus Fritillaria: F. graeca, F. rhodocanakis and F. spetsiotica. Fritillaria graeca is a Greek endemic taxon and its distribution includes Peloponnisos, C & E Sterea Ellas, C Evia and in proximity to Sterea Ellas, Salamis and Kea islands, while the stenoendemic F. rhodocanakis and F. spetsiotica are mainly found on Idra and Spetses islands respectively. The last two taxa are included in the Red Data Book of Rare and Threatened Plants of Greece, while F. rhodocanakis is also included in the IUCN Red List. Hybridization among them is a common phenomenon in the areas where they coexist, leading to an array of morphologically and karyologically intermediate forms. The current study presents the taxa’s karyomorphometric analysis for the first time and reveals hybrids’ cytological variety, including differences in marker chromosomes, polyploidy and the number of B-chromosomes.     

Antibody Specificities Associated with Neutralization Breadth in Plasma from Human Immunodeficiency Virus Type 1 Subtype C-Infected Blood Donors

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.The generation of an antibody response capable of neutralizing a broad range of viruses remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Despite multiple efforts in the design of immunogens capable of inducing such humoral responses, little progress has been made (18, 20, 39). The sequence variability of the virus, as well as masking mechanisms exhibited by the envelope glycoprotein, has further hindered this pursuit (6, 22). It is known that while the majority of HIV-infected individuals mount a strong neutralization response against their own virus within the first 6 to 12 months of infection, breadth is observed in only a few individuals years later (5, 10, 15, 26, 33, 40, 41). However, very little is known about the specificities of the antibodies that confer this broad cross-neutralization. It is plausible that broadly cross-neutralizing (BCN) plasmas contain antibodies that target conserved regions of the envelope glycoprotein, as exemplified by a number of well-characterized broadly neutralizing monoclonal antibodies (MAbs). The b12 MAb recognizes the CD4 binding site (CD4bs), and 2G12 binds to surface glycans (7, 42, 44, 56). The 447-52D MAb recognizes the V3 loop, and 17b, E51, and 412d bind to CD4-induced (CD4i) epitopes that form part of the coreceptor binding site (13, 21, 51, 54). Finally, the MAbs 2F5, 4E10, and Z13e1 recognize distinct linear sequences in the gp41 membrane-proximal external region (MPER) (36, 57). The targets of these neutralizing MAbs provide a rational starting point for examining the complex nature of polyclonal plasma samples.Several groups have addressed the need to develop methodologies to elucidate the presence of certain neutralizing-antibody specificities (1, 8, 9, 29, 30, 43, 55). A number of these studies reported that the BCN antibodies in plasma can in some cases be adsorbed using gp120 immobilized on beads (1, 9, 29, 30, 43). Furthermore, the activities of some of these anti-gp120 neutralizing antibodies could be mapped to the CD4bs, as the D368R mutant gp120 failed to adsorb them (1, 29, 30, 43).Antibodies to CD4i epitopes are frequently found in HIV-1-infected individuals and are thought to primarily target the coreceptor binding site, which includes the bridging sheet and possibly parts of the V3 region. Decker and colleagues (8) showed that MAbs to HIV-1 CD4i epitopes can neutralize HIV-2 when pretreated with soluble CD4 (sCD4), indicating that the CD4i epitope is highly conserved among different HIV lineages. The poor accessibility of CD4i epitopes, however, has precluded this site from being a major neutralizing-antibody target (24), although a recent study suggested that some of the cross-neutralizing activity in polyclonal sera mapped to a CD4i epitope (30).Another site that has attracted considerable attention as a target for cross-neutralizing antibodies is the MPER, a linear stretch of 34 amino acids in gp41. Anti-MPER antibodies have been detected in the plasma of HIV-infected individuals by using chimeric viruses with HIV-1 MPER grafted into a simian immunodeficiency virus or an HIV-2 envelope glycoprotein (15, 55). These studies concluded that 2F5- and 4E10-like antibodies were rarely found in HIV-1-infected plasmas; however, other specificities within the MPER were recognized by around one-third of HIV-1-infected individuals (15). More recently, 4E10-like and 2F5-like antibodies (30, 43), as well as antibodies to novel epitopes within the MPER (1), have been shown to be responsible for neutralization breadth in a small number of plasma samples. The anti-MPER MAb 4E10 has been shown to react to autoantigens, leading to the suggestion that their rarity in human infection is due to the selective deletion of B cells with these specificities (17, 35). Furthermore, a recent study found an association between anti-MPER and anti-cardiolipin (CL) antibodies, although an association with neutralization was not examined (31).A recent study by Binley and coworkers used an array of methodologies to determine the antibody specificities present in subtype B and subtype C plasma samples with neutralization breadth (1). While antibodies to gp120, some of which mapped to the CD4bs, and to MPER were identified, most of the neutralizing activity in the BCN plasma could not be attributed to any of the known conserved envelope epitopes. Furthermore, it is not clear how common these specificities are among HIV-1-positive plasmas and whether they are only associated with BCN activity.In this study, we investigated a large collection of HIV-1-infected plasmas obtained from the South African National Blood Services. We aimed to determine if there is a relationship between the presence of certain antibody specificities, such as those against CD4i epitopes, MPER, or the CD4bs, and the neutralizing activities present in these plasmas. Furthermore, we evaluated the presence of various autoreactive antibodies and analyzed whether they might be associated with neutralization breadth.     

