Transmembrane signaling: tumor promoter distribution

Diacylglycerol plays a critical role in transmembrane signaling by activating protein kinase C (PKC). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) mimics that action, and in the human erythrocyte, TPA-activated PKC phosphorylates membrane proteins. Although molecular aspects of this process have been investigated, details of the interaction of TPA with plasma membranes remain elusive. Because TPA is hydrophobic, it has been assumed that it associates with the lipid bilayer. However, there is no direct evidence for its transbilayer distribution. Because knowledge of its location would limit molecular models proposed to explain its mode of action, we have used membrane-splitting techniques, based on freeze-fracture of planar cell monolayers, to quantify transmembrane partitioning of [3H]TPA. Under conditions where PKC-mediated phosphorylation was stimulated by [3H]TPA and where more than 90% of the [3H]TPA was associated with the human red cell plasma membrane, two-thirds of the TPA partitioned with the cytoplasmic leaflet after bilayer splitting. This represents the first direct topographic localization of TPA in a biological membrane and supports the hypothesis that the mechanism of TPA activation requires its association with the cytoplasmic leaflet of the bilayer.     

Detailed analysis of an amplified region at chromosome 11q13 in malignant tumors.

We have examined the amplification unit at chromosome band 11q13 in 12 primary tumors and one cancer cell line (A431) with 15 DNA markers. The amplified region and size varied from one tumor to another; the smallest amplicon was estimated to be 700 kb long and the largest was 4.5 Mb long at maximum, on the basis of a physical map. Furthermore, the DNA amplified in tumors was not always continuous, because in three cases one locus within the amplicon was not amplified. The amplified region common to all 13 cancers consisted of 500 kb of DNA which incorporated eight defined loci (BCL-1, cCI11-524, cCI11-283, cCI11-234, HBI-1, cCI11-454, HSTF1, and INT2). As two of them, cCI11-524 and cCI11-454, were found to contain DNA sequences conserved in other species, one or both of these loci might encode the gene(s) that may be associated with progression of these tumors.     

Detection of Polypeptides Involved in Early Stages of Nodulation in Soybean Roots

Soluble proteins extracted from the roots of nodulating soybean[Glycine max (L.) Merr. cv. T202] and from roots of the non-nodulatingisoline rj₁ of cv. T202 (cv. T201), which had been inoculatedwith Bradyrhizobium japonicum, were analyzed by two-dimensionalpolyacrylamide gel electrophoresis and silver staining, in anattempt to identify polypeptides involved in early stages ofnodulation. Almost identical patterns of polypeptides were generatedby extracts of 3-day-old roots of uninoculated T201 and T202and of inoculated T201 and T202, but a unique spot, correspondingto a polypeptide of 38 kDa was detected in the case of inoculatedroots of T202. Western blotting analysis using "inoculated-T202-rootspecific" antiserum, prepared by titration of antiserum againstproteins from inoculated T202 roots with proteins from inoculatedT201 roots, revealed spots corresponding to polypeptides of26,27, and 33 kDa that were detectable only in the extractsof roots of inoculated T202. However, no unique polypeptidespots were detected in the case of roots of inoculated T201and T202, as compared with those from uninoculated T201 andT202 roots by Western blotting analysis using "inoculated-T201-rootspecific" antiserum prepared by titration of antiserum againstproteins from inoculated T201 roots with proteins from uninoculatedT201 roots. (Received May 27, 1991; Accepted September 30, 1991)     

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.     

Comparative analysis of the complete mitochondrial genomes of three rockfishes (Scorpaeniformes,Sebastiscus) and insights into the phylogenetic relationships of Sebastidae

Mitochondrial genome is a powerful molecule marker to provide information for phylogenetic relationships and revealing molecular evolution in ichthyological studies. Sebastiscus species, a marine rockfish, are of essential economic value. However, the taxonomic status and phylogenetic relationships of Sebastidae have been controversial so far. Here, the mitochondrial genomes (mitogenomes) of three species, S. tertius, S. albofasciatus, and S. marmoratus, were systemically investigated. The lengths of the mitogenomes’ sequences of S. tertius, S. albofasciatus, and S. marmoratus were 16910, 17056, and 17580 bp, respectively. It contained 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNA (tRNA) genes, and one identical control region (D-loop) among the three species. The genetic distance and K_a/K_s ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, which were most likely due to the difference in their living environment. The phylogenetic tree was constructed by Bayesian Inference (BI) and Maximum Likelihood (ML). Most interestingly, the results indicated that Sebastidae and Scorpaenidae were grouped into a separate branch, so the taxonomic status of Sebastidae should be classified into subfamily Sebastinae. Our results may lead to a taxonomic revision of Scorpaenoidei.     

Persistence of extended-spectrum β-lactamase plasmids among Enterobacteriaceae in commercial broiler farms

To clarify the persistence of extended-spectrum β-lactamase (ESBL) producers, 13 plasmids from two broiler farms were analyzed. On the farm not using antimicrobials, one plasmid from Klebsiella pneumoniae isolated from a day-old chick was similar to that from Escherichia coli isolated a year later, with the deletion of two transposons. On the farm using antimicrobials, most circulating plasmids (eight out of nine) in a flock of 40-days-old chicks were identical, although one from K. pneumoniae had a deletion of a transposon carrying a class 1 integron containing aadA2 and dfrA12. Thus, ESBL plasmids persisted in the farms with or without antimicrobial agent use.     

RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major pathways that account for survival after post-replicational DNA damage. TLS functions by filling gaps on a daughter strand that remain after DNA replication caused by damage on the mother strand, while HR can repair gaps and breaks using the intact sister chromatid as a template. The RAD18 gene, which is conserved from lower eukaryotes to vertebrates, is essential for TLS in Saccharomyces cerevisiae. To investigate the role of RAD18, we disrupted RAD18 by gene targeting in the chicken B-lymphocyte line DT40. RAD18(-/-) cells are sensitive to various DNA-damaging agents including ultraviolet light and the cross-linking agent cisplatin, consistent with its role in TLS. Interestingly, elevated sister chromatid exchange, which reflects HR- mediated post-replicational repair, was observed in RAD18(-/-) cells during the cell cycle. Strikingly, double mutants of RAD18 and RAD54, a gene involved in HR, are synthetic lethal, although the single mutant in either gene can proliferate with nearly normal kinetics. These data suggest that RAD18 plays an essential role in maintaining chromosomal DNA in cooperation with the RAD54-dependent DNA repair pathway.