Structure and chromosome mapping of the human SIX4 and murine Six4 genes

Six4, a member of the homeobox gene subfamily (Six), is expressed in a developmentally regulated fashion, and supposed to be involved in embryogenesis. We cloned the human SIX4 and murine Six4 genomic DNAs and determined their structures. The structure, including the 5' upstream region of both genes, was well conserved suggesting the conserved function and regulation of these genes. Human SIX4 was mapped to chromosome 14q23.     

Plagiochilines C,D, E and F,four novel secoaromadendrane-type sesquiterpene hemiacetals from Plagiochila asplenioides and Plagiochila semidecurrens

Four novel secoaromadendrane-type sesquiterpene hemiacetals, plagiochilines C, D, E and F, together with the previously known (?)-bicyclogermacrene, have been isolated from the liverwort, Plagiochila asplenioides and their structures have been spectroscopically elucidated. From Plagiochila semidecurrens plagiochilines A and C have been obtained along with (?)-bicyclogermacrene and its related sesquiterpene hydrocarbons.     

Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein

The Escherichia coli SeqA protein recognizes the 11 hemimethylated G-^mA-T-C sites in the oriC region of the chromosome, and prevents replication over-initiation within one cell cycle. The crystal structure of the SeqA C-terminal domain with hemimethylated DNA revealed the N⁶-methyladenine recognition mechanism; however, the mechanism of discrimination between the hemimethylated and fully methylated states has remained elusive. In the present study, we performed mutational analyses of hemimethylated G-^mA-T-C sequences with the minimal DNA-binding domain of SeqA (SeqA_71–181), and found that SeqA_71–181 specifically binds to hemimethylated DNA containing a sequence with a mismatched ^mA:G base pair [G-^mA(:G)-T-C] as efficiently as the normal hemimethylated G-^mA(:T)-T-C sequence. We determined the crystal structures of SeqA_71–181 complexed with the mismatched and normal hemimethylated DNAs at 2.5 and 3.0 Å resolutions, respectively, and found that the mismatched ^mA:G base pair and the normal ^mA:T base pair are recognized by SeqA in a similar manner. Furthermore, in both crystal structures, an electron density is present near the unmethylated adenine, which is only methylated in the fully methylated state. This electron density, which may be due to a water molecule or a metal ion, can exist in the hemimethylated state, but not in the fully methylated state, because of steric clash with the additional methyl group.     

Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.     

DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil

A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Geranyl chalcone derivatives with antifungal and radical scavenging properties from the leaves of Artocarpus nobilis

Antifungal activity guided fractionation of the n-butanol extract from the methanol extract of the leaves of Artocarpus nobilis furnished 2',4',4-trihydroxy-3'-geranylchalcone (1), 2 ',4',4-trihydroxy-3'-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]chalcone (2), 2',4',4-trihydroxy-3'-[2-hydroxy-7-methyl-3-methylene-6-octaenyl]chalcone (3), 2',3,4,4'-tetrahydroxy-3'-geranylchalcone (4), 2',3,4,4'-tetrahydroxy-3'-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]chalcone (5). The chalcones 3 and 5 are new natural products whereas 1 and 2 are reported first time from the family Moraceae. All these compounds showed good fungicidal activity against Cladosporium cladosporioides and high radical scavenging activity towards the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical in TLC bio-autography method.     

Chemosystematics of the hepaticae

Most liverworts (Hepaticae) contain oil bodies which are composed of lipophilic terpenoids and aromatic compounds. The chemosystematics of 36 families of the Jungermannidae and seven families of the Marchantiidae of the Hepaticae are discussed using terpenoid and aromatic components.