Intracellular vesicle movement, cAMP and myosin II in Dictyostelium

Dictyostelium amoebae were analyzed before and after rapid addition of 10(-6) M cAMP for cellular motility, dynamic shape changes, and intracellular particle movement. Before cAMP addition, amoebae moved in a persistent anterior fashion and were elongate with F-actin localized predominantly in the anterior pseudopod. Intracellular particles moved rapidly and anteriorly. Within seconds after 10(-6) M cAMP addition, cells stopped translocating, pseudopod formation ceased, intracellular particle movement was depressed, and F-actin was lost from the pseudopod and concomitantly relocalized in the cell cortex. After 10 seconds, expansion zones reappeared but were small and no longer anteriorly localized. Vesicle movement partially rebounded but was no longer anteriorly directed. The myosin II null mutant HS2215 exhibited both depressed cellular translocation and vesicle movement. The addition of cAMP to HS2215 cells did not result in any detectable change in the random, depressed movement of particles. The results with HS2215 suggest that myosin II is essential for (1) rapid cellular translocation, (2) cellular polarity, (3) rapid particle movement, (4) anteriorly directed particle movement, and (5) the cAMP response. Electron micrographs suggest that at least half of the particles examined in this study contain in turn smaller membrane bound vesicles or multilamellar membrane bodies. The possible role of these vesicles is discussed.     

The mATPase histochemical profile of rat type IIX fibres: correlation with myosin heavy chain immunolabelling

Summary In the present study we report a novel histochemical method which, by sequential pre-incubations in alkaline and acidic media, selectively differentiates muscle fibres expressing myosin heavy chain IIX, on the basis of a specific profile for myofibrillar actomyosin ATPase (mATPase) activity. The enzyme reactions were tested for specificity by means of anti-myosin heavy chain monoclonal antibodies, which were characterized on Western blots of muscle homogenates. Enzyme histochemical reactions with the traditional pH buffers were compared to those of the new method and, in conjunction with the immunoreactions, used to confirm the relationship between MyHC expression and the distinct profiles for mATPase. Imrnunohistochemical reactions demonstrated that the new method only differentiates those fibres expressing myosin heavy chain IIX. The method revealed a continuum in which the intermediate staining intensities corresponded to hybrid fibres expressing myosin heavy chain IIX in combination with either the IIA or IIB forms. Quantitative histochemistry and immunohistochemistry (by image analysis), used to examine the relationship between staining intensities for mATPase and amounts of myosin heavy chain IIX expression, revealed that the new method discriminates well between hybrid fibres expressing variable amounts of the IIX isoform (r² = 0.93).     

Evidence for a phospholipid requirement of chitin synthase inSchizophyllum commune

Evidence for a lipid dependence of membrane-associated chitin synthase inSchizophyllum commune is based on the following observations: Arrhenius plots of the temperature dependence of this enzyme showed deflections from linearity that are characteristic for lipid-affected membrane-bound enzymes. The activity of chitin synthase dissociated by digitonin decreased at increasing digitonin/protein ratios and could be restored by addition of egg lecithin. After further delipification by sucrose gradient centrifugation, enzyme activity progressively decreased, banded at higher densities, and was less effectively restored by lecithin. The activity of dissociated chitin synthase was also restored by soybean phosphatidylcholine and low concentrations of phosphatidylinositol and phosphatidylserine. At higher concentrations, phosphatidylinositol and phosphatidylserine were inhibitory. Lysophosphatidylcholine and phosphatidylethanolamine were slightly stimulatory, whereas no effect resulting from ergosterol was observed.     

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.     

Role of β-carotene in the reaction centres of Photosystems I and II of spinach chloroplasts prepared in non-polar solvents

Spinach chloroplasts have been prepared nonaqueously using non-polar solvents (n-hexane, CCl₄, n-heptane) and the β-carotene content extracted in a controlled manner. This procedure is reproducible and does not result in large structural or spectral changes of the chloroplasts. The organisation of the chlorophyll-proteins is unaltered, as fragmentation with digitonin results in the appearance of the same fractions as found previously for aqueously-prepared chloroplasts, including the pink zone containing cytochromes f and b₆ in the ratio 1:2. The chloroplasts possess both Photosystem I activity (P-700 photo-bleaching, and NADP⁺ photoreduction) and Photosystem II activity (parabenzoquinone reduction with Mn²⁺ as electron donor, and chlorophyll fluorescence induction). Use of moderate intensity red illumination has allowed a study of the role of β-carotene in photochemistry separate from its roles in energy transfer and photoprotection.Removal of the fraction of β-carotene closely associated with the Photo-system I reaction centre caused the rate of NADP⁺ photoreduction to fall to a low, but significantly non-zero level. Thus, in the complete absence of β-carotene, photochemistry can still be observed, however the specific association of β-carotene with the reaction centre is required for maximal rates. We propose that β-carotene bound at the reaction centre decreases the rate of transfer of excitation energy away from the reaction centre, and increases the rate of photochemistry. It is possible that this occurs via formation of an exciplex between ground state β-carotene and chlorophyll in the first excited state.     

Association between the ACCN1 gene and multiple sclerosis in Central East Sardinia

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3' untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3' UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.     

Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

Background  

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.