Hundreds of small RNAs of approximately 22 nucleotides, collectively named microRNAs (miRNAs), have been discovered recently in animals and plants. Although their functions are being unravelled, their mechanism of biogenesis remains poorly understood. miRNAs are transcribed as long primary transcripts (pri-miRNAs) whose maturation occurs through sequential processing events: the nuclear processing of the pri-miRNAs into stem-loop precursors of approximately 70 nucleotides (pre-miRNAs), and the cytoplasmic processing of pre-miRNAs into mature miRNAs. Dicer, a member of the RNase III superfamily of bidentate nucleases, mediates the latter step, whereas the processing enzyme for the former step is unknown. Here we identify another RNase III, human Drosha, as the core nuclease that executes the initiation step of miRNA processing in the nucleus. Immunopurified Drosha cleaved pri-miRNA to release pre-miRNA in vitro. Furthermore, RNA interference of Drosha resulted in the strong accumulation of pri-miRNA and the reduction of pre-miRNA and mature miRNA in vivo. Thus, the two RNase III proteins, Drosha and Dicer, may collaborate in the stepwise processing of miRNAs, and have key roles in miRNA-mediated gene regulation in processes such as development and differentiation.

Human umbilical cord blood (UCB) has been regarded as an alternative source for cell transplantation and cell therapy because of its hematopoietic and nonhematopoietic (mesenchymal) potential. Although there has been debate about whether mesenchymal stem cells (MSCs) are invariably present in UCB, several reports showed that MSC‐like cells could be consistently derived from human UCB and, moreover, could differentiate into various cells of a mesodermal origin. However, it remains unclear whether these UCB‐derived MSCs are also capable of differentiating into skeletal muscle cells. In this study, we isolated MSCs from human UCB and induced them to differentiate into skeletal muscle cells. During cell culture expansion, UCB‐derived mononuclear cells gave rise to adherent layers of fibroblast‐like cells expressing MSC‐related antigens such as SH2, SH3, α‐smooth muscle actin, CD13, CD29, and CD49e. More important, when these UCB‐derived MSCs were incubated in promyogenic conditions for up to 6 weeks, they expressed myogenic markers in accordance with myogenic differentiation pattern. Both flow cytometric and reverse transcriptase–polymerase reaction analyses showed that two early myogenic markers, MyoD and myogenin, were expressed after 3 days of incubation but not after 2 weeks. At week 6, more than half of UCB‐derived MSCs expressed myosin heavy chain, a late myogenic marker. Our results demonstrate that UCB‐derived MSCs possess a potential of skeletal myogenic differentiation and also imply that these cells could be a suitable source for skeletal muscle repair and a useful tool of muscle‐related tissue engineering.

No abstract

Protein tyrosine phosphatase (PTP) is a family of enzymes important for regulating cellular phosphorylation state. The oxidation and consequent inactivation of several PTPs by H 2 O 2 are well demonstrated. It is also shown that recovery of enzymatic activity depends on the availability of cellular reductants. Among these redox-regulated PTPs, PTEN, Cdc25 and low molecular weight PTP are known to form a disul¢de bond between two cysteines, one in the active site and the other nearby, during oxidation by H 2 O 2 . The disul¢de bond likely confers e⁄ciency in the redox regulation of the PTPs and protects cysteine-sulfenic acid of PTPs from further oxidation. In this review, through a comparative analysis of the oxidation process of Yap1 and PTPs, we propose the mechanism of disul¢de bond formation in the PTPs. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

No abstract

The eye colour mutant sepia (se1) is defective in PDA {6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and characterize the gene encoding PDA synthase and show that it is the structural gene for sepia. Based on previously reported information [Wiederrecht, Paton and Brown (1984) J. Biol. Chem. 259, 2195-2200; Wiederrecht and Brown (1984) J. Biol. Chem. 259, 14121-14127; Andres (1945) Drosoph. Inf. Serv. 19, 45; Ingham, Pinchin, Howard and Ish-Horowicz (1985) Genetics 111, 463-486; Howard, Ingham and Rushlow (1988) Genes Dev. 2, 1037-1046], we isolated five candidate genes predicted to encode GSTs (glutathione S-transferases) from the presumed sepia locus (region 66D5 on chromosome 3L). All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro. The molecular mass of recombinant CG6781 was estimated to be 28 kDa by SDS/PAGE and 56 kDa by gel filtration, indicating that it is a homodimer under native conditions. Sequencing of the genomic region spanning CG6781 revealed that the se1 allele has a frameshift mutation from 'AAGAA' to 'GTG' at nt 190-194, and that this generates a premature stop codon. Expression of the CG6781 open reading frame in an se1 background rescued the eye colour defect as well as PDA synthase activity and drosopterins content. The extent of rescue was dependent on the dosage of transgenic CG6781. In conclusion, we have discovered a new catalytic activity for an Omega class GST and that CG6781 is the structural gene for sepia which encodes PDA synthase.

Mesenchymal stem cells (MSCs) retain both self-renewal and multilineage differentiation capabilities. Despite wide therapeutic potential, many aspects of human MSCs, particularly the molecular parameters to define the stemness, remain largely unknown. Using high-density oligonucleotide microarrays, we obtained the differential gene expression profile between a fraction of mononuclear cells of human umbilical cord blood (UCB) and its MSC subpopulation. Of particular interest was a subset of 47 genes preferentially expressed at 50-fold or higher in MSCs, which could be regarded as a molecular foundation of human MSCs. This subset contains numerous genes encoding collagens, other extracellular matrix or related proteins, cytokines or growth factors, and cytoskeleton-associated proteins but very few genes for membrane and nuclear proteins. In addition, a direct comparison of this microarray-generated transcriptome with the published serial analysis of gene expression data suggests that a molecular context of UCB-derived MSCs is more or less similar to that of bone marrow-derived cells. Altogether, our results will provide a basis for studies on molecular mechanisms controlling core properties of human MSCs. Stem Cells 2005;23:584-593

No abstract