KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.     

Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.     

Molecular cloning of cDNA encoding human DNA helicase Q1 which has homology to Escherichia coli Rec Q helicase and localization of the gene at chromosome 12p12.

A complementary DNA encoding DNA-dependent ATPase Q1 possessing DNA helicase activity, which is the major DNA-dependent ATPase in human cell extracts, was cloned from a cDNA library of human KB cells. The predicted amino acid sequence has seven consecutive motifs conserved in the RNA and DNA helicase super family and DNA helicase Q1 belongs to DEXH helicase family. A homology search indicated that helicase Q1 had 47% homology in the seven conserved regions with Escherichia coli RecQ protein. Three RNA bands of 4.0, 3.3, and 2.2 kilobases were detected in HeLa cells by Northern blotting. Analysis of the genomic DNA indicated the presence of a homologous gene in mouse cells. The DNA helicase Q1 gene was localized on the short arm of human chromosome 12 at 12p12.     

Isolation and characterization of a novel mycovirus infecting an edible mushroom,Grifola frondosa

Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.     

Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

We report an allele-specific sequencing method using allele-specific long-range polymerase chain reaction (PCR) to determine if multiple (specifically, more than three) single nucleotide polymorphisms (SNPs) are located on the same allele. We sequenced the glucocorticoid receptor (GR) gene as a model and detected four nucleotide changes, including two novel variations, in intron 4 and exons 6, 8, and 9 alpha in four of the investigated cell lines. The terminal SNPs (intron 4 and exon 9 alpha) were separated by 19 kb. Following SNP identification, the first round PCR allele-specific primers are designed at the both distal SNP sites (intron 4 and exon 9 alpha), placing the SNP positions at the primer 3'-end. Using these first round PCR products as template, the second round PCR was performed to separately amplify exons 6 and 8. These second round PCR products were subsequently sequenced. The sequencing results showed that the four SNPs were located on the same allele, i.e., forming a haplotype. This allele-specific long-range PCR/sequencing (ALP/S) method is rapid and applicable to the allelic assignment for more than three SNPs.     

nanos1: a mouse nanos gene expressed in the central nervous system is dispensable for normal development

A mouse nanos (nanos1) gene was cloned and its function was examined by generating a gene-knockout mouse. The nanos1 gene encodes an RNA-binding protein, which contains a putative zinc-finger motif that exhibits similarity with other nanos-class genes in vertebrates and invertebrates. Although nanos1 is not detected in primordial germ cells, it is observed in seminiferous tubules of mature testis. Interestingly, maternally expressed nanos1 is observed in substantial amounts in oocytes, but the amount of maternal RNA is rapidly reduced after fertilization, and the transient zygotic nanos1 expression is observed in eight-cell embryos. At 12.5 days postcoitum, nanos1 is re-expressed in the central nervous system and the expression continues in the adult brain, in which the hippocampal formation is the predominant region. The nanos1 -deficient mice develop to term without any detectable abnormality and they are fertile. No significant neural defect is observed in terms of their behavior to date.     

Facile Synthesis of (22R, 23R)-Homobrassinolide

A synthesis of (22R,23R)-homobrassinolide is described. The LC and the chemical correlation studies for the oxidation product of a stigmasterol-like side chain with osmium tetroxide are mentioned. A stereochemical view for the mechanism of osmium tetroxide oxidation of the side chain is proposed.     

The role of carbon flux and biometric observations in constraining a terrestrial ecosystem model: a case study in disturbed forests in East Asia

The process of confining unnecessary freedom is a step toward advanced ecosystem modeling. This study demonstrates the importance of carbon flux and biometric observation in constraining a terrestrial ecosystem model with a simple optimization scheme. At the selected sites from AsiaFlux network, a simultaneous optimization scheme for both carbon flux and biomass was compared with carbon flux-oriented and biomass-oriented optimization schemes using the Biome-BGC model. The optimization scheme oriented to either carbon flux or biomass provided simulation results that were consistent with observations, but with reduced performance in unconstrained variables. The simultaneous optimization scheme yielded results that were consistent with observations for both carbon flux and biomass. By comparing long-term projections simulated by three schemes, it was found that the optimization oriented only to carbon flux has limited performance because misrepresented biomass significantly affected a projection of carbon exchange through heterotrophic respiration. From these experiments, we found that (1) a process model like Biome-BGC is capable of reproducing both carbon flux and biomass within acceptable proximity, (2) constraining biomass is importance not just because it is one of carbon cycle components, but also it significantly affects simulations of carbon flux. Thus, it is important to invest more effort to improve simulation of biomass as well as carbon flux.