Primary cilia of inv/inv mouse renal epithelial cells sense physiological fluid flow: bending of primary cilia and Ca2+ influx

Primary cilia are hypothesized to act as a mechanical sensor to detect renal tubular fluid flow. Anomalous structure of primary cilia and/or impairment of increases in intracellular Ca2+ concentration in response to fluid flow are thought to result in renal cyst formation in conditional kif3a knockout, Tg737 and pkd1/pkd2 mutant mice. The mutant inv/inv mouse develops multiple renal cysts like kif3a, Tg737 and pkd1/pkd2 mutants. Inv proteins have been shown to be localized in the renal primary cilia, but response of inv/inv cilia to fluid stress has not been examined. In the present study, we examined the mechanical response of primary cilia to physiological fluid flow using a video microscope, as well as intracellular Ca2+ increases in renal epithelial cells from normal and inv/inv mice in response to flow stress. Percentages of ciliated cells and the length of primary cilia were not significantly different between primary renal cell cultures from normal and inv/inv mutant mice. Localization of inv protein was restricted to the base of primary cilia even under flow stress. Inv/inv mutant cells had similar bending mechanics of primary cilia in response to physiological fluid flow compared to normal cells. Furthermore, no difference was found in intracellular Ca2+ increases in response to physiological fluid flow between normal and inv/inv mutant cells. Our present study suggests that the function of the inv protein is distinct from polaris (the Tg737 gene product), polycystins (pkd1 and pkd2 gene products).     

Systematic screening and identification of antigens recognized by monoclonal antibodies raised against the developing lateral olfactory tract

During development, olfactory bulb axons navigate a complex microenvironment composed of myriad molecules to construct a bundle called the lateral olfactory tract. The axons themselves also express thousands of different molecules. In the present study, we produced and characterized six monoclonal antibodies that label the lateral olfactory tract and its surroundings in a unique pattern. The labeling profiles suggested that the antigen molecules recognized by each antibody are heterogeneously distributed around the developing lateral olfactory tract. We developed an efficient screening method to identify the antigen molecules by combining expression of a cDNA library in COS-7 cells and the subsequent immunohistochemical staining of the cells. The systematic screening successfully identified specific cDNA clones for all of the monoclonal antibodies, which highly probably coded for the antigen molecules, and therefore unveiled the molecular nature of local components that embrace the developing lateral olfactory tract in mice.     

Potent anti-R5 human immunodeficiency virus type 1 effects of a CCR5 antagonist, AK602/ONO4128/GW873140, in a novel human peripheral blood mononuclear cell nonobese diabetic-SCID, interleukin-2 receptor gamma-chain-knocked-out AIDS mouse model

We established human peripheral blood mononuclear cell (PBMC)-transplanted R5 human immunodeficiency virus type 1 isolate JR-FL (HIV-1(JR-FL))-infected, nonobese diabetic-SCID, interleukin 2 receptor gamma-chain-knocked-out (NOG) mice, in which massive and systemic HIV-1 infection occurred. The susceptibility of the implanted PBMC to the infectivity and cytopathic effect of R5 HIV-1 appeared to stem from hyperactivation of the PBMC, which rapidly proliferated and expressed high levels of CCR5. When a novel spirodiketopiperazine-containing CCR5 inhibitor, AK602/ONO4128/GW873140 (molecular weight, 614), was administered to the NOG mice 1 day after R5 HIV-1 inoculation, the replication and cytopathic effects of R5 HIV-1 were significantly suppressed. In saline-treated mice (n = 7), the mean human CD4(+)/CD8(+) cell ratio was 0.1 on day 16 after inoculation, while levels in mice (n = 8) administered AK602 had a mean value of 0.92, comparable to levels in uninfected mice (n = 7). The mean number of HIV-RNA copies in plasma in saline-treated mice were approximately 10(6)/ml on day 16, while levels in AK602-treated mice were 1.27 x 10(3)/ml (P = 0.001). AK602 also significantly suppressed the number of proviral DNA copies and serum p24 levels (P = 0.001). These data suggest that the present NOG mouse system should serve as a small-animal AIDS model and warrant that AK602 be further developed as a potential therapeutic for HIV-1 infection.     

