Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These “muscle-specific” isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and/or heart are affected.

Nesprins are a family of nuclear transmembrane proteins anchored via Sun proteins to the nuclear membrane. Analysis of nesprins during human muscle development revealed an increase in nesprin-1-giant during early myogenesis in vitro. During the transition from immature to mature muscle fibres in vivo, nesprin-2 partly replaced nesprin-1 at the nuclear envelope and short nesprin isoforms became dominant. Sun1 and Sun2 proteins remained unchanged during this fibre maturation. In emerin-negative skin fibroblasts, nesprin-2-giant was relocated from the nuclear envelope to the cytoplasm, not to the endoplasmic reticulum, while nesprin-1 remained at the nuclear envelope. In emerin-negative keratinocytes lacking nesprin-1, nesprin-2 remained at the nuclear envelope. HeLa cell nuclear envelopes lacked nesprin-1, which was the dominant form in myoblasts, while a novel 130kD nesprin-2 isoform dominated Ntera-2 cells. The results suggest the possibility of isoform-specific and tissue-specific roles for nesprins in nuclear positioning.

The neurogenic loci of Drosophila are required for proper partitioning of ectodermal cells into epidermal versus neural lineages. The loci appear to encode components of a developmental pathway involving cellular communication. In an effort to understand the role of the neurogenic locus mastermind in these processes, we have characterized its expression and sequence. The locus produces a number of transcripts that accumulate ubiquitously during early embryogenesis but more specifically in the central nervous system during later stages. Sequence analysis of a major cDNA product predicts an unusual protein containing an abundance of amino acid homopolymers and charge clusters typical of regulatory molecules. Nearly half of the mass of the predicted protein derives from only three amino acids: glutamine, glycine, and asparagine. Immunohistochemical studies of the protein in cell culture and early embryos show that the protein accumulates predominately in the nucleus.

Persons occupationally exposed to nonhuman primates (NHPs) can be persistently infected with simian foamy virus (SFV). The clinical significance and person-to-person transmissibility of zoonotic SFV infection is unclear. Seven SFV-infected men responded to annual structured interviews and provided whole blood, oral, and urogenital specimens for study. Wives were tested for SFV infection. Proviral DNA was consistently detected by PCR in PBMCs of infected men and inconsistently in oral or urogenital samples. SFV was infrequently cultured from their PBMCs and throat swabs. Despite this and a long period of intimate exposure (median 20 years), wives were SFV negative. Most participants reported nonspecific symptoms and diseases common to aging. However, one of two persons with mild thrombocytopenia had clinically asymptomatic nonprogressive, monoclonal natural killer cell lymphocytosis of unclear relationship to SFV. All participants worked with NHPs before 1988 using mucocutaneous protection inconsistently; 57% described percutaneous injuries involving the infecting NHP species. SFV likely transmits to humans through both percutaneous and mucocutaneous exposures to NHP body fluids. Limited follow-up has not identified SFV-associated illness and secondary transmission among humans.

Plakoglobin (c-catenin) is a homolog of b-catenin with similar dual adhesive and signaling functions. The adhesive function of these proteins is mediated by their interactions with cadherins, whereas their signaling activity is regulated by association with various intracellular partners. In this respect, b-catenin has a well-defined oncogenic activity through its role in the Wnt signaling pathway, whereas plakoglobin acts as a tumor/metastasis suppressor through mechanisms that remain unclear. We previously expressed plakoglobin in SCC9 squamous carcinoma cells (SCC9-P) and observed a mesenchymalto-epidermoid transition. Comparison of the protein and RNA profiles of parental SCC9 cells and SCC9-P transfectants identified various differentially expressed proteins and transcripts, including the nonmetastatic protein 23 (Nm23). In this study, we show that Nm23-H1 mRNA and Nm23-H2 protein are increased after plakoglobin expression. Coimmunoprecipitation and confocal microscopy studies using SCC9-P and various epithelial cell lines with endogenous plakoglobin expression revealed that Nm23 interacts with plakoglobin, cadherins and a-catenin. Furthermore, Nm23-H2 is the primary isoform involved in these interactions, which occur prominently in the cytoskeleton-associated pool of cellular proteins. In addition, we show that plakoglobinNm23 interaction requires the N-terminal (a-catenin interacting) domain of plakoglobin. Our data suggest that by increasing the expression and stability of Nm23, plakoglobin has a role in regulating the metastasis suppressor activity of Nm23, which may further provide a potential mechanism for the tumor/metastasis suppressor function of plakoglobin itself.

