Tissue-specific replicating capacity of a chimeric poliovirus that carries the internal ribosome entry site of hepatitis C virus in a new mouse model transgenic for the human poliovirus receptor

Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new mouse model (MPVRTg25-61) that is transgenic for human PV receptor (hPVR; CD155) was generated in order to obtain a higher expression level of hPVR in the liver than those of hPVRTg mouse lines generated by us so far. The transgene used was constructed by combining a putative regulatory region of the mouse PVR homolog and the whole structural region of the hPVR gene. Virus 2A-369 replicated well in the liver of MPVRTg25-61 but not in the brain, whereas control Mahoney virus replicated well both in the liver and in the brain. The data suggest that the HCV IRES works more efficiently in the liver than in the brain and that PV IRES works well both in the liver and in the brain. The results support the notion that tissue-specific activity of IRES may be reflected in tissue tropism of a virus whose specific translation initiation is driven by IRES, that is, an IRES-dependent virus tropism.     

Ovarian dynamics and their associations with peripheral concentrations of gonadotropins,ovarian steroids,and inhibin during the estrous cycle in goats

Ovarian changes determined by daily transrectal ultrasound and its relationship with FSH, LH, estradiol-17beta, progesterone, and inhibin were investigated in six goats for three consecutive interovulatory intervals. Estrous cycles were synchronized using two injections of prostaglandin F2alpha analogue 11 days apart. All follicles 3 mm or greater in diameter and corpora lutea were measured daily. A follicular wave was defined as one or more follicles growing to 5 mm or greater in diameter. The day that the follicles reached 3 mm in diameter was defined as the day of wave emergence, and the first wave after ovulation was defined as wave 1. During the interovulatory interval (mean +/- SEM, 21.3 +/- 0.4 days; n = 18), follicular waves emerged at 0.3 +/- 0.5, 6.5 +/- 0.2, and 12.1 +/- 0.4 days for wave 1, wave 2, and wave 3, respectively, in goats with three waves of follicular development and at -0.6 +/- 0.3, 4.7 +/- 0.2, 9.4 +/- 0.5, and 13.4 +/- 0.5 days for wave 1, wave 2, wave 3, and wave 4, respectively, in goats with four waves of follicular development (Day 0 = the day of ovulation). The mean diameter of the largest follicle of the ovulatory wave was significantly larger than those of the largest follicles of the other waves. Corpora lutea could be identified ultrasonically at Day 3 postovulation and attained 12.1 +/- 0.3 mm in diameter on Day 8. Transient increases in plasma concentrations of FSH were detected around the day of follicular wave emergence. The level of FSH was negatively correlated with that of inhibin. These results demonstrated that follicular waves occurred in goats and that the predominant follicular wave pattern was four waves with ovulation from wave 4. These results also suggested that the emergence of follicular waves was closely associated with increased secretion of FSH.     

Characterization of rat follistatin-related gene: effects of estrous cycle stage and pregnancy on its messenger RNA expression in rat reproductive tissues

Follistatin-related gene (FLRG) was first identified as a target of a chromosomal translocation in a human B-cell leukemia. Because FLRG protein binds to activins and bone morphogenetic proteins, FLRG is postulated to be a regulator of these growth factors. However, physiological aspects of FLRG are unclear. To elucidate the physiology of FLRG, we examined expression of FLRG in reproductive tissues of the rat. FLRG mRNA was abundantly expressed in the placenta. FLRG mRNA was also expressed in the ovary, uterus, testis, lung, adrenal gland, pituitary, kidney, small intestine, and heart. During the second half of pregnancy, expression of FLRG in the placenta continuously increased, whereas follistatin mRNA levels decreased from Day 12 to Day 14 and remained low thereafter. FLRG was also expressed in decidua. Levels of decidual FLRG mRNA remained low from Day 12 to Day 16 and then noticeably increased until Day 20. In contrast, follistatin mRNA was highly expressed in the decidua on Day 12, continuously decreased until Day 16, and then remained at relatively low levels thereafter. During the rat estrous cycle, levels of ovarian FLRG mRNA fluctuated diurnally, with highest levels during daytime, and did not change relative to the day of the estrous cycle. The present results suggest that FLRG may play a role in the regulation of reproductive events.     

Solution structure of epiregulin and the effect of its C-terminal domain for receptor binding affinity

Epiregulin (EPR), a novel member of epidermal growth factor (EGF) family, is a ligand for ErbB-1 and ErbB-4 receptors. The binding affinity of EPR for the receptors is lower than those of other EGF-family ligands. The solution structure of EPR was determined using two-dimensional nuclear magnetic resonance spectroscopy. The secondary structure in the C-terminal domain of EPR is different from other EGF-family ligands because of the lack of hydrogen bonds. The structural difference in the C-terminal domain may provide an explanation for the reduced binding affinity of EPR to the ErbB receptors.     

Chemical structure and immunobiological activity of lipid A from Prevotella intermedia ATCC 25611 lipopolysaccharide

The novel chemical structure and immunobiological activities of Prevotella intermedia ATCC 25611 lipid A were investigated. A lipopolysaccharide (LPS) preparation of P. intermedia was extracted using a phenol-chloroform-petroleum ether method, after which its purified lipid A was prepared by weak acid hydrolysis followed by chromatographic separations. The lipid A structure was determined by mass spectrometry and nuclear magnetic resonance to be a diglucosamine backbone with a phosphate at the 4-position of the non-reducing side sugar, as well as five fatty acids containing branched long chains. It was similar to that of Bacteroides fragilis and Porphyromonas gingivalis, except for the phosphorylation site. P. intermedia lipid A induced weaker cytokine production and NF-kappaB activation in murine cells via Toll-like receptor (TLR) 4 as compared to Escherichia coli synthetic lipid A (compound 506). Our results indicate that P. intermedia lipid A activates cells through a TLR4-dependent pathway similar to E. coli-type lipid A, even though these have structural differences.     

