Highly potent PDE4 inhibitors with therapeutic potential

The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Ariflo 1, which is an orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a-b, 11b-14b, and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hydroxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy residue with a difluoromethoxy residue led to an increase of in vivo potency. Structure-activity relationships are presented. Single-dose rat pharmacokinetic data for 11b, 12b, and 17a are also presented.     

Visfatin is present in bovine mammary epithelial cells, lactating mammary gland and milk, and its expression is regulated by cAMP pathway

Visfatin was originally identified as a growth factor for immature B cells, and recently demonstrated to bind insulin receptor. Visfatin mRNA and protein were detected by RT-PCR and Western blot analysis in cloned bovine mammary epithelial cells, lactating bovine mammary gland and human breast cancer cell line, MCF-7. Immunocytochemical staining localized the visfatin protein in the cytosol and nucleus of both cells. Quantitative-RT-PCR analysis revealed that the expression of the visfatin mRNA was significantly elevated when treated with forskolin (500 microM), isopreterenol (1-10 microM) and dibutyric cyclic AMP (1 mM) for 24 h, and significantly reduced when treated with insulin (5-50 ng/ml) and dexsamethasone (0.5-250 nM) for 24 h. These results indicate that mammary epithelial cells express the visfatin protein and secrete them into the milk.     

GB virus C infection in Indonesian HIV‐positive patients

GB virus C (GBV‐C), a human virus of the Flaviviridae family that is structurally and epidemiologically closest to hepatitis C virus (HCV), has been reported to confer beneficial outcomes in HIV‐positive patients. However, the prevalence of GBV‐C in HIV‐positive individuals in Indonesia is unknown. Since GBV‐C is more prevalent in anti‐HCV positive patients than in anti‐HCV negative subjects, transmission of GBV‐C and HCV could be by the same method. This study examined the prevalence and molecular characteristics of GBV‐C infection in HIV patients in Yogyakarta, Indonesia. The prevalence of GBV‐C among HIV patients (n = 125, median age 31 years) based on the 5′UTR region was 111/125 (88.8%), including 39/48 (81.3%) and 72/77 (93.5%) HIV‐infected patients with and without HCV infection, respectively. GBV‐C isolates were of genotype 2a, 3 and 6 in 58.3%, 12.6% and 28.4% of patients, respectively. Patients with genotype 3 were significantly younger than those with genotypes 2a or 6 (P = 0.001 and P = 0.012, respectively). Genotypes 3 and 6 were significantly associated with injection drug use (P = 0.004 and P = 0.002, respectively) and HCV co‐infection (P < 0.001 for both genotypes), indicating a shared transmission route with HCV. In conclusion, the prevalence of GBV‐C among HIV‐positive patients in Indonesia is high, and three genotypes were detected, namely genotype 2a, 3 and 6.     

Interfamily Transfer of Dual NB-LRR Genes Confers Resistance to Multiple Pathogens

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens.     

Selection of a reference gene for studies on lipid‐related aquatic adaptations of toothed whales (Grampus griseus)

Toothed whales are one group of marine mammals that has developed special adaptations, such as echolocation for predation, to successfully live in a dynamic aquatic environment. Their fat metabolism may differ from that of other mammals because toothed whales have acoustic fats. Gene expression in the metabolic pathways of animals can change with respect to their evolution and environment. A real‐time quantitative polymerase chain reaction (RT‐qPCR) is a reliable technique for studying the relative expressions of genes. However, since the accuracy of RT‐qPCR data is totally dependent on the reference gene, the selection of the reference gene is an essential step. In this study, 10 candidate reference genes (ZC3H10, FTL, LGALS1, RPL27, GAPDH, FTH1, DCN, TCTP, NDUS5, and UBIM) were initially tested for amplification efficiency using RT‐qPCR. After excluding DCN, the remaining nine genes, which are nearly 100% efficient, were selected for the gene stability analysis. Stable reference genes across eight different fat tissue, liver, and muscle samples from Grampus griseus were identified by four algorithms, which were provided in Genorm, NormFinder, BestKeeper, and Delta CT. Finally, a RefFinder comprehensive ranking was performed based on the stability values, and the nine genes were ranked as follows: LGALS1 > FTL > GAPDH > ZC3H10 > FTH1 > NDUS5 > TCTP > RPL27 > UBIM. The LGALS1 and FTL genes were identified as the most stable novel reference genes. The third‐ranked gene, GAPDH, is a well‐known housekeeping gene for mammals. Ultimately, we suggest the use of LGALS1 as a reliable novel reference gene for genomics studies on the lipid‐related aquatic adaptations of toothed whales.     

