The role of annuloplasty in mitral valve repair.

Mitral valve repair surgery has progressed dramatically since its inception over 40 years ago. As techniques have evolved, complicated mitral valve reconstruction has become commonplace, with durable late results. Likewise, the value of concomitant annuloplasty during valve repair has been firmly established as contributing to late valve repair durability. This review discusses the evolution of annuloplasty techniques and the physiologic reasoning behind various approaches.

The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases.

Receptor tyrosine kinases (RTKs) transduce signals via cytoplasmic adaptor proteins to downstream signaling components. We have identified loss-of-function mutations in dshc, the Drosophila homolog of the mammalian adaptor protein SHC. A point mutation in the phosphotyrosine binding (PTB) domain completely abolishes DSHC function and provides in vivo evidence for the function of PTB domains. Unlike other adaptor proteins, DSHC is involved in signaling by only a subset of RTKs: dshc mutants show defects in Torso and DER but not Sevenless signaling, which is confirmed by epistasis experiments. We show by double-mutant analysis that the adaptors DOS, DRK, and DSHC act in parallel to transduce the Torso signal. Our results suggest that DSHC confers specificity to receptor signaling.

Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification.

Work in different organisms revealed that the vasa gene product is essential for germline specification. Here, we describe the asymmetric segregation of zebrafish vasa RNA, which distinguishes germ cell precursors from somatic cells in cleavage stage embryos. At the late blastula (sphere) stage, vasa mRNA segregation changes from asymmetric to symmetric, a process that precedes primordial germ cell proliferation and perinuclear localization of Vasa protein. Analysis of hybrid fish between Danio rerio and Danio feegradei demonstrates that zygotic vasa transcription is initiated shortly after the loss of unequal vasa mRNA segregation. Blocking DNA replication indicates that the change in vasa RNA segregation is dependent on a maternal program. Asymmetric segregation is impaired in embryos mutant for the maternal effect gene nebel. Furthermore, ultrastructural analysis of vasa RNA particles reveals that vasa RNA, but not Vasa protein, localizes to a subcellular structure that resembles nuage, a germ plasm organelle. The structure is initially associated with the actin cortex, and subsequent aggregation is inhibited by actin depolymerization. Later, the structure is found in close proximity of microtubules. We previously showed that its translocation to the distal furrows is microtubule dependent. We propose that vasa RNA but not Vasa protein is a component of the zebrafish germ plasm. Triggered by maternal signals, the pattern of germ plasm segregation changes, which results in the expression of primordial germ cell-specific genes such as vasa and, consequently, in germline fate commitment.

The molecular motor dynein is involved in targeting swallow and bicoid RNA to the anterior pole of Drosophila oocytes.

Localization of bicoid (bcd) messenger RNA to the anterior pole of the Drosophila oocyte requires the exuperantia ( exu), swallow (swa) and staufen (stau) genes. We show here that Swa protein transiently co-localizes with bcd RNA in mid-oogenesis. Swa also localizes to the anterior pole of the oocyte in the absence of bcd RNA. This localization does not require Exu, but depends on intact microtubules. In mutant ovaries with duplicated polarity of microtubules, Swa and bcd RNA are ectopically localized at the posterior pole, as well as being present at the anterior pole. We identify dynein light chain-1 (Ddlc-1), a component of the minus-end-directed microtubule motor cytoplasmic dynein, as a Swa-binding protein. We propose that Swa acts as an adaptor for the dynein complex and thereby enables dynein to transport bcd RNA along microtubules to their minus ends at the anterior pole of the oocyte.

Inhibition of protein dephosphorylation results in the accumulation of splicing snRNPs and coiled bodies within the nucleolus.

Coiled bodies are conserved subnuclear organelles that contain splicing snRNPs, a subset of nucleolar antigens, and the autoantigen p80 coilin. Most nuclei contain one to five nucleoplasmic coiled bodies, often with one or more located at the nucleolar periphery. Here we show that exposure of mammalian cells to low levels of the specific Ser/Thr protein phosphatase inhibitor, okadaic acid, results in the accumulation of p80 coilin and splicing snRNPs within nucleoli. Mutation of a single serine residue in p80 coilin to aspartate (S202D) also causes coiled bodies and splicing snRNPs to localize within nucleoli when the mutant is transiently transfected and expressed in HeLa cells. Neither okadaic acid nor the S202D coilin mutant causes nucleolar accumulation of serine-arginine-domain proteins. These data indicate that protein dephosphorylation is required to allow normal formation of nucleoplasmic coiled bodies and point to p80 coilin as a substrate whose phosphorylation state may regulate snRNP-nucleolar interactions. The data are consistent with a trafficking mechanism whereby splicing snRNPs cycle through the nucleolus.