Background: The global HIV/AIDS pandemic and guidelines for initiating anti-retroviral therapy (ART)and opportunistic infection prophylaxis demand affordable, reliable, and accurate CD4 testing. A simple innovative approach applicable to existing technology that has been successfully applied in resourcechallenged settings, PanLeukogated CD4 (PLG), could offer solutions for cost saving and improved precision.Methods: Day-old whole blood from 99 HIV+ donors was simultaneously studied in five North-American laboratories to compare the performance of their predicate methods with the dual-platform PLG method. The predicate technology included varying 4-color CD45/CD3/CD4/CD8 protocols on different flow cytometers. Each laboratory also assayed eight replicate specimens of day-old blood from 10 to 14 local donors. Bias and precision of predicate and PLG methods was studied between-and within-participating laboratories.Results: Significantly (P < 0.0001) improved between-laboratory precision/coefficient of variation (CV%) was noted using the PLG method (overall median 9.3% vs. predicate median CV 13.1%). Within-laboratory precision was also significantly (P < 0.0001) better overall using PLG (median

Myotonic dystrophy (DM) is a multisystemic disorder caused by an inherited CTG repeat expansion which affects three genes encoding the DM protein kinase (DMPK), a homeobox protein Six5 and a protein containing WD repeats. Using a panel of 16 monoclonal antibodies against several different DMPK epitopes we detected DMPK, as a single protein of approximately 80 kDa, only in skeletal muscle, cardiac muscle and, to a lesser extent, smooth muscle. Many earlier reports of DMPK with different sizes and tissue distributions appear to be due to antibody cross-reactions with more abundant proteins. One such antibody, MANDM1, was used to isolate two related protein kinases, MRCK alpha and beta, from a human brain cDNA library and the shared epitope was located at the catalytic site of DMPK using a phage-displayed random peptide library. The peptide library also identified an epitope shared between DMPK and a 55 kDa muscle-specific protein. The results suggest that effects of the repeat expansion on the DMPK gene may be responsible for muscle and heart features of DM, whereas clinical changes in other tissues may be due to effects on the other two genes.

Myotonic dystrophy (DM1) is caused by the expansion of a CTG repeat in the noncoding region of a protein kinase, DMPK, expressed in skeletal and cardiac muscles. The aim of the present study was to determine the effects of very large CTG expansions on DMPK expression and skeletal muscle development. In fetuses suffering from the severe congenital form of DM1 with large CTG expansions (1800 to 3700 repeats), the skeletal muscle level of DMPK was reduced to 57% of control levels and a similar reduction was observed in cultured DM1 muscle cells relative to control cultures. These results are consistent with greatly reduced DMPK expression from the mutant allele and normal expression from the unaffected allele in this autosomal dominant disorder. In normal fetuses, DMPK protein levels increased dramatically between 9 and 16 weeks and remained high throughout the remaining gestation period. DM1 fetuses showed impaired skeletal muscle development, characterized by a persistence of embryonic and fetal myosin heavy chains and almost total absence of slow myosin heavy chains at the end of gestation. DMPK expression, however, was similar in both fast and slow fibers from normal adult muscle. The reduced DMPK and the delayed slow fiber maturation in congenital DM1 may be two separate consequences of nuclear retention of DMPK RNA transcripts with expanded CUG repeats.