Nonsynonymous single-nucleotide polymorphisms of the human apoptosis-related endonuclease--DNA fragmentation factor beta polypeptide, endonuclease G, and Flap endonuclease-1--genes show a low degree of genetic heterogeneity

DNA fragmentation factor beta (DFFB) polypeptide, endonuclease G (EndoG), and Flap endonuclease-1 (FEN-1) are responsible for DNA fragmentation, a hallmark of apoptosis. Although the human homologs of these genes show three, four, and six nonsynonymous single-nucleotide polymorphisms (SNPs), respectively, data on their genotype distributions in populations worldwide are limited. In this context, the objectives of this study were to elucidate the genetic heterogeneity of all these SNPs in wide-ranging populations, and thereby to clarify the genetic background of these apoptosis-related endonucleases in human populations. We investigated the genotype distribution of their SNPs in 13 different populations of healthy Asians, Africans, and Caucasians using novel genotyping methods. Among the 13 SNPs in the 3 genes, only 3 were found to be polymorphic: R196K and K277R in the DFFB gene, and S12L in the EndoG gene. All 6 SNPs in the FEN-1 gene were entirely monoallelic. Although it remains unclear whether each SNP would exert any effect on endonuclease functions, these genes appear to exhibit low degree of genetic heterogeneity with regard to nonsynonymous SNPs. These findings allow us to conclude that human apoptosis-related endonucleases, similarly to other human DNase genes, revealed previously, are well conserved at the protein level during the course of human evolution.     

Genetic diversity in the Japanese rosy bitterling, Rhodeus ocellatus kurumeus (Cyprinidae)

Comparison of meristic characters (pored lateral line scales, vertebrae, and fin rays), and PCR-RFLP analysis in the D-loop and ND1 regions of mitochondrial DNA were performed to estimate the genetic diversity in local populations of the Japanese rosy bitterling, Rhodeus ocellatus kurumeus. In terms of meristic characters, the Fukuoka population was the largest in both range and variance of the number of pored lateral line scales and vertebrae (abdominal and caudal), and Osaka was the second, whereas the Kagawa population showed the smallest range and variance in these characters. In PCR-RFLP analysis, 11 haplotypes (3 in Fukuoka, 2 in Okayama, 2 in Kagawa, and 4 in Osaka) were observed, and nucleotide sequence divergence (NSD) was approximately two times larger in ND1 (mean, 0.61%) than in D-loop (mean, 0.31%). In the neighbor-joining (NJ) tree, based upon the NSD value in ND1, haplotypes were arranged into four clades, which corresponded to the locality of each haplotype. The Fukuoka population was conspicuously apart from the other populations (mean, 0.90% in NSD), but the remaining three showed a similar genetic distance with each other (mean, 0.48%–0.52% in NSD). In haplotype diversity of mtDNA, half the stations in Osaka and all in Kagawa were monomorphic. Especially, two haplotypes endemic to Kagawa were randomly distributed, irrespective of drainages. Rhodeus o. kurumeus in Fukuoka inhabits small rivers and creeks (open water systems), while that in Kagawa and Osaka lives in small ponds (closed water systems). Taking the information of morphology, mtDNA, and habitat into consideration, the low genetic diversity in Kagawa and Osaka populations of R. o. kurumeus is thought to be mainly the result of the isolation of their habitat. Received: January 14, 2001 / Revised: June 14, 2001 / Accepted: July 1, 2001     

Microarray analysis of uterine gene expression in mouse and human pregnancy

Improved care of infants born prematurely has increased their survival. However, the incidence of preterm labor has not changed. To understand the processes involved in preterm labor, we used oligonucleotide microarrays to study gene expression in murine and human uterus during pregnancy. The induction of enzymes for prostaglandin synthesis was used as a marker for important changes during pregnancy because prostaglandins strongly contribute to both human and murine labor. We identified 504 genes that changed at least 2-fold between d 13.5 and 19.0 in the gravid mouse uterus. In the pregnant human myometrium, we found 478 genes that changed at least 2-fold in either term or preterm labor compared with preterm nonlabor specimens and 77 genes that significantly varied in both preterm and term labor. Patterns of gene regulation within functional groups comparing human preterm and term labor were similar, although the magnitude of change often varied. Surprisingly, few genes that changed significantly throughout pregnancy were the same in the mouse and human. These data suggest that functional progesterone withdrawal in human myometrium may not be the primary mechanism for labor induction, may implicate similar mechanisms for idiopathic preterm and term labor in humans, and may identify novel targets for further study.     

Simple derivation of the function of genetic information and correction of the proof

The problem of predicting time to extinction in stochastics population models is approached in two ways. First, a finite Markov chain approximation is used to give the distribution of time to extinction and shown to predict simulation results accurately. Second, an approximate numerical integration technique is found to give good relative predictions of persistence using much less computer time. The relevance of the two approaches to real problems is discussed.