The dynamics of coiled bodies in the nucleus of adenovirus-infected cells.

The coiled body is a specific intranuclear structure of unknown function that is enriched in splicing small nuclear ribonucleoproteins (snRNPs). Because adenoviruses make use of the host cell-splicing machinery and subvert the normal subnuclear organization, we initially decided to investigate the effect of adenovirus infection on the coiled body. The results indicate that adenovirus infection induces the disassembly of coiled bodies and that this effect is probably secondary to the block of host protein synthesis induced by the virus. Furthermore, coiled bodies are shown to be very labile structures, with a half-life of approximately 2 h after treatment of HeLa cells with protein synthesis inhibitors. After blocking of protein synthesis, p80 coilin was detected in numerous microfoci that do not concentrate snRNP. These structures may represent precursor forms of the coiled body, which goes through a rapid cycle of assembly/disassembly in the nucleus and requires ongoing protein synthesis to reassemble.

Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus.

Coiled bodies are conserved subnuclear domains found in both plant and animal cells. They contain a subset of splicing snRNPs and several nucleolar antigens, including Nopp140 and fibrillarin. In addition, autoimmune patient sera have identified a coiled body specific protein, called p80 coilin. In this study we show that p80 coilin is ubiquitously expressed in human tissues. The full-length human p80 coilin protein correctly localizes in coiled bodies when exogenously expressed in HeLa cells using a transient transfection assay. Mutational analysis identifies separate domains in the p80 coilin protein that differentially affect its subnuclear localization. The data show that p80 coilin has a nuclear localization signal, but this is not sufficient to target the protein to coiled bodies. The results indicate that localization in coiled bodies is not determined by a simple motif analogous to the NLS motifs involved in nuclear import. A specific carboxy-terminal deletion in p80 coilin results in the formation of pseudo-coiled bodies that are unable to recruit splicing snRNPs. This causes a loss of endogenous coiled bodies. A separate class of mutant coilin proteins are shown to localize in fibrillar structures that surround nucleoli. These mutants also lead to loss of endogenous coiled bodies, produce a dramatic disruption of nucleolar architecture and cause a specific segregation of nucleolar antigens. The structural change in nucleoli is accompanied by the loss of RNA polymerase I activity. These data indicate that p80 coilin plays an important role in subnuclear organization and suggest that there may be a functional interaction between coiled bodies and nucleoli.

Molecular analysis of the coiled body.

There is increasing interest in studying how specific metabolic activities within the nucleus are organised into functional domains. The best known example is the nucleolus where rRNA genes are transcribed and rRNA processed and assembled into ribosomal units. Other subnuclear domains have been known for many years through morphological studies but are only recently being analysed at the molecular level. Here we focus on an evolutionarily conserved nuclear domain, called the coiled body, which contains splicing snRNPs. We review recent literature on the coiled body and discuss a possible model for its biological function.

Determining the effect of inducible protein phosphorylation on the DNA-binding activity of transcription factors.

Inducible phosphorylation or dephosphorylation of transcription factors is an important mechanism of signal-dependent gene regulation in eukaryotic cells. This paper describes a combination of techniques that can be used to study the effect of this covalent protein modification on the DNA-binding activity of transcription factors. The protein of interest is genetically tagged with oligohistidine to allow rapid purification on nickel-chelate columns after being transiently overexpressed in eukaryotic tissue culture cells. Phosphorylation-dependent DNA-binding activity is determined in a blotting assay including an in situ dephosphorylation step. Studies on the protooncogene-encoded transcription factor c-Jun employing this assay revealed a TPA-inducible protein dephosphorylation event that strongly increases the DNA-binding potential of the protein. Our data confirm the results published recently by others and provide a rapid, efficient, sensitive, and well-controlled experimental system to analyze the phosphorylation-regulated modulations in the DNA-binding activity of transcription factors.

2'-O-alkyloligoribonucleotides, synthesis and applications in molecular biology.

Oligo(2'-O-alkylribonucleotides) have been synthesized in which alkyl is methyl, allyl and butyl. The various phosphoramidite monomers of 2'-O-alkyl uridine, cytidine, adenosine, guanosine, inosine and 2,6-diaminopurine riboside have been synthesized from a minimum of key intermediates. Extra protection of the lactam function in uracil and hypoxanthine proves useful. The high stability of the oligomers combined with incorporation of non-radioactive reporter groups such as fluorophores, biotin and 2,4-dinitrophenylamino (DNP) moities renders them as excellent antisense tools for studying RNA processing, for locating and visualising RNA and RNP complexes in cells, for examining splicing complexes by electron microscopy and for the affinity chromatography of RNA or RNP complexes.