Production of poly-β-hydroxybutyrate by thermophilic cyanobacterium, Synechococcus sp. MA19, under phosphate-limited conditions

Synechococcus sp. MA19, grown autotrophically under phosphate-limited conditions at 50 °C, produced poly--hydroxybutyrate (PHB) when intracellular phosphate content was 0.043–0.076mmol per g of cellular components. In the culture for 260h using Ca₃(PO₄)₂ as a phosphate source, strain MA19 accumulated PHB at 55% (w/w) of the dry cells and the amount of PHB produced was 2.4gl^–1 which was almost twice that without Ca₃(PO₄)₂ addition.     

Bisphenol A inhibits testicular functions and increases luteinizing hormone secretion in adult male rats

Effects of a xenobiotic estrogen, bisphenol A (BPA), on reproductive functions were investigated using adult male rats. BPA was dissolved into sesame oil and injected s.c. every day (1 mg/rat) for 14 days. Animals were killed by decapitation after the final administration of BPA, and the trunk blood, pituitary, and testes were collected. Plasma concentrations of prolactin were dramatically increased and pituitary contents of prolactin were slightly increased in the BPA group compared to the control group. Plasma concentrations of testosterone were decreased and plasma concentrations of LH were increased in BPA-treated rats compared to control rats. Testicular contents of inhibin were decreased in BPA-treated rats compared to control rats, although plasma concentrations of inhibin were not changed after administration of BPA. The testicular response to hCG for progesterone and testosterone release was decreased in BPA-treated rats. Administration of BPA did not change the pituitary response to luteinizing hormone-releasing hormone (LH-RH) in castrated male rats treated with testosterone. Male sexual behavior also was not changed as a result of BPA treatment. These results suggest that BPA directly inhibits testicular functions and the increased level of plasma LH is probably due to a reduction in the negative feedback regulation by testosterone. The testis is probably a more sensitive site for BPA action than the hypothalamus-pituitary axis.     

La(3+) and Gd(3+) induce shape change of giant unilamellar vesicles of phosphatidylcholine

Lanthanides such as La(3+) and Gd(3+) are well known to have large effects on the function of membrane proteins such as mechanosensitive ionic channels and voltage-gated sodium channels, and also on the structure of phospholipid membranes. In this report, we have investigated effects of La(3+) and Gd(3+) on the shape of giant unilamellar vesicle (GUV) of dioleoylphosphatidylcholine (DOPC-GUV) and GUV of DOPC/cholesterol by the phase-contrast microscopy. The addition of 10-100 microM La(3+) (or Gd(3+)) through a 10-microm diameter micropipette near the DOPC-GUV (or DOPC/cholesterol-GUV) triggered several kinds of shape changes. We have found that a very low concentration (10 microM) of La(3+) (or Gd(3+)) induced a shape change of GUV such as the discocyte via stomatocyte to inside budded shape transformation, the two-spheres connected by a neck to prolate transformation, and the pearl on a string to cylinder (or tube) transformation. To understand the effect of these lanthanides on the shape of the GUV, we have also investigated phase transitions of 30 microM dipalmitoylphosphatidylcholine-multilamellar vesicle (DPPC-MLV) by the ultra-sensitive differential scanning calorimetry (DSC). The chain-melting phase transition temperature and the L(beta') to P(beta') phase transition temperature of DPPC-MLV increased with an increase in La(3+) concentration. This result indicates that the lateral compression pressure of the membrane increases with an increase in La(3+) concentration. Thereby, the interaction of La(3+) (or Gd(3+)) on the external monolayer membrane of the GUV induces a decrease in its area (A(ex)), whereas the area of the internal monolayer membrane (A(in)) keeps constant. Therefore, the shape changes of the GUV induced by these lanthanides can be explained reasonably by the decrease in the area difference between two monolayers (DeltaA=A(ex)-A(in)).     

Viability of plant hairy roots is sustained without propagation in low sugar medium kept at ambient temperature

The effect of sucrose concentration in the medium on the growth and resumption ability to form lateral roots was investigated using the hairy roots of pak-bung and tobacco. It was found that the growth evaluated by root tip elongation of pak-bung and tobacco hairy roots was suppressed in the medium having an initial sucrose concentration of <2.5kg/m(3), and that the resumption abilities of both the hairy roots could be preserved when the hairy roots were kept at an initial sucrose concentration of 2.5kg/m(3) under ambient temperature conditions. The values of maintenance energy for pak-bung and tobacco hairy roots were determined to be 0.11 and 0.12 per day, respectively, from the total sugar consumption rates. Under the oligotrophic condition of the sucrose concentration of 2.5kg/m(3), the hairy roots were considered to exist as resting cells with maintenance metabolism, and the minimum demand for the energy source to ensure survival of the cells was met because the cells hardly multiplied and sugar consumption was not significant. In addition, long-term storage of pak-bung hairy roots in the liquid medium with 2.5kg/m(3) sucrose was performed at 25 degrees C. It was demonstrated that the hairy roots could maintain their resumption abilities without a serious loss of viability over 600 days and that the number of budding lateral roots per unit length of the main roots remained a value of 72 roots/m after the 600-day storage.