15-Cis-carotenoids found in the reaction center of a green sulfur bacterium Chlorobium tepidum and in the Photosystem I reaction center of a cyanobacterium Synechococcus vulcanus

Carotenoids were extracted, at 4 °C in complete darkness and under nitrogen atmosphere, from the reaction center (RC) of a green-sulfur bacterium and the Photosystem (PS) I RC of a cyanobacterium; each extract was subjected to high-performance liquid chromatography (HPLC) using an apparatus equipped with a two-dimensional diode-array detector in order to spectroscopically identify cis–trans carotenoids while performing HPLC analysis. In the extract from the RC of Chlorobium tepidum, 15-cis and all-trans--carotenes as well as 13-cis-, 15-cis- and all- trans-chlorobactenes (in the order of elution) were identified, whereas in the extract from the PS I RC of Synechococcus vulcanus, 15-cis-, all-trans- and 9-cis--carotenes were found. Thus, the universal presence of 15- cis carotenoids in the 'iron sulfur-type' RCs has been shown in addition to the previous cases of the 'quinone-type' RCs.     

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

Measuring Airway Surface Liquid Depth in Ex Vivo Mouse Airways by X-Ray Imaging for the Assessment of Cystic Fibrosis Airway Therapies

In the airways of those with cystic fibrosis (CF), the leading pathophysiological hypothesis is that an ion channel defect results in a relative decrease in airway surface liquid (ASL) volume, producing thick and sticky mucus that facilitates the establishment and progression of early fatal lung disease. This hypothesis predicts that any successful CF airway treatment for this fundamental channel defect should increase the ASL volume, but up until now there has been no method of measuring this volume that would be compatible with in vivo monitoring. In order to accurately monitor the volume of the ASL, we have developed a new x-ray phase contrast imaging method that utilizes a highly attenuating reference grid. In this study we used this imaging method to examine the effect of a current clinical CF treatment, aerosolized hypertonic saline, on ASL depth in ex vivo normal mouse tracheas, as the first step towards non-invasive in vivo ASL imaging. The ex vivo tracheas were treated with hypertonic saline, isotonic saline or no treatment using a nebuliser integrated within a small animal ventilator circuit. Those tracheas exposed to hypertonic saline showed a transient increase in the ASL depth, which continued for nine minutes post-treatment, before returning to baseline by twelve minutes. These findings are consistent with existing measurements on epithelial cell cultures, and therefore suggest promise for the future development of in vivo testing of treatments. Our grid-based imaging technique measures the ASL depth with micron resolution, and can directly observe the effect of treatments expected to increase ASL depth, prior to any changes in overall lung health. The ability to non-invasively observe micron changes in the airway surface, particularly if achieved in an in vivo setting, may have potential in pre-clinical research designed to bring new treatments for CF and other airway diseases to clinical trials.     

Severe hypercholesterolemia,impaired fat tolerance,and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.     

Daily growth increments in otoliths of wild-caught honmoroko Gnathopogon caerulescens

Otolith growth increments in wild-caught alizarin complex one (ALC)-marked honmoroko Gnathopogon caerulescens were examined to verify the veracity of the age determination method in cyprinids. ALC-marked G. caerulescens recaptured from their natural environment had lapilli increment counts outside the ALC ring mark that had formed on a daily basis during the juvenile stage. This apparently being the first direct evidence of daily periodicity of otolith increment formation in wild-caught cyprinids.     

Molecular cloning of urea transporters from the kidneys of baleen and toothed whales

Urea transport in the kidney is important for the production of concentrated urine. This process is mediated by urea transporters (UTs) encoded by two genes, UT-A (Slc14a2) and UT-B (Slc14a1). Our previous study demonstrated that cetaceans produce highly concentrated urine than terrestrial mammals, and that baleen whales showed higher concentrations of urinary urea than sperm whales. Therefore, we hypothesized that cetaceans have unique actions of UTs to maintain fluid homeostasis in marine habitat. Kidney samples of common minke (Balaenoptera acutorostrata), sei (B. borealis), Bryde's (B. brydei) and sperm whales (Physeter macrocephalus) were obtained to determine the nucleotide sequences of mRNAs encoding UT. The sequences of 2.5-kb cDNAs encode 397-amino acid proteins, which are 90-94% identical to the mammalian UT-A2s. Two putative glycosylation sites are conserved between the whales and the terrestrial mammals, whereas consensus sites for protein kinases are not completely conserved; only a single protein kinase A consensus site was identified in the whale UT-A2s. Two protein kinase C consensus sites are present in the baleen whale UT-A2s, however, a single protein kinase C consensus site was identified in the sperm whale UT-A2. These different phosphorylation sites of whale UT-A2s may result in the high concentrations of urinary urea in whales, by reflecting their urea permeability.     

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of β-actin and β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick β-actin and mouse β-tubulin, respectively. Both hybridizable β-actin and βtubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both β-actin and β-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of β-actin and β-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.