Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation‐contraction coupling

Excitation‐contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca²⁺ channels, dihydropyridine receptor (DHPR; voltage‐dependent L‐type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine‐rich domain‐containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α_1S subunit known as the II–III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II–III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II–III loop. Therefore, this work demonstrates an isoform‐specific interaction between STAC3 and the II–III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.     

Trypanosoma cruzi specific antibody response in immunized C3H(He) mice during infection

C3H(He) mice previously immunized with live culture derived Corpus Christi strain T. cruzi are significantly protected (up to 100% survival) against challenge by Brazil strain blood trypanosomes. The antibody response, directed against the Brazil strain or the Corpus Christi strain, in these mice has been observed by comparing sera from mice immunized only, infected only, or immunized and infected. The anti- T. cruzi titers determined by both direct agglutination (DA) and indirect fluorescence (IFA) were routinely found to be highest for immunized and infected mice with immunized mice and infected mice following in decreasing order. The use of mercaptoethanol treatment of sera (DA) and isotope specific second antibody (IFA) showed that IgG is the major parasite specific immunoglobulin response through infection. Evidence of cross-reacting antigens on the two parasite strains was found. By both DA and IFA, 11 of 18 anti-Brazil strain monoclonal antibodies were found to react (IFA titers of 320 or greater) with both parasite strains. No evidence of localization of cross-reacting antigens (using mouse antisera) or antigenic determinants (using monoclonal antibodies) was found in that uniform fluorescence over the parasite was observed in all IFA tests.     

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Neuronal autoantibody titers in the course of small-cell lung carcinoma and platinum-associated neuropathy

The aims of this study were to investigate, in patients with newly diagnosed small-cell lung carcinoma (SCLC), whether or not there may be a relationship between the presence, type or titer of circulating neuronal autoantibodies and (i) the extent of SCLC dissemination at presentation, (ii) the development of peripheral neuropathy during platinum chemotherapy, (iii) survival time. We studied stored serum from 58 patients with uncomplicated SCLC who had participated in two trials conducted by the North Central Cancer Treatment Group (NCCTG); 29 had extensive disease and 29 had limited disease. No patient had neuropathy or other neurological or paraneoplastic problems at the time of enrollment but each group included 14 or 15 patients respectively who developed peripheral neuropathy in the course of chemotherapy. We tested five consecutive serum specimens from each patient in blinded fashion by (i) an indirect immunofluorescence assay optimized to detect neuron-restricted nuclear and cytoplasmic antibodies (triple substrate of mouse cerebellum, gut and kidney), and (ii) immunoprecipitation assays to detect neuronal Ca²⁺-channel-binding antibodies (N-type and P/Q-type). Sera that were positive by immunofluorescence were analyzed further by Western blotting. Neuronal autoantibodies were significantly more frequent in patients who had limited SCLC at presentation (12/29 or 41% positive) than in those with extensive SCLC (5/29 or 17% positive, P = 0.02). Neuronal autoantibodies of nuclear or cytoplasmic specificity were found in 50% of the seropositive patients with limited SCLC (21% of the total group), but in no patient with extensive SCLC (P = 0.01). The frequency of neuronal autoantibodies did not differ significantly among patients who did and did not develop peripheral neuropathy. Titers fell progressively during chemotherapy and did not rise again when peripheral neuropathy became clinically evident. This argues against a synergism between drug toxicity and neuronal autoimmunity as the mechanism of platinum-associated peripheral neuropathy. Seropositivity for neuronal autoantibodies did not affect the survival of patients with either limited or extensive SCLC. It is conceivable that the immunosuppression attendant on combined cisplatin/etoposide therapy cancels a pre-existing protective antitumor immune response (presumably cytotoxic-T-cell-mediated) for which the nuclear and cytoplasmic paraneoplastic IgG autoantibodies serve as a surrogate marker. Testing of this hypothesis would require the survival of seropositive and seronegative patients to be compared in a larger trial, using a therapeutic modality that does not compromise immunocompetence. Received: 20 November 1998 / Accepted: 6 January 1999     

Identification of nonsense mutations in Neurospora: Application to the complex arg-6 locus

Summary The arg-6 locus of Neurospora crassa encodes two enzymes of arginine synthesis, acetylglutamate kinase and acetylglutamyl phosphate reductase. Mutants lacking one or the other enzyme fall into two different complementation groups; a large non-complementing group lacks both enzymes. We wished to survey over 50 alleles for suppressibility by a nonsense suppressor. We compared two methods of assessing suppressibility. One, based on trans-action of a nonsense suppressor, was simple, but not efficient in detecting all suppressible alleles. The other, based on crosses involving a marker linked to the locus surveyed was very efficient in methodology and is suited to all cases, such as the arg-6 locus, in which allelic crosses are sterile. The data indicate that the arg-6 locus encodes a bifunctional protein.     

ART: mathematics and applications. A report on the mathematical foundations and on the applicability to real data of the algebraic reconstruction techniques

Some properties of the algebraic reconstruction techniques (ART) for reconstructing objects from their projections (e.g. electron micrographs) are discussed. Some generalizations of previously published ART algorithms are given. In particular, ART is extended to handle weighted projection data. An early conjecture about ART is proved for one of the algorithms: it converges to the most uniform solution of the constraint equations provided by the projection data. Other convergence properties of the ART algorithms are discussed and proved. Some new ART algorithms are described. These are believed to converge to optimal reconstructions consistent with the projection data. The importance of choosing the correct ray widths in case of real projection data is demonstrated, and a method for calculating correct ray widths is given. A method is proposed for estimating the optimal number of iterations in a reconstruction. The performance of ART on real data is demonstrated both in the absence of and in the presence of noise.     

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli . We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gels. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE^22–88 fragment forms hetero-oligomers with MinE⁺ when the proteins are co-expressed. In contrast, the MinE^36–88 fragment does not form MinE⁺/MinE^36–88 hetero-oligomers, although MinE^36–88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE⁺ in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of minicelling by expression of MinE^36–88 in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE^36–88 and the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.     

Application of Methods for Mammalian Chromosome Spreads to the Cells of Cestodes

Tapeworm cells obtained by physical maceration between ground-glass surfaces are incubated for 3 hr in Hanks' balanced salt solution (BSS) supplemented with colchicine to a concentration of 10-⁴ M. After washing in BSS, the cells are incubated for 10 min in 1/4 strength BSS then centrifuged 10 min. Fixation of the intact button of cells (or alternatively, by squirting the cells directly into the fixative) in Carnoy's alcohol-chloroform-acetic acid (6;3:1) for 30 min follows, and cells, dispersed and washed in the fixative, are flattened by dropping the suspension on clean, water-wet slides which are then air-dried and stained with Giemsa diluted 1 ml;47 ml with distilled water to which 2 ml of buffer—M/15 KH₂PO₄, 32 ml, mixed with M/15 Na₂HPO₄, 68 ml—is added. After staining 15 min and washing in distilled water, slides are air-dried and mounted with resin. Well separated and well stained chromosomes have resulted.     

Soil properties and geomorphic processes influence vegetation composition,structure, and function in the Cerrado Domain

Plant and Soil - The Cerrado of central Brazil—the world’s largest Neotropical savanna – is comprised of a mosaic of highly heterogeneous vegetation growing on an extremely...