Fasting-induced suppression of pulsatile luteinizing hormone secretion is related to body energy status in ovariectomized goats

The effect of energy status on the response of luteinizing hormone (LH) pulse frequency to acute short-term energy deficiency created by fasting in estradiol-treated ovariectomized Shiba goats was studied in two experiments. In experiment 1, eight goats whose mean body weight (BW) was 25.6 +/- 5.8 (mean +/- S.D.)kg were fed 500 g hay cubes daily for 1 week. Then they were fasted for 3 days. Blood samples were collected for 4 h at 6 min intervals on the last day of feeding, first, second and third day of fasting for LH analysis. The goats were divided into light (<24 kg, n = 4) and heavy (> or =24 kg, n = 4) groups for data analysis. There was no difference in LH pulse frequency between the last day of feeding and each day of fasting in the heavy group. LH pulse frequency was significantly (P < 0.05) suppressed on the second day (3.3 +/- 1.3 pulses/4 h) and on the third day (2.3 +/- 1.9 pulses/4 h) relative to the day prior to fasting (4.8 +/- 1.5 pulses/4 h) in the light group. In experiment 2, BW plus a body mass index (gBMI: (body weight (kg)/withers height (m)/body length (m)) x 10) were measured to define energy status. Nine goats (BW, 25.6 +/- 5.8 kg) were fed 500 g hay cubes daily for a week and then fasted for 3 days. Then they were divided into two groups offered either a maintenance (n = 4) or a restricted (n = 5) level of feeding for 4 weeks. The restricted level of feeding was 30% of maintenance requirement based on the BW recorded weekly. The feeding level was then adjusted to maintain BW for a further week followed by 3 day fasting for restricted animals. Blood samples were collected for 6 h at 10 min intervals on the day prior to fasting and on third day of fasting before and after the dietary manipulation. BW (26.6 +/- 2.2 to 26.8 +/- 3.8 kg) and gBMI (8.4 +/- 0.4 to 7.8 +/- 0.3) remained constant over the period prior to fasting for the maintenance animals but were significantly lower (P < 0.05) after 4 weeks for the restricted goats (BW, 26.3 +/- 2.1 to 21.5 +/- 2.4 kg; gBMI, 8.4 +/- 0.9 to 6.9 +/- 0.7). There was no significant difference in the LH pulse frequency between feeding and fasting day in both sampling periods in the maintenance group. In the restricted group, LH pulse frequency was not suppressed by fasting in the first sampling period (6.8 +/- 2.9 to 5.2 +/- 2.5 pulses/6 h), whereas it tended to be suppressed (4.8 +/- 3.1 to 1.6 +/- 2.3 pulses/6 h; P < 0.06) and was significantly (P < 0.05) correlated to body weight (r = 0.70) and gBMI (r = 0.81) after the dietary manipulation. These results suggest that the suppressive effect of short-term energy restriction (fasting) on pulsatile LH secretion is related to body energy status.     

Genetic analysis of rabies virus isolates in the Philippines

To determine the genetic characteristics of the rabies virus in the Philippines, 59 rabies virus isolates were obtained from domestic rabid dogs and their partial nucleotide sequences of nucleoprotein (N) gene were compared. Based on comparison with reported sequences, phylogenetic analysis revealed that all isolates from the Philippines had close genetic relations and formed two subgroups. The Philippines isolates belonged to a different lineage from other Asian isolates but were closer to them than to terrestrial isolates and laboratory strains. Several specific nucleotide and amino acid substitutions were observed among the Philippines isolates. Our results suggest that rabies viruses in the Philippines might have a characteristic evolution.     

Investigation of parenchymal cell differentiation in organotypic slice culture of mouse fetal liver under administration of sodium butyrate

The fetal mouse liver tissues in our organotypic slice culture were spread and flattened for at least 3 weeks; small, round cells were distributed in the center and polygonal cells were seen in the periphery. Ultrastructurally, polygonal cells showed abundant rough endoplasmic reticulum and mitochondria. They expressed albumin (ALB) and α-fetoprotein (AFP) for at least 3 weeks, and Cx32-immunoreactivity was also seen in a plaque on the cells. Many proliferating cell nuclear antigen (PCNA)-positive cells were observed at the periphery, and there were scattered CK-19-positive cells. The spreading of the fetal liver tissue in organotypic slice culture was reduced in medium containing sodium butyrate (SB). The expression of ALB was well maintained in polyglonal cells of the SB(+) group 3 weeks after culture and AFP-immunoreactivity was decreased in the SB(+) group. The concentration of ALB in the medium was significantly higher in the SB(+) than in the SB(-) group. CK-19-positive cells in the SB(+) group were increased in number more than those in the SB(-) group. PCNA-positive cells were less numerous in the SB(+) group, and Cx32-positive plaques were increased. SB can help immature hepatocytes to differentiate into the mature type and the cholangiocytic lineage, reducing their proliferation. These findings suggest that parenchymal cells in our organotypic slice culture of the fetal mouse liver can maintain structure and function as in vivo for the long term, and SB is shown to be a differentiation inducer of parenchymal cells in the slice culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cholesterol modulates interaction between an amphipathic class A peptide, Ac-18A-NH2, and phosphatidylcholine bilayers

