Isolation of human alpha-fetoprotein in two fractionation steps and demonstration of homogeneity.

Human alpha-fetoprotein (hAFP) has been isolated from cord serum in 40% yield using an isolation procedure consisting of only two major steps: affinity chromatography followed by preparative polyacrylamide gel electrophoresis (PAGE). The final product appeared homogeneous on the basis of five independent criteria for purity. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) demonstrated a single polypeptide chain with molecular weight of 71,000. The protein exhibited an apparent isoelectric point (pI') of 4.85, molecular radius of 3.0 nm and a valence (net H+/molecule) of 21.9 derived from computation of analytical PAGE data. The two-step isolation procedure made it possible for a single operator to isolate milligram amounts of hAFP in a matter of weeks.     

The Dystrophin Complex Controls BK Channel Localization and Muscle Activity in Caenorhabditis elegans

Genetic defects in the dystrophin-associated protein complex (DAPC) are responsible for a variety of pathological conditions including muscular dystrophy, cardiomyopathy, and vasospasm. Conserved DAPC components from humans to Caenorhabditis elegans suggest a similar molecular function. C. elegans DAPC mutants exhibit a unique locomotory deficit resulting from prolonged muscle excitation and contraction. Here we show that the C. elegans DAPC is essential for proper localization of SLO-1, the large conductance, voltage-, and calcium-dependent potassium (BK) channel, which conducts a major outward rectifying current in muscle under the normal physiological condition. Through analysis of mutants with the same phenotype as the DAPC mutants, we identified the novel islo-1 gene that encodes a protein with two predicted transmembrane domains. We demonstrate that ISLO-1 acts as a novel adapter molecule that links the DAPC to SLO-1 in muscle. We show that a defect in either the DAPC or ISLO-1 disrupts normal SLO-1 localization in muscle. Consistent with observations that SLO-1 requires a high calcium concentration for full activation, we find that SLO-1 is localized near L-type calcium channels in muscle, thereby providing a mechanism coupling calcium influx with the outward rectifying current. Our results indicate that the DAPC modulates muscle excitability by localizing the SLO-1 channel to calcium-rich regions of C. elegans muscle.     

A family of yeast proteins mediating bidirectional vacuolar amino acid transport

Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal gamma-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport system h described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles.     

Effects of IL-8, Gro-alpha, and LTB(4) on the adhesive kinetics of LFA-1 and Mac-1 on human neutrophils

Firm adhesion ofrolling neutrophils on inflamed endothelium is dependent on₂ (CD18)-integrins and activating stimuli. LFA-1 (CD11a/CD18) appears to be more important than Mac-1 (CD11b/CD18) inneutrophil emigration at inflammatory sites, but little is known of therelative binding characteristics of these two integrins underconditions thought to regulate firm adhesion. The present studyexamined the effect of chemoattractants on the kinetics of LFA-1 andMac-1 adhesion in human neutrophils. We found that subnanomolarconcentrations of interleukin-8, Gro-, and leukotriene B₄ (LTB₄) induced rapid and optimal rates ofLFA-1-dependent adhesion of neutrophils to intercellular adhesionmolecule (ICAM)-1-coated beads. These optimal rates of LFA-1 adhesionwere transient and decayed within 1 min after chemoattractantstimulation. Mac-1 adhesion was equally rapid initially but continuedto rise for 6 min after stimulation. A fourfold higher density ofICAM-1 on beads markedly increased the rate of binding to LFA-1 but did not change the early and narrow time window for the optimal rate ofadhesion. Using well-characterized monoclonal antibodies, we showedthat activation of LFA-1 and Mac-1 by Gro- was completely blocked byanti-CXC chemokine receptor R2, but activation of these integrins byinterleukin-8 was most effectively blocked by anti-CXC chemokinereceptor R1. The topographical distribution of beads also reflectedsignificant differences between LFA-1 and Mac-1. Beads bound to Mac-1translocated to the cell uropod within 4 min, but beads bound to LFA-1remained bound to the lamellipodial regions at the same time. Thesekinetic and topographical differences may indicate distinct functionalcontributions of LFA-1 and Mac-1 on neutrophils.

     

Microarray analysis of shear stressed endothelial cells

The cDNA microarray is an extremely beneficial tool for study of differential gene expression in the cardiovascular system. This technique is used in many different applications including drug discovery, environmental science, and the effects of mechanical forces on vascular cell phenotype. The paper reviews work by others, and describes our study on effects of shear stress on vascular endothelial cells. These microarray studies verified earlier findings using Northern and polymerase chain reaction (PCR) analyses in this area; and also found previously unidentified differentially expressed genes, leading to new hypotheses regarding how cells and tissues respond to biochemical and mechanical stimuli.     

Clonal structure and hybrid susceptibility to a smut pathogen in microscale hybrid zones of northern wetland Carex (Cyperaceae)

Interspecific hybrid taxa, especially those with the potential for clonal spread, may play important roles in community dynamics and plant-pathogen interactions. This study combines the mapping of clonal structure for two rhizomatous sedges (Carex limosa, C. rariflora) and their nearly sterile interspecific hybrid with an investigation of the relationship between these taxa and a nonsystemic floral smut pathogen (Anthracoidea limosa) in six subarctic fens in Nouveau-Québec, Canada. We used allozyme polymorphisms in 14 of 18 putative loci to confirm hybrid identification and to distinguish among genotypes for mapping. The incidence of A. limosa was 5-20 times greater on hybrids than on parental taxa across all sites at two spatial scales (intensive extent = 10.5 m(2), extensive extent = entire fens). Spatial autocorrelation was detected in smut incidence; however, its statistical removal did not alter the strong association between hybrids and smut infection. Smut incidence on both C. limosa and hybrids was greater when they were growing in areas of high hybrid density. Our study provides evidence that disease can help maintain boundaries between species. We suggest explanations for hybrid susceptibility and provide evidence for a model in which hybrids act as a source for reinfection for all three taxa during subsequent years.     

Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-dependent protein kinase

The growth and behavior of higher organisms depend on the accurate perception and integration of sensory stimuli by the nervous system. We show that defects in sensory perception in C. elegans result in abnormalities in the growth of the animal and in the expression of alternative behavioral states. Our analysis suggests that sensory neurons modulate neural or neuroendocrine functions, regulating both bodily growth and behavioral state. We identify genes likely to be required for these functions downstream of sensory inputs. Here, we characterize one of these genes as egl-4, which we show encodes a cGMP-dependent protein kinase. We demonstrate that this cGMP-dependent kinase functions in neurons of C. elegans to regulate multiple developmental and behavioral processes including the orchestrated growth of the animal and the expression of particular behavioral states.