Sequence analysis of HSPA1A and HSPA1B in a multi-ethnic study population.

Two copies of the Hsp70 gene, HSPA1A and HSPA1B, are located on chromosome 6p21. The coding regions of HSPA1A and HSPAIB are intronless and nearly identical, however, promoter and 3' UTR sequences are different. The coding regions and putative promoter regions of HSPAIA and HSPAIB were re-sequenced in an affected and unaffected asthma screening panel of US Caucasians, African Americans, and US Hispanics (n = 72) to identify polymorphisms. HSPAIA and HSPAIB were each amplified in two separate whole-gene fragments. The polymorphisms identified were compared to those reported in dbSNP. Nine polymorphisms (one novel) were identified in HSPAIA, five of which are coding. Fourteen polymorphisms (five novel) were identified in HSPAIB, five of which are coding. One polymorphism (Asp110Glu GAG > GAC) was found in both genes. Two-thirds of the polymorphisms reported in dbSNP were not identified in our screening panel. Although similar in sequence, HSPAIA and HSPAIB do not share common patterns of polymorphisms.     

Opportunities and limitations in assessing the multifunctionality of agriculture within the CAPRI model

In recent years the objectives of agricultural policy have shifted from a principal focus on production and income towards agriculture's provision of public goods summarized by the term ‘multifunctionality’. Agricultural sector models, which are important tools for policy advice, need to be adjusted in order to maintain their relevance and reliability in accordance with policy changes. This paper investigates the strengths and limitations of incorporating multifunctionality indicators in the agricultural sector model Common Agricultural Policy Regional Impact Analysis (CAPRI) by reviewing the existing literature and incorporating such indicators in the model. Multifunctionality indicators are developed and implemented for four selected aspects of multifunctionality: food security, landscape, environmental concerns and rural viability. By running different policy reform scenarios, it is shown that indicators closely related to the underlying economic variables of the sector model may provide useful to describe the effects of policy reforms on agriculture's multifunctionality. However, these indicators do not completely cover the selected aspects of multifunctionality. In order to yield a broader coverage, this paper proposes to strengthen interdisciplinary research by linking agricultural sector models with other model systems like farm-based economical–ecological models, regional economic models or landscape information systems.     

Analysis of transposon insertion mutants highlights the diversity of mechanisms underlying male progamic development in Arabidopsis

To identify genes with essential roles in male gametophytic development, including postpollination (progamic) events, we have undertaken a genetic screen based on segregation ratio distortion of a transposon-borne kanamycin-resistance marker. In a population of 3359 Arabidopsis Ds transposon insertion lines, we identified 20 mutants with stably reduced segregation ratios arising from reduced gametophytic transmission. All 20 mutants showed strict cosegregation of Ds and the reduced gametophytic transmission phenotype. Among these, 10 mutants affected both male and female transmission and 10 mutants showed male-specific transmission defects. Four male and female (ungud) mutants and 1 male-specific mutant showed cellular defects in microspores and/or in developing pollen. The 6 remaining ungud mutants and 9 male-specific (seth) mutants affected pollen functions during progamic development. In vitro and in vivo analyses are reported for 5 seth mutants. seth6 completely blocked pollen germination, while seth7 strongly reduced pollen germination efficiency and tube growth. In contrast, seth8, seth9, or seth10 pollen showed reduced competitive ability that was linked to slower rates of pollen tube growth. Gene sequences disrupted in seth insertions suggest essential functions for putative SETH proteins in diverse processes including protein anchoring, cell wall biosynthesis, signaling, and metabolism.     

A single Gbeta subunit locus controls cross-talk between protein kinase C and G protein regulation of N-type calcium channels

The modulation of N-type calcium channels is a key factor in the control of neurotransmitter release. Whereas N-type channels are inhibited by Gbetagamma subunits in a G protein beta-isoform-dependent manner, channel activity is typically stimulated by activation of protein kinase C (PKC). In addition, there is cross-talk among these pathways, such that PKC-dependent phosphorylation of the Gbetagamma target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gbeta(1)-mediated responses. The molecular mechanisms that control this G protein beta subunit subtype-specific regulation have not been described. Here, we show that G protein inhibition of N-type calcium channels is critically dependent on two separate but adjacent approximately 20-amino acid regions of the Gbeta subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from those implicated previously in Gbetagamma signaling to other effectors such as G protein-coupled inward rectifier potassium channels, phospholipase beta(2), and adenylyl cyclase, thus raising the possibility that the specificity for G protein signaling to calcium channels might rely on unique G protein structural determinants. In addition, we identify a highly specific locus on the Gbeta(1) subunit that serves as a molecular detector of PKC-dependent phosphorylation of the G protein target site on the N-type channel alpha(1) subunit, thus providing for a molecular basis for G protein-PKC cross-talk. Overall, our results significantly advance our understanding of the molecular details underlying the integration of G protein and PKC signaling pathways at the level of the N-type calcium channel alpha(1) subunit.     

Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit

Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.     

Profiling the expression of mitogen-induced T-cell proteins by using multi-membrane dot-blotting

High throughput technologies are standard methods for analysis of the proteome. Multi-layer multi-well plate dot-blotting system (MLDot) technology is a high-throughput dot blotting system that provides a simple, cost-effective approach for protein expression profiling in multiple samples. In contrast to traditional dot blot, MLDot uses a layered stack of thin, sieve-like membranes in place of a single nitrocellulose membrane. Therefore, up to 10 membranes can be prepared from the samples arrayed in a single 96-well plate. We describe the ability of MLDot to detect the predicted changes in protein expression following multiple mitogen treatment of T-cells. We compare the levels of the phopshorylated forms of CREB, Jun, and Akt in Jurkat T-cells as detected by MLDot to those measured by a gel-based assay. We also describe the ability of MLDot to detect differences in the levels of phosphorylated Akt in Jurkat cells as compared to primary lymphocytes.     

Genomewide significant linkage to recurrent, early-onset major depressive disorder on chromosome 15q

A genome scan was performed on the first phase sample of the Genetics of Recurrent Early-Onset Depression (GenRED) project. The sample consisted of 297 informative families containing 415 independent affected sibling pairs (ASPs), or, counting all possible pairs, 685 informative affected relative pairs (555 ASPs and 130 other pair types). Affected cases had recurrent major depressive disorder (MDD) with onset before age 31 years for probands or age 41 years for other affected relatives; the mean age at onset was 18.5 years, and the mean number of depressive episodes was 7.3. The Center for Inherited Disease Research genotyped 389 microsatellite markers (mean spacing of 9.3 cM). The primary linkage analysis considered allele sharing in all possible affected relative pairs with the use of the Z(lr) statistic computed by the ALLEGRO program. A secondary logistic regression analysis considered the effect of the sex of the pair as a covariate. Genomewide significant linkage was observed on chromosome 15q25.3-26.2 (Zlr=4.14, equivalent LOD = 3.73, empirical genomewide P=.023). The linkage was not sex specific. No other suggestive or significant results were observed in the primary analysis. The secondary analysis produced three regions of suggestive linkage, but these results should be interpreted cautiously because they depended primarily on the small subsample of 42 male-male pairs. Chromosome 15q25.3-26.2 deserves further study as a candidate region for susceptibility to MDD.     

Decadal changes in summer mortality in U.S. cities

Recent studies suggest that anthropogenic climate warming will result in higher heat-related mortality rates in U.S. cities than have been observed in the past. However, most of these analyses assume that weather-mortality relationships have not changed over time. We examine decadal-scale changes in relationships between human mortality and hot, humid weather for 28 U.S. cities with populations greater than one million. Twenty-nine years of daily total mortality rates, age-standardized to account for underlying demographic changes, are related to afternoon apparent temperatures ( T(a)) and organized by decade for each city. Threshold T(a) values, or the T(a) at and above which mortality is significantly elevated, are calculated for each city, and the mortality rates on days when the threshold T(a) was exceeded are compared across decades. On days with high T(a), mortality rates were lower in the 1980s and 1990s than in the 1960s and 1970s in a majority of the cities. Regionally, northeastern and northern interior cities continue to exhibit elevated, albeit reduced, death rates on warm, humid days in the 1980s and 1990s, while most southern cities do not. The overall decadal decline in mortality in most cities is probably because of adaptations: increased use of air conditioning, improved health care, and heightened public awareness of the biophysical impacts of heat exposure. This finding of a more muted mortality response of the U.S. populace to high T(a) values over time raises doubts about the validity of projections of future U.S. mortality increases linked to potential greenhouse warming.