Autoimmune thyroid disease in anti-Ro/SS-A-positive children with annular erythema: report of two cases.

Anti-Ro/SS-A-associated recurrent annular erythema is a rare disorder, and represents a cutaneous manifestation of primary Sjögren's syndrome (SS). We report two childhood cases complicated with autoimmune thyroid disease, one with Graves' disease and the other with autoimmune thyroiditis. Both children were positive for anti-Ro/SS-A and anti-La/SS-B antibodies.

Proteomic analysis of the TGF-beta signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression.

Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-beta (TGF-beta) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-beta-induced growth inhibition, there seems to be a Smad4-independent TGF-beta signaling pathway.

Zoonotic risk of Toxocara canis infection through consumption of pig or poultry viscera.

The potential zoonotic risk of Toxocara canis infections from consumption of swine or poultry viscera containing larvae was assessed using a pig model. Two groups of six pigs were fed either fresh swine viscera (group FS) or poultry viscera (FP) containing around 3500 Toxocara larvae. Another two groups of six pigs were fed swine viscera (PS) or poultry viscera (PP) preserved at 4 degrees C for 1 week.

Alternative reading frame protein (ARF)-independent function of CARF (collaborator of ARF) involves its interactions with p53: evidence for a novel p53-activation pathway and its negative feedback control.

CARF, a collaborator of ARF (alternative reading frame protein), was cloned as a novel ARF-binding protein from a yeast-interaction screen. It potentiated ARF-mediated p53 function, and also caused a moderate increase in p53 activity in the absence of ARF. We herein report the molecular mechanism of ARF-independent function of CARF.

Control of siRNA expression using the Cre-loxP recombination system.

Gene silencing mediated by RNA interference (RNAi) was first discovered in Caenorhabditis elegans, and was subsequently recognized in various other organisms. In mammalian cells, RNAi can be induced by small interfering RNAs (siRNAs). In earlier studies, our group developed a vector-based system for expression of siRNA under control of a polymerase III promoter, the U6 promoter, which can induce RNAi in living cells.

Reduction in mortalin level by its antisense expression causes senescence-like growth arrest in human immortalized cells.

Background: Human normal cells have active p53 and pRB tumor suppressor pathways and undergo telomere shortening at each cell division. When immortalized, these exhibit functional inactivation of one or both tumor suppressor pathways and activation of telomere-maintaining mechanisms. Regulation of immortalization promoting molecular pathways by other genes is poorly understood and is an essential component of cancer therapeutics.

Generation of an shRNAi expression library against the whole human transcripts.

RNA interference is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, homologous to the gene, and is receiving attention as a powerful tool for reverse genetics in the post-genome era. Throughout our current research to generate an siRNA expression library for the whole human genome, we face many technical difficulties. We present here the strategies for overcoming some of the difficulties, including the development of genetically stable and highly active siRNA expression vectors, the selection procedure of the favorable target sites, and the efficient and low cost procedure for constructing an siRNA expression library.

World of small RNAs: from ribozymes to siRNA and miRNA.

RNAs, besides bridging genetic information to proteins, the major determinants of bio-structures and functions, serve as active regulators of gene expression. Initiated nearly 20 years ago with ribozymes (the small RNAs with catalytic activity providing fine tuning of gene expression and function, used as molecular scissors and tools for gene discovery), an era of more complex and coordinated gene regulation by small RNAs, siRNA, and miRNA has recently started. Simple nucleotide complementarity results in highly ordered and regulated events, such as assembly of RNA and proteins, resulting in gene silencing either by mRNA degradation or suppression of translation.

Transglutaminase-mediated N- and C-terminal fluorescein labeling of a protein can support the native activity of the modified protein.

Fluorescein and its analogs are among the best fluorophores to label proteins and the labeling generally involves chemical modification of a translated protein. Using this methodology, labeling at a specific position remains difficult. It is known that the guinea pig liver transglutaminase (TGase)-catalyzed enzymatic modification method can allow terminal-specific fluorophore labeling of a protein by monodansylcadaverine.

A novel maxizyme vector targeting a bcr-abl fusion gene induced specific cell death in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Patients with Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) generally have a poor prognosis and would benefit from the development of new therapeutic approaches. We previously demonstrated that an allosterically controllable ribozyme, maxizyme (Mz), can induce apoptosis in chronic myelogenous leukemia (CML) cells. Ph(+) ALL cells harbor a bcrabl fusion gene (e1a2) encoding a 190-kDa fusion protein (p190) involved in disease pathogenesis.

A small modulatory dsRNA specifies the fate of adult neural stem cells.

Discovering the molecular mechanisms that regulate neuron-specific gene expression remains a central challenge for CNS research. Here, we report that small, noncoding double-stranded (ds) RNAs play a critical role in mediating neuronal differentiation. The sequence defined by this dsRNA is NRSE/RE1, which is recognized by NRSF/REST, known primarily as a negative transcriptional regulator that restricts neuronal gene expression to neurons.

