Desmuslin, an intermediate filament protein that interacts with α-dystrobrevin and desmin

Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, α1-syntrophin, and the sarcoglycan complex. The precise role of α-dystrobrevin in skeletal muscle has not yet been determined. To study α-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and α-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.

Characterization of Zinc Uptake, Binding, and Translocation in Intact Seedlings of Bread and Durum Wheat Cultivars

Durum wheat (Triticum turgidum L. var durum) cultivars exhibit lower Zn efficiency than comparable bread wheat (Triticum aestivum L.) cultivars. To understand the physiological mechanism(s) that confers Zn efficiency, this study used 65Zn to investigate ionic Zn2+ root uptake, binding, and translocation to shoots in seedlings of bread and durum wheat cultivars. Time-dependent Zn2+ accumulation during 90 min was greater in roots of the bread wheat cultivar. Zn2+ cell wall binding was not different in the two cultivars. In each cultivar, concentration-dependent Zn2+ influx was characterized by a smooth, saturating curve, suggesting a carrier-mediated uptake system. At very low solution Zn2+ activities, Zn2+ uptake rates were higher in the bread wheat cultivar. As a result, the Michaelis constant for Zn2+ uptake was lower in the bread wheat cultivar (2.3 μm) than in the durum wheat cultivar (3.9 μm). Low temperature decreased the rate of Zn2+ influx, suggesting that metabolism plays a role in Zn2+ uptake. Ca inhibited Zn2+ uptake equally in both cultivars. Translocation of Zn to shoots was greater in the bread wheat cultivar, reflecting the higher root uptake rates. The study suggests that lower root Zn2+ uptake rates may contribute to reduced Zn efficiency in durum wheat varieties under Zn-limiting conditions.

Characterization of Cadmium Binding, Uptake, and Translocation in Intact Seedlings of Bread and Durum Wheat Cultivars

High Cd content in durum wheat (Triticum turgidum L. var durum) grain grown in the United States and Canada presents potential health and economic problems for consumers and growers. In an effort to understand the biological processes that result in excess Cd accumulation, root Cd uptake and xylem translocation to shoots in seedlings of bread wheat (Triticum aestivum L.) and durum wheat cultivars were studied. Whole-plant Cd accumulation was somewhat greater in the bread wheat cultivar, but this was probably because of increased apoplastic Cd binding. Concentration-dependent 109Cd2+-influx kinetics in both cultivars were characterized by smooth, nonsaturating curves that could be dissected into linear and saturable components. The saturable component likely represented carrier-mediated Cd influx across root-cell plasma membranes (Michaelis constant, 20–40 nm; maximum initial velocity, 26–29 nmol g−1 fresh weight h−1), whereas linear Cd uptake represented cell wall binding of 109Cd. Cd translocation to shoots was greater in the bread wheat cultivar than in the durum cultivar because a larger proportion of root-absorbed Cd moved to shoots. Our results indicate that excess Cd accumulation in durum wheat grain is not correlated with seedling-root influx rates or root-to-shoot translocation, but may be related to phloem-mediated Cd transport to the grain.

Constitutive receptor activation by Crouzon syndrome mutations in fibroblast growth factor receptor (FGFR)2 and FGFR2/Neu chimeras.

Crouzon syndrome is an autosomal dominant condition primarily characterized by craniosynostosis. This syndrome has been associated with a variety of amino acid point mutations in the extracellular domain of fibroblast growth factor receptor 2 (FGFR2). FGFR2/Neu chimeras were generated by substituting the extracellular domain of Neu with that of FGFR2 containing the following Crouzon mutations: Tyr-340-->His; Cys-342-->Tyr; Cys-342-->Arg; Cys-342-->Ser; Ser-354-->Cys: and delta17 (deletion of amino acids 345-361). Each of the mutant chimeric FGFR2/Neu constructs stimulated focus formation in NIH 3T3 cells, indicating that Crouzon mutations can stimulate signal transduction through a heterologous receptor tyrosine kinase. In vitro kinase assay results indicate that FGFR2 receptors containing Crouzon mutations have increased tyrosine kinase activity and, when analyzed under nonreducing conditions, exhibited disulfide-bonded dimers. Thus the human developmental abnormality Crouzon syndrome arises from constitutive activation of FGFR2 due to aberrant intermolecular disulfide-bonding. These results together with our earlier observation that achondroplasia results from constitutive activation of the related receptor FGFR3, leads to the prediction that other malformation syndromes attributed to FGFRs, such as Pfeiffer syndrome and Thanatophoric dysplasia, also arise from constitutive receptor activation.

Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart.

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

Fine mapping of colon tumor susceptibility (Scc) genes in the mouse, different from the genes known to be somatically mutated in colon cancer.

The predisposition to colon cancer is multigenetically controlled in animals and probably also in humans. We have analyzed the multigenic control of susceptibility to 1,2-dimethylhydrazine-induced colon tumors in mice by using a set of 20 homozygous CcS/Dem recombinant congenic strains, each of which contains a different random subset of approximately 12.5% of genes from the susceptible strain STS/A and 87.5% of genes from the relatively resistant strain BALB/cHeA. Some CcS/Dem strains received the alleles from the susceptible strain STS/A at one or more of the multiple colon tumor susceptibility loci and are susceptible, whereas others are resistant. Linkage analysis shows that these susceptibility genes are different from the mouse homologs of the genes known to be somatically mutated in human colon cancer (KRAS2, TP53, DCC, MCC, APC, MSH2, and probably also MLH1). Different subsets of genes control tumor numbers and size. Two colon cancer susceptibility genes, Scc1 and Scc2, map to mouse chromosome 2. The Scc1 locus has been mapped to a narrow region of 2.4 centimorgans (90% confidence interval).

Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13.

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library. Human StAR, coexpressed in COS-1 cells with cytochrome P450scc and adrenodoxin, increased pregnenolone synthesis > 4-fold. A major StAR transcript of 1.6 kb and less abundant transcripts of 4.4 and 7.5 kb were detected in ovary and testis. Kidney had a lower amount of the 1.6-kb message. StAR mRNA was not detected in other tissues including placenta. Treatment of granulosa cells with 8-bromo-adenosine 3',5'-cyclic monophosphate for 24 hr increased StAR mRNA 3-fold or more. The structural gene encoding StAR was mapped using somatic cell hybrid mapping panels to chromosome 8p. Fluorescence in situ hybridization placed the StAR locus in the region 8p11.2. A StAR pseudogene was mapped to chromosome 13. We conclude that StAR expression is restricted to tissues that carry out mitochondrial sterol oxidations subject to acute regulation by cAMP and that StAR mRNA levels are regulated by cAMP.

Glycosylation of the c-Myc transactivation domain.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic posttranslational modification composed of a single monosaccharide, GlcNAc, glycosidically composed of a single monosaccharide, GlcNAc, glycosidically linked to the side-chain hydroxyl of serine or threonine residues. Although O-GlcNAc occurs on a myriad of nuclear and cytoplasmic proteins, only a few have thus far been identified. These O-GlcNAc-bearing proteins are also modified by phosphorylation and form reversible multimeric complexes. Here we present evidence for O-GlcNAc glycosylation of the oncoprotein c-Myc, a helix-loop-helix/leucine zipper phosphoprotein that heterodimerizes with Max and participates in the regulation of gene transcription in normal and neoplastic cells. O-GlcNAc modification of c-Myc is shown by three different methods: (i) demonstration of lectin binding to in vitro translated protein using a protein-protein interaction mobility-shift assay; (ii) glycosidase or glycosyltransferase treatment of in vitro translated protein analyzed by lectin affinity chromatography; and (iii) direct characterization of the sugar moieties on purified recombinant protein overexpressed in either insect cells or Chinese hamster ovary cells. Analyses of serial deletion mutants of c-Myc further suggest that the O-GlcNAc site(s) are located within or near the N-terminal transcription activation/malignant transformation domain, a region where mutations of c-Myc that are frequently found in Burkitt and AIDS-related lymphomas cluster.