Evolution and biogeography of the endemic Roucela complex (Campanulaceae: Campanula) in the Eastern Mediterranean

At the intersection of geological activity, climatic fluctuations, and human pressure, the Mediterranean Basin – a hotspot of biodiversity – provides an ideal setting for studying endemism, evolution, and biogeography. Here, we focus on the Roucela complex (Campanula subgenus Roucela), a group of 13 bellflower species found primarily in the eastern Mediterranean Basin. Plastid and low‐copy nuclear markers were employed to reconstruct evolutionary relationships and estimate divergence times within the Roucela complex using both concatenation and species tree analyses. Niche modeling, ancestral range estimation, and diversification analyses were conducted to provide further insights into patterns of endemism and diversification through time. Diversification of the Roucela clade appears to have been primarily the result of vicariance driven by the breakup of an ancient landmass. We found geologic events such as the formation of the mid‐Aegean trench and the Messinian Salinity Crisis to be historically important in the evolutionary history of this group. Contrary to numerous past studies, the onset of the Mediterranean climate has not promoted diversification in the Roucela complex and, in fact, may be negatively affecting these species. This study highlights the diversity and complexity of historical processes driving plant evolution in the Mediterranean Basin.     

Discovery and SAR of isonicotinamide BACE-1 inhibitors that bind beta-secretase in a N-terminal 10s-loop down conformation

A series of low-molecular weight 2,6-diamino-isonicotinamide BACE-1 inhibitors containing an amine transition-state isostere were synthesized and shown to be highly potent in both enzymatic and cell-based assays. These inhibitors contain a trans-S,S-methyl cyclopropane P(3) which bind BACE-1 in a 10s-loop down conformation giving rise to highly potent compounds with favorable molecular weight and moderate to high susceptibility to P-glycoprotein (P-gp) efflux.     

Factors influencing in the response of <i>Schizolobium parahybum</i> (Vell) Blake to <i>Ceratocystis paradoxa</i> and <i>C. moniliformis</i>

In the Ecuadorian coast one of the most destructive diseases of the pachaco is vascular wilt or stem rot caused by Ceratocystis complex, so the aim of this study was to determine the factors that affect the efficiency of the reaction of bark pachaco to this disease. This research was conducted under laboratory conditions, using trees pachaco S38, S41, S98, AE-1, AE-2 and AE-3, and pathogenic species Ceratocystis paradoxa and C. moniliformis. The method utilized was tissue stem bark,with bark sections with 4.5 cm², and a suspension of 3x10⁴ units infection and remained in a humid chamber for 96 hours at 25 ± 5 °C. Were determined grades of resistance/ susceptibility using a scale from 0 to 4, depending on the amount of mycelia and peritecio in each plant sample. Three factors were used: four colonies obtained by several transfers from each fungal specie, four ages of colonies of each fungal specie and four volumes of inoculum applied (units of infection), using for each experiment separately Completely Randomized Design with 4 replications factorial arrangement. For comparison between treatment means was used Tukey test at 5% probability of error. For future trials using this technique, you could use 30-day colonies for C. paradoxa and 40 days for C. moniliformis, and an application volume of 100 μL/cm², it would improve the level of response for the formation of perithecium and mycelia in samples cortex.