Novel relationship between the antifungal activity and cytotoxicity of marine-derived metabolite xestoquinone and its family

Xestoquinone and related metabolites (the xestoquinone family) occur in marine sponges and are known to show a variety of biological activities. In this study, the first comprehensive evaluation of antifungal activity was performed for xestoquinone and nine natural and unnatural analogues in comparison with their cytotoxicity. The cytotoxicity against two human squamous cell carcinoma cell lines, A431 and Nakata, indicated that the terminal quinone structure of the polycyclic molecules was important (xestoquinone, etc.) and that the presence of a ketone group at C-3 of the opposite terminus dramatically diminished the activity (halenaquinone, etc.). In contrast, a ketone group at C-3 enhanced the antifungal activity against the plant pathogen, Phytophthora capsici, regardless of the presence of a quinone moiety. The cytotoxicity and antifungal activity of the xestoquinone family were negatively correlated with each other.     

Survival of Deep-Sea Shrimp (Alvinocaris sp.) During Decompression and Larval Hatching at Atmospheric Pressure

We report successful larval hatching of deep-sea shrimp after decompression to atmospheric pressure. Three specimens of deep-sea shrimp were collected from an ocean depth of 1157 m at cold-seep sites off Hatsushima Island in Sagami Bay, Japan, using a pressure-stat aquarium system. Phylogenetic analysis of Alvinocaris sp. based on cytochrome c oxidase subunit gene sequences confirmed that these species were a member of the genus Alvinocaris. All 3 specimens survived to reach atmospheric pressure conditions after stepwise 63-day decompression. Two of the specimens contained eggs, which hatched after 10 and 16 days, respectively, of full decompression. Although no molting of the shrimp larvae was observed during 74 days of rearing under atmospheric pressure, the larvae developed conventional dark-adapted eyes after 15 days.     

Estrogen, insulin, and dietary signals cooperatively regulate longevity signals to enhance resistance to oxidative stress in mice

To investigate the biological significance of a longevity mutation found in daf-2 of Caenorhabditis elegans, we generated a homologous murine model by replacing Pro-1195 of insulin receptors with Leu using a targeted knock-in strategy. Homozygous mice died in the neonatal stage from diabetic ketoacidosis, whereas heterozygous mice showed the suppressed kinase activity of the insulin receptor but grew normally without spontaneously developing diabetes during adulthood. We examined heterozygous insulin receptor mutant mice for longevity phenotypes. Under 80% oxygen, mutant female mice survived 33.3% longer than wild-type female mice, whereas mutant male mice survived 18.2% longer than wild-type male mice. These results suggested that mutant mice acquired more resistance to oxidative stress, but the benefit of the longevity mutation was more pronounced in females than males. Manganese superoxide dismutase activity in mutant mice was significantly upregulated, suggesting that the suppressed insulin signaling leads to an enhanced antioxidant defense. To analyze the molecular basis of the gender difference, we administered estrogen to mutant mice. It was found that the survival of mice under 80% oxygen was extended when they were administered estradiol. In contrast, mutant and wild-type female mice showed shortened survivals when their ovaries were removed. The influence of estrogen is remarkable in mutant mice compared with wild-type mice, suggesting that estrogen modulates insulin signaling in mutant mice. Furthermore, we showed additional extension of survival under oxidative conditions when their diet was restricted. Collectively, we show that three distinct signals; insulin, estrogen, and dietary signals work in independent and cooperative ways to enhance the resistance to oxidative stress in mice.     

Odorant binding initially occurring at the central pocket in bovine odorant-binding protein

Why bovine odorant-binding protein (OBPb), among OBP family, assumes a dimeric structure has been unclear. Here we clarified, by measuring the fluorescence of intrinsic tryptophan and tyrosine residues of intact OBPb and OBPb whose C-terminal 10 amino acids were deleted, that odorant enters the central pocket formed by the dimerization when OBPb first encounters odorant, and odorant with high affinity with OBPb subsequently enters the internal cavity (suggested binding site), releasing the pre-bound odorant. The internal cavity-bound odorant can be released by the binding of other odorants at another internal cavity or at the central pocket, depending on the binding odorants. Due to this mechanism enabled by the dimerization, OBPb is more reactive than other monomeric OBPs.     

Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.