Cholesterol (Chol) in phosphatidylcholine large unilamellar vesicles (PC LUV) modulated interaction of the bilayers with a class A amphipathic peptide, Ac-18A-NH2: Chol increased the peptide binding capacity and reduced the affinity together with the peptide-induced leakage of calcein from LUV. Similar effects of Chol have been observed on the interaction of LUV with apoA-I [Saito, H., Miyako, Y., Handa, T., and Miyajima, K. (1997) J. Lipid Res. 38, 287-294]. Circular dichroism (CD) spectra of the peptide indicated a similar helical structure formation in LUV with and without Chol. The fluorescence spectral shift, quantum yield, anisotropy, and acrylamide-quenching of the peptide Trp indicated that in PC:Chol (3:2) LUV, Ac-18A-NH2 was located in a more polar membrane environment with increased motional freedom and greater accessibility to the aqueous medium. Fluorescence energy transfer from the Trp indole ring to acceptors situated at different depths in the bilayers revealed that the amphipathic peptide penetrated the hydrophobic interior of PC bilayers, while the peptide was located at the polar zwitterionic surface in PC:Chol LUV. The inclusion of Chol causes the headgroup separation of PC at the surface of LUV and increases the binding maximum of the wedge-shaped amphipathic peptide without disrupting the membrane structure. In addition, the rigidifying effect of Chol on PC acyl chains prevents the penetration of the peptide into the bilayer interior. These findings imply that Chol in membranes affects the binding and motional freedom of exchangeable plasma apolipoproteins containing class A amphipathic sequences, e.g., apoA-I and apoCs.     

Refinement of wingless expression by a wingless- and notch-responsive homeodomain protein,defective proventriculus

Pattern formation during animal development is often induced by extracellular signaling molecules, known as morphogens, which are secreted from localized sources. During wing development in Drosophila, Wingless (Wg) is activated by Notch signaling along the dorsal-ventral boundary of the wing imaginal disc and acts as a morphogen to organize gene expression and cell growth. Expression of wg is restricted to a narrow stripe by Wg itself, repressing its own expression in adjacent cells. This refinement of wg expression is essential for specification of the wing margin. Here, we show that a homeodomain protein, Defective proventriculus (Dve), mediates the refinement of wg expression in both the wing disc and embryonic proventriculus, where dve expression requires Wg signaling. Our results provide evidence for a feedback mechanism that establishes the wg-expressing domain through the action of a Wg-induced gene product.     

Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens)

A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.     

Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity

DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G(2)/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.     

Detection of genetic alterations induced by low-dose X rays: analysis of loss of heterozygosity for TK mutation in human lymphoblastoid cells

To elucidate the genetic influence of low-dose ionizing radiation at the chromosome level, we exposed human lymphoblastoid TK6-20C cells to 10 cGy of X rays. The TK mutation frequency was 5.7 +/- 1.3 x 10(-6) at the background level and 6.9 +/- 2.8 x 10(-6) after X irradiation. Although this small increase was not statistically significant (P = 0.40), we applied multilocus analysis using 4 TK locus markers and 12 microsatellite loci spanning chromosome 17 for TK mutants exhibiting loss of heterozygosity (LOH). The analysis demonstrated a clear effect of low-dose ionizing radiation. We observed radiation-specific patterns in the extent of hemizygous LOH in 14 TK mutants among the 92 mutants analyzed. The deleted regions in these patterns were larger than they were in the control mutants, where those restricted to the TK locus. Surprisingly, the radiation-specific LOH patterns were not observed among the 110 nonirradiated TK mutants in this study. They were identified previously in TK6 cells exposed to 2 Gy of X rays. We consider these hemizygous LOH mutants to be a result of end-joining repair of X-ray-induced DNA double-strand breaks.