Absorbance spectral change of the cytochrome P-450S21-phenylisocyanide complex upon binding of reduced NADPH-cytochrome-P-450 reductase.

Reduction of cytochrome P-450S21 (SF) (SF, substrate-free; purified from bovine adrenocortical microsomes) with sodium dithionite (Na2S2O4) in the presence of phenylisocyanide produced a ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex with Soret absorbance maxima at 429 and 456 nm. On the other hand, when a preformed ferric cytochrome P-450S21 (SF)-NADPH-cytochrome-P-450 reductase (Fp2) complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum of the ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex changed drastically, as characterized by an increase in absorbance intensity at 429 nm and a decrease at 456 nm. Similar spectral changes were observed by addition of reduced Fp2 to the preformed ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex. Experiments to reduce a ferric cytochrome P-450S21 (SF)-phenylisocyanide complex with sodium dithionite in the presence of various amounts of Fp2 showed that; (1), the spectral change reached maxima for both absorption increase at 429 nm and decrease at 456 nm when cytochrome P-450S21 and Fp2 were previously mixed at the cytochrome P-450S21:Fp2 ratio of 1:5; (2), the spectral change was suppressed in 300 mM potassium phosphate buffer (pH 7.4). These results suggest that the absorbance spectral change is due to a conformational change around the heme moiety induced by association with reduced Fp2.     

Impairment of Mobility in Endodermal Cells by FAK Deficiency

Focal adhesion kinase (FAK) is a novel nonreceptor protein tyrosine kinase that localizes in focal adhesions. It is expressed in a variety of cell types, and we reported earlier that its deficiency causes a decrease of mobility in mesodermal cells with enhanced formation of focal adhesions. With embryoid bodies generated from embryonic stem cells, we also observed a decrease of mobility in FAK-deficient endodermal cells with enhanced focal adhesion formation.     

Coordinated Regulation of the Genes Participating in Starch Biosynthesis by the Rice Floury-2 Locus

The recessive floury-2 (flo-2) locus of rice (Oryza sativa L.), which is located on chromosome 4, causes a strong reduction in expression of the gene encoding an isoform of branching enzyme RBE1 in immature seeds 10 d after flowering. Mapping of the RBE1 gene demonstrated the localization on rice chromosome 6, suggesting that the wild-type Floury-2 (Flo-2) gene regulates RBE1 gene expression in trans. However, reduced expression of the genes encoding some other starch-synthesizing enzymes, including another isoform of branching enzyme RBE3 and granule-bound starch synthase, was also found in the flo-2 seeds. In spite of the low level of RBE1 gene expression in the immature seeds of the flo-2 mutants, the RBE1 gene was equally expressed in the leaves of the wild type and flo-2 mutants. Thus, these results imply that the Flo-2 gene may co-regulate expression of some of the genes participating in starch synthesis possibly in a developing seed-specific manner.     

Construction of Libraries for Methylation Sites by In-gel Competitive Reassociation (IGCR)

The in-gel competitive reassociation (IGCR) procedure was successfullyapplied to construct a comprehensive library enriched in DNAfragments containing C^5mCGG sequences from mouse liver and braingenomic DNA. For IGCR, methylation-insensitive restriction enzyme(Msp I) digests were used as target DNA and methylation-sensitiverestriction enzyme (Hpa II) digests as competitor DNA. Southernblot analysis indicated that 60 to 70% of the clones in thelibrary were derived from the methylated sites and overall enrichmentwas 200- to 1000-fold. IGCR was further applied to constructa library for the sites differentially methylated between brainand liver DNA. In the library, approximately 20% of the HpaII sites exhibited different degrees of methylation betweenthese tissues.     

Further confirmation of the presence of DNA in the pyrenoid core of the siphonous green algal genus Caulerpa (Caulerpales, Ulvophyceae, Chlorophyta)

We re-examined the distribution of chloroplast DNA (ct-DNA) in the pyrenoid core of Caulerpa okamurae Weber van Bosse and C. lentillifera J. Agardh by fluorescence microscopy after staining the squashes and Technovit sections with DNA fluorochromes such as 4′6-diamidino-2-phenylmdole (DAPI), ethidium bromide, Hoechst 33258 and chromomycin A3. All fluorochromes stained specifically the pyrenoid core on the squashes and Technovit sections. In addition, we present new data on the localization of ct-DNA in the pyrenoid core of two other species of the genus Caulerpa: C. cactoides (Turner) Agardh and C. geminata Harvey.     

A gene at 59 minutes on the Escherichia coli chromosome encodes a lipoprotein with unusual amino acid repeat sequences.

We report a 1.432-kb DNA sequence at 59 min on the Escherichia coli chromosome that connects the published sequences of the pcm gene for the isoaspartyl protein methyltransferase and that of the katF or rpoS (katF/rpoS) gene for a sigma factor involved in stationary-phase gene expression. Analysis of the DNA sequence reveals an open reading frame potentially encoding a polypeptide of 379 amino acids. The polypeptide sequence includes a consensus bacterial lipidation sequence present at residues 23 to 26 (Leu-Ala-Gly-Cys), four octapeptide proline- and glutamine-rich repeats of consensus sequence QQPQIQPV, and four heptapeptide threonine- and serine-rich repeats of consensus sequence PTA(S,T)TTE. The deduced amino acid sequence, especially in the C-terminal region, is similar to that of the Haemophilus somnus LppB lipoprotein outer membrane antigen (40% overall sequence identity; 77% identity in last 95 residues). The LppB lipoprotein binds Congo red dye and has been proposed to be a virulence determinant in H. somnus. Utilizing a plasmid construct with the E. coli gene under the control of a phage T7 promoter, we demonstrate the lipidation of this gene product by the incorporation of [3H]palmitic acid into a 42-kDa polypeptide. We also show that treatment of E. coli cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 46-kDa precursor. We thus designate the protein NlpD (new lipoprotein D). E. coli cells overexpressing NlpD bind Congo red dye, suggesting a common function with the H. somnus LppB protein. Disruption of the chromosomal E. coli nlpD gene by insertional mutagenesis results in decreased stationary-phase survival after 7 days.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds

To manipulate the quantity and quality of storage components in Brassica napus seeds, we have constructed an antisense gene for the storage protein napin. The antisense gene was driven by the 5-flanking region of the B. napus napin gene to express antisense RNA in a seed-specific manner. Seeds of transgenic plants with antisense genes often contained reduced amounts of napin. In some transgenic plants, no accumulation of napin was observed. However, the total protein content of transgenic and wild-type seeds did not differ significantly. Seeds lacking napin accumulated 1.4 to 1.5 times more cruciferin than untransformed seeds, although the oleosin content was not affected. Fatty acid content and composition in the seeds of transgenic plants were also analyzed by gas chromatography. Though the total fatty acid content of the transformants was the same as that of non-transformants, there was a reduction in 18:1 contents and a concomitant increase of 18:2 in seeds with reduced napin levels. This observed change in fatty acid composition was inherited in the next generation.     

Primary culture of rat hepatocytes using membrane-supported collagen sandwich with hormone-free medium

Summary Primary culture of rat hepatocytes in hormone-free medium using membrane-supported collagen sandwich maintained their cellular morphology and expressed albumin secretion for about 3 weeks in vitro. It was reconfirmed that mimicking the cellular environment in vivo was effective for cellular maintenance.