LIM kinase-2 targeting as a possible anti-metastasis therapy.

Background: Metastatic properties of tumors involve movement of cancerous cells from one place to another and tissue invasion. Metastatic cells have altered cell adhesion and movement that can be examined by in vitro chemotaxis assays. The Rho/ROCK/LIM kinase pathway is one of the major signaling pathways involved in tumor metastasis.

RNAi expression vectors in plant cells.

Suppression by double-stranded RNA (dsRNA) of expression of a target gene is known as RNA interference (RNAi). Tobacco BY-2 cell suspension has been used as a model cultured plant cell, because it is possible to produce populations of tobacco BY-2 cell suspensions that are uniform and divide synchronously for functional gene analysis. Here, we describe a method to induce RNAi by introducing a hairpin-type dsRNA expression vector into BY-2 cells via electroporation.

RNAi expression vectors in mammalian cells.

RNA interference (RNAi) is a recently developed technique for gene silencing by introducing dsRNA into cells, and it is shown to work in mammalian cells when siRNAs are used. Several groups have developed vector-based siRNA expression systems that can induce RNAi in living cells. These vector systems use a pol III promoter, such as U6 or H1, and are classified into two groups based on the form of expressed RNAs: tandem-type and hairpin-type.

Maxizyme technology.

Ribozymes are small and versatile nucleic acids that can cleave RNAs at specific sites. These molecules have great potential to be used as effective gene-therapeutic agents. However, because of the limitation for cleavable sequences within the target mRNA, in some cases conventional ribozymes have failed to exhibit precise cleavage specificity.

Functional gene discovery using hybrid ribozyme libraries.

Hybrid ribozymes that couple the cleavage activity of hammerhead ribozymes with the unwinding activity of RNA helicases are powerful tools in the study of cell genetics and pharmaceutical drug development. They are useful for targeting a specific gene as well as screening functional genes to show phenotypic alterations. By randomizing the binding arms within the ribozymes, we can create a library of ribozymes that are capable of cleaving any mRNA.

Helicase-attached novel hybrid ribozymes.

Ribozymes have potential as therapeutic agents and in functional studies of genes of interest. The activities of ribozymes in vivo depend on the accessibility of ribozymes to a cleavage site in the target RNA. At present, the selection of a target site for ribozymes is often based on a computer-aided structural analysis of the target RNA or trial-and-error experiments in which vast numbers of ribozymes are tested systematically.

Ribozyme expression systems.

We have succeeded in constructing an effective system for the expression of ribozymes under the control of a human tRNAVal promoter, which ensures a high level of production of ribozymes in vivo. The engineered tRNAVal-driven ribozymes, based on computer-predicted secondary structure, were relatively stable and were transported to the cytoplasm, where they could be colocalized with their target RNA. The activity of the exported ribozymes was significantly higher than that of ribozymes that remained in the nucleus.

Venous-drainage-guided selective hepatectomy: a novel approach to liver surgery.

Selective hepatectomy under the guidance of hepatic venous drainage has not yet been developed because hepatic venous occlusion alone produces no visible congested area. Now that this area can be identified by simultaneous occlusion of the hepatic vein and artery, venous-drainage-guided selective hepatectomy is considered feasible. Because the congested area becomes dysfunctional or atrophic due to the absence of portal blood supply, it can be regarded as a first candidate for preventive resection in livers that may bear latent tumors.

Expression of siRNA from a single transcript that includes multiple ribozymes in mammalian cells.

RNA interference (RNAi) has been developed recently as a powerful tool for silencing of mRNAs in various organisms. In mammalian cells, the introduction of small interfering RNAs (siRNAs) can inhibit gene expression in a sequence-specific manner without induction of the nonspecific degradation that is activated by long double-stranded RNA (dsRNA) (>30 nt). Here, we report a method for generating siRNAs in mammalian cells using a self-cleaving ribozyme-expressing vector.

Strategies for generation of an siRNA expression library directed against the human genome.

RNA interference (RNAi) is a phenomenon whereby expression of an individual gene is specifically silenced by the introduction of a double-stranded RNA (dsRNA) whose sequence is homologous to that of the gene in question. The generation of a small interfering RNA (siRNA) expression library directed against the entire human genome is a project that requires solutions to many difficult technical problems. We present here some strategies for solving some of these problems, including the development of genetically stable and highly active siRNA expression vectors, a procedure for selection of favorable target sites, and an efficient and inexpensive procedure for constructing an siRNA expression library.

Modified peptide selection in vitro by introduction of a protein-RNA interaction.

The ribosome display system is a very effective and powerful tool for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. The system depends on the stability of ribosome-mRNA complexes that have been formed as a result of the removal of a stop codon. To assess the general applicability of the system, we examined the stability of ribosome-mRNA complexes in the presence and absence of a stop codon, as well as in the presence and the absence of an additional interaction between the translated peptide and its mRNA within the ribosome-mRNA complex.

Mitogen-activated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase.

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively.