Functional evolution of duplicated odorant-binding protein genes, Obp57d and Obp57e, in Drosophila

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in the sensing of odors, tastes, and pheromones. Although a large number of OBP genes have been identified in insect genomes, their molecular functions and biological roles have been clarified in limited cases. Two OBP genes, Obp57d and Obp57e, were involved in the evolution of host-plant preference in Drosophila sechellia. Comparative analyses of the Obp57d/e genomic sequences from 27 closely related species suggested that the two genes arose by tandem gene duplication and functionally diverged from each other. In this study, the functional evolution of Obp57d and Obp57e was examined by in vitro binding assays using recombinant proteins synthesized in a bacterial system. Compared to the ancestral Dpse\OBP57de, Dmel\OBP57d was more specialized to tridecanoic acid while Dmel\OBP57e was generalized regarding their binding affinity, suggesting that the two OBP genes underwent subfunctionalization and neofunctionalization. A behavioral analysis using knockout flies supported that the biological role is different between OBP57d and OBP57e in vivo. Site-directed mutagenesis of the evolutionarily conserved amino acids revealed that these residues play an important role in protein folding. These findings provide a clue to understanding how the repertoire of OBP genes is maintained in a genome under natural selection.     

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue,bone marrow,and skeletal muscle tissue

The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.     

Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.     

Endothelium-dependent vasorelaxation effect of rutin-free tartary buckwheat extract in isolated rat thoracic aorta

The aim of this study was to point out the potential of tartary buckwheat on vascular functions. A nonabsorbed fraction of hot-water extract of tartary buckwheat on a SP70 column (TBSP-T), which was free from rutin, was used for this aim. In a contractile experiment using Sprague-Dawley rat thoracic aorta rings contracted by 1.0 microM phenylephrine (PE) or 50 mM KCl, TBSP-T evoked a significant vasorelaxation [EC50 (mg/ml): PE; 2.2; KCl, 1.9]. By a further fractionation of TBSP-T by liquid-liquid partitioning into basic, neutral and acidic fractions, a marked enhancement of vasorelaxation effect was observed only for acidic fraction (EC50, 0.25 mg/ml). The action of acidic fraction was significantly attenuated in endothelium-denuded aortic rings and in the presence of nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 microM). The fraction also enhanced the cyclic guanosine monophosphate (cGMP) production in aortic rings contracted with PE [cGMP (pmol/mg protein): PE, 7.2+/-2.3; PE+Acidic fraction, 35+/-8]. These results indicate that acidic fraction could mediate NO/cGMP pathways, thereby exerting endothelium-dependent vasorelaxation action. In conclusion, tartary buckwheat was proven to regulate vascular tones and have latent acidic candidates except for rutin.     

Albumin suppresses vascular endothelial growth factor via alteration of hypoxia-inducible factor/hypoxia-responsive element pathway

Reduction of vascular endothelial growth factor (VEGF) expression plays a crucial role in chronic kidney disease (CKD). In order to clarify a cause of VEGF suppression in CKD, we examined an interaction between proteinuria and VEGF. Rat proximal tubular cells were subjected to hypoxia with or without albumin to mimic proteinuric conditions, and VEGF expression was assessed by real-time quantitative PCR and enzyme-linked immunosorbent assays. Albumin significantly reduced VEGF expression under hypoxia. Luciferase activity controlled by hypoxia-responsive element (HRE) was suppressed by albumin, demonstrating suppression of the hypoxia-inducible factor (HIF)/HRE pathway. Studies utilizing a proteasome inhibitor and a prolyl hydroxylase inhibitor showed that mechanisms of HIF/HRE pathway suppression by albumin load did not involve degradation of HIF protein levels. Further, albumin did not change HIF mRNA levels. Our data, for the first time, suggest a clear ‘link’ between proteinuria and hypoxia, the two principal pathogenic factors for CKD progression.     

Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145-amino acid residues with extensive alpha-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.     

Grazing impact of microzooplankton on a diatom bloom in a mesocosm as estimated by pigment-specific dilution technique

To investigate the impact of microzooplankton grazing on phytoplankton bloom in coastal waters, an enclosure experiment was conducted in Saanich Inlet, Canada during the summer of 1996. Daily changes in the microzooplankton grazing rate on each phytoplankton group were investigated with the growth rates of each phytoplankton group from the beginning toward the end of bloom using the dilution technique with high-performance liquid chromatography (HPLC). On Day 1 when nitrate and iron were artificially added, chlorophyll a concentration was relatively low (4.3 μg l⁻¹) and 19′-hexanoyloxyfucoxanthin-containing prymnesiophytes were predominant in the chlorophyll biomass. However, both the synthetic rates and concentrations of 19′-hexanoyloxyfucoxanthin declined before bloom, suggesting that 19′-hexanoyloxyfucoxanthin-containing prymnesiophytes weakened. Chlorophyll a concentration peaked at 23 μg l⁻¹ on Day 4 and the bloom consisted of the small chain-forming diatoms Chaetoceros spp. (4 μm in cell diameter). Diatoms were secondary constituents in the chlorophyll biomass at the beginning of the experiment, and the growth rates of diatoms (fucoxanthin) were consistently high (>0.5 d⁻¹) until Day 3. Microzooplankton grazing rates on each phytoplankton group remarkably increased except on alloxanthin-containing cryptophytes after the nutrient enrichments, and peaked with >0.6 d⁻¹ on Day 3, indicating that >45% of the standing stock of each phytoplankton group was removed per day. Both the growth and mortality rates of alloxanthin-containing cryptophytes were relatively high (>1 and >0.5 d⁻¹, respectively) until the bloom, suggesting that a homeostatic mechanism might exist between predators and their prey. Overall, microzooplankton grazing showed a rapid response to the increase in phytoplankton abundance after the nutrient enrichments, and affected the magnitude of the bloom significantly. High grazing activity of microzooplankton contributed to an increase in the abundance of heterotrophic dinoflagellates with 7-24 μm in cell size, the fraction of large-sized (>10 μm) chlorophyll a, and stimulated the growth of larger-sized ciliates after the bloom.     

Galectin-9 in physiological and pathological conditions

We first cloned galectin-9 (Gal-9)/ecalectin as a T cell-derived eosinophil chemoattractant. Gal-9 plays a role in not only accumulation but also activation of eosinophils in experimental allergic models and human allergic patients, because Gal-9 induces eosinophil chemoattraction in vitro and in vivo and activates eosinophils in many aspects. Gal-9 requires divalent galactoside-binding activity but not the linker peptide of Gal-9 