A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Cloning of the macrolide antibiotic biosynthesis gene acyA, which encodes 3-O-acyltransferase, from Streptomyces thermotolerans and its use for direct fermentative production of a hybrid macrolide antibiotic.

A gene encoding the macrolide modification enzyme 3-O-acyltransferase (acyA) was cloned by chromosome walking onto the carbomycin biosynthetic region in Streptomyces thermotolerans TH475, with the 3' region of the gene encoding the macrolide modification enzyme 4"-O-acyltransferase (acyB1) as a probe. A shortened fragment (1.8 kb) containing acyA was subcloned with pIJ350. A high-level tylosin producer, Streptomyces fradiae MBBF, transformed with the plasmid could produce a hybrid macrolide, 3-O-acetyltylosin, most efficiently.     

Cytotoxic screening of medicinal and edible plants in Okinawa,Japan, and identification of the main toxic constituent of Rhodea japonica (Omoto)

The cytotoxic activity of ethanol extracts from 53 parts of 36 species of medicinal and edible plants cultivated in Okinawa was measured by using K562 human leukemia cells by a flow cytometric method. Two extracts from Rhodea japonica and Hypericum chinense were cytotoxic at a concentration of 10 microg/ml. The main cytotoxic constituent of Rhodea japonica was isolated and identified to be rhodexin A, which has been isolated as a cardetonic agent of the plant. The IC(50) value for rhodexin A against the growth of K562 cells was 19 nM, this activity being much stronger than that of ouabain (IC(50), 60 nM).     

Isolation and characterization of the mouse ortholog of the Fukuyama-type congenital muscular dystrophy gene

Fukuyama-type congenital muscular dystrophy (FCMD) is a severe autosomal-recessive muscular dystrophy accompanied by brain malformation. Previously, we identified the gene responsible for FCMD through positional cloning. Here we report the isolation of its murine ortholog, Fcmd. The predicted amino acid sequence of murine fukutin protein encoded by Fcmd is 90% identical to that of its human counterpart. Radiation hybrid mapping localized the gene to 2.02 cR telomeric to D4Mit272 on chromosome 4. Northern blot analysis revealed ubiquitous expression of Fcmd in adult mouse tissues. Through in situ hybridization, we observed a wide distribution of Fcmd expression throughout embryonic development, most predominantly in the central and peripheral nervous systems. We also detected high Fcmd expression in the ventricular zone of proliferating neurons at 13.5 days post-coitum. Brain malformation in FCMD patients is thought to result from defective neuronal migration. Our data suggest that neuronally expressed Fcmd is likely to be important in the development of normal brain structure.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.     

Establishment of germline-competent embryonic stem cell lines from the MSM/Ms strain

MSM/Ms is an inbred mouse strain established from the Japanese wild mouse, Mus musculus molossinus, which has been phylogenetically distinct from common laboratory mouse strains for about 1 million years. The nucleotide substitution rate between MSM/Ms and C57BL/6 is estimated to be 0.96%. MSM/Ms mice display unique characteristics not observed in the commonly used laboratory strains, including an extremely low incidence of tumor development, high locomotor activity, and resistance to high-fat-diet-induced diabetes. Thus, functional genomic analyses using MSM/Ms should provide a powerful tool for the identification of novel phenotypes and gene functions. We report here the derivation of germline-competent embryonic stem (ES) cell lines from MSM/Ms blastocysts, allowing genetic manipulation of the M. m. molossinus genome. Fifteen blastocysts were cultured in ES cell medium and three ES lines, Mol/MSM-1, -2, and -3, were established. They were tested for germline competency by aggregation with ICR morulae and germline chimeras were obtained from all three lines. We also injected Mol/MSM-1 ES cells into blastocysts of ICR or C57BL/6 × BDF1 mice and found that blastocyst injection resulted in a higher production rate of chimeric mice than did aggregation. Furthermore, Mol/MSM-1 subclones electroporated with a gene trap vector were also highly efficient at producing germline chimeras using C57BL/6 × BDF1 blastocyst injection. This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome.     

Genetic analysis of craniofacial traits in the medaka

Family and twin studies suggest that a substantial genetic component underlies individual differences in craniofacial morphology. In the current study, we quantified 444 craniofacial traits in 100 individuals from two inbred medaka (Oryzias latipes) strains, HNI and Hd-rR. Relative distances between defined landmarks were measured in digital images of the medaka head region. A total of 379 traits differed significantly between the two strains, indicating that many craniofacial traits are controlled by genetic factors. Of these, 89 traits were analyzed via interval mapping of 184 F(2) progeny from an intercross between HNI and Hd-rR. We identified quantitative trait loci for 66 craniofacial traits. The highest logarithm of the odds score was 6.2 for linkage group (LG) 9 and 11. Trait L33, which corresponds to the ratio of head length to head height at eye level, mapped to LG9; trait V15, which corresponds to the ratio of snout length to head width measured behind the eyes, mapped to LG11. Our initial results confirm the potential of the medaka as a model system for the genetic analysis of complex traits such as craniofacial morphology.     

Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis

Chicken B lymphocyte precursors and DT40 cells diversify their immunoglobulin-variable (IgV) genes through homologous recombination (HR)-mediated Ig gene conversion. To identify DNA polymerases that are involved in Ig gene conversion, we created DT40 clones deficient in DNA polymerase eta (poleta), which, in humans, is defective in the variant form of xeroderma pigmentosum (XP-V). Poleta is an error-prone translesion DNA synthesis polymerase that can bypass UV damage-induced lesions and is involved in IgV hypermutation. Like XP-V cells, poleta-disrupted (poleta) clones exhibited hypersensitivity to UV. Remarkably, poleta cells showed a significant decrease in the frequency of both Ig gene conversion and double-strand break-induced HR when compared to wild-type cells, and these defects were reversed by complementation with human poleta. Our findings identify a DNA polymerase that carries out DNA synthesis for physiological HR and provides evidence that a single DNA polymerase can play multiple cellular roles.     

On the Molecular Weight of Polypeptide Chain of Sweet Potato β-Amylase

A number of N-acyl-L-proline derivatives were synthesized and their biological activities were investigated by using lettuce (Lactuca sativa L. cv. Sacramento) seedling test. A wide variety of these compounds promoted root growth at 25°C both under light and in darkness. Of the compounds tested, N-(2-ftuorobenzoyl)-L-proline methyl ester (4) showed the highest activity and caused a 270% increase in the root elongation compared to the control. N-(2-Naphthoyl)-L-proline methyl ester (14) promoted the root growth, while N-(1-naphthoyl)-L-proline methyl ester inhibited it. L-Proline, benzoic acid, and 2-naphthoic acid had no significant effect on lettuce seedlings. Compounds 4 and 14, and N-(2-chlorobenzoyl)-L-proline methyl ester (7) reduced the inhibitory effect of 1 ppm ABA on the root growth, while the D-isomer of 4 was less activite than compound 4. Compounds 4, 7, and 14 did not show any rescue-activity for the complete inhibition of germination that was caused by treating 10 ppm of ABA.