Response of a neotenic goby,ice goby (Leucopsarion petersii), to thyroid hormone and thiourea treatments

In order to clarify the mechanisms of neoteny in the ice goby (Leucopsarion petersii), we examined effects of thyroid hormone and thiourea (TU) treatments on their neotenic characteristics and the pituitary-thyroid axis. Adult ice goby were exposed to 3, 5, 3'-triiodothyronine (T3, 0.1 ppm), TU (inhibitor of thyroid hormone synthesis, 30 ppm), or the combination of the two for 2 weeks. Observations of whole body T3 levels, thyroid follicles and TSH immunoreactive cells in the pituitary suggests the presence of a functioning thyroidal system. However, all of the neotenic features did not disappear in T3-treated fish, suggesting the absence of T3 responsiveness in peripheral tissues. These results indicate the similarity between neoteny of the ice goby and obligatory-type neoteny of urodeles.     

A decreased expression of angiopoietin-like 3 is protective against atherosclerosis in apoE-deficient mice

KK/Snk mice (previously KK/San) possessing a recessive mutation (hypl) of the angiopoietin-like 3 (Angptl3) gene homozygously exhibit a marked reduction of VLDL due to the decreased Angptl3 expression. Recently, we proposed that Angptl3 is a new class of lipid metabolism modulator regulating VLDL triglyceride (TG) levels through the inhibition of lipoprotein lipase (LPL) activity. In this study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of hypl mutation against hyperlipidemia and atherosclerosis in apolipoprotein E knockout (apoEKO) mice. ApoEKO mice with hypl mutation (apoEKO-hypl) exhibited a significant reduction of VLDL TG, VLDL cholesterol, and plasma apoB levels compared with apoEKO mice. Hepatic VLDL TG secretion was comparable between both apoE-deficient mice. Turnover studies revealed that the clearance of both [3H]TG-labeled and 125I-labeled VLDL was significantly enhanced in apoEKO-hypl mice. Postprandial plasma TG levels also decreased in apoEKO-hypl mice. Both LPL and hepatic lipase activities in the postheparin plasma increased significantly in apoEKO-hypl mice, explaining the enhanced lipid metabolism. Furthermore, apoEKO-hypl mice developed 3-fold smaller atherogenic lesions in the aortic sinus compared with apoEKO mice. Taken together, the reduction of Angptl3 expression is protective against hyperlipidemia and atherosclerosis, even in the absence of apoE, owing to the enhanced catabolism and clearance of TG-rich lipoproteins.     

Contribution of capsaicin-sensitive sensory neurons to stress-induced increases in gastric tissue levels of prostaglandins in rats

We examined whether capsaicin-sensitive sensory neurons might be involved in the increase in the gastric tissue level of prostaglandins, thereby contributing to the reduction of water immersion restraint stress (WIR)-induced gastric mucosal injury in rats. Gastric tissue levels of calcitonin gene-related peptide (CGRP), 6-keto-PGF1alpha, and PGE2 were transiently increased 30 min after WIR. These increases were significantly inhibited by subcutaneous injection of capsazepine (CPZ), a vanilloid receptor antagonist, and by functional denervation of capsaicin-sensitive sensory neurons induced by the administration of high-dose capsaicin. The administration of capsaicin (orally) and CGRP (intravenously) significantly enhanced the WIR-induced increases in the gastric tissue level of prostaglandins 30 min after WIR, whereas CGRP-(8-37), a CGRP receptor antagonist, significantly inhibited them. Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of nitric oxide (NO) synthase (NOS), and that with indomethacin inhibited the WIR-induced increases in gastric tissue levels of prostaglandins, whereas either pretreatment with aminoguanidine (AG), a selective inhibitor of the inducible form of NOS, or that with NS-398, a selective inhibitor of cyclooxygenase (COX)-2, did not affect them. CPZ, the functional denervation of capsaicin-sensitive sensory neurons, and CGRP-(8-37) significantly increased gastric MPO activity and exacerbated the WIR-induced gastric mucosal injury in rats subjected to 4-h WIR. The administration of capsaicin and CGRP significantly increased the gastric tissue levels of prostaglandins and inhibited both the WIR-induced increases in gastric MPO activity and gastric mucosal injury 8 h after WIR. These effects induced by capsaicin and CGRP were inhibited by pretreatment with L-NAME and indomethacin but not by pretreatment with AG and NS-398. These observations strongly suggest that capsaicin-sensitive sensory neurons might release CGRP, thereby increasing the gastric tissue levels of PGI2 and PGE2 by activating COX-1 through activation of the constitutive form of NOS in rats subjected to WIR. Such activation of capsaicin-sensitive sensory neurons might contribute to the reduction of WIR-induced gastric mucosal injury mainly by inhibiting neutrophil activation.     

Heat shock induces intestinal-type alkaline phosphatase in rat IEC-18 cells

We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43 degrees C induced the expression of IAP-I and HSP72 mRNAs time dependently (<60 min) but did not induce expression of IAP-II, tissue nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by the RT-PCR method. To confirm the identity of the IAP-I gene, we sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in IEC-18 cells, but it became detectable as a 62-kDa band on a Western blot after heat shock. IAP-I was also induced by sodium arsenite, which generates reactive oxygen species and is an inducer of members of the HSP family. Glutathione suppressed activating protein-1 and cAMP response element-binding protein activation caused by heat shock but did not suppress the expression of IAP-I. These results suggest that cellular stress induces the elevation of IAP-I mRNA and protein synthesis. IAP-I may play an important role as a dephosphorylating enzyme under stress conditions.     

Detection thresholds for phenyl ethyl alcohol using serial dilutions in different solvents

Detection thresholds are typically obtained by presenting a subject with serial dilutions of an odorant. Many factors, including the solvent used to dilute the odorant, can influence the measurement of detection thresholds. Differences have been reported in detection thresholds for phenyl ethyl alcohol (PEA) when different solvents are used. In this study we used gas chromatography (GC) to investigate further the effect of solvent on odor detection thresholds. We used a single ascending method and serial dilutions of PEA in four different solvents--liquid paraffin (LP), mineral oil (MO), propylene glycol (PG) and dipropylene glycol (DPG)--to determine the PEA thresholds for 31 adult subjects. For each solvent, we prepared eight serial log base 10 step dilutions (1-8), with corresponding liquid PEA concentrations of 6.3 x 10(1)-6.3 x 10(-6) (% v/v). We found that the threshold concentrations for PEA in LP (step 6.5) and PEA in MO (step 5.5) were significantly lower (P < 0.05) than for PEA in PG (step 4.0) and DPG (step 4.0) We then used GC to measure both the liquid and gas PEA concentrations for the dilution steps prepared with LP and PG. Although there were large threshold differences in the liquid concentrations of PEA in LP and PG, the headspace gas concentrations of PEA were the same. These results demonstrate the importance of determining the gas concentration of odorant stimuli when performing odor threshold measurements, in particular when comparing odor detection thresholds obtained using different solvents.     

Molecular detection and direct enumeration of methanogenic Archaea and methanotrophic Bacteria in domestic solid waste landfill soils

Methane oxidizing and producing activities of cover soil (10, 30 cm depth) and burial waste (1, 3 m depth) were evaluated: top cover soil (10 cm) had the highest methane oxidizing activity, while 1 m depth buried waste showed the highest methane producing potential. All the sequences of the 1 m sample were found to be closely related to 16S rDNAs of mainly hydrogenotrophic methanogens known, such as genera Methanosarcina, Methanoculleus, and Methanobacterium. We developed a modified fluorescence in situ hybridization (FISH) direct counting method for landfill samples, resulting in the detection of approx. 1% of total cells as archaeal cells (presumably methanogens). However, probe-positive cells could not be found with probes for methanotrophs by the methods.     

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.     

Enantioresolution and absolute configurations of chiral meta-substituted diphenylmethanols as determined by X-ray crystallographic and 1H NMR anisotropy methods

meta-Substituted diphenylmethanols were enantioresolved by the method of chiral phthalic acid yielding enantiopure alcohols. Their absolute configurations were unambiguously determined by X-ray crystallography of chiral phthalate esters and/or by the (1)H NMR anisotropy method using 2-methoxy-2-(1-naphthyl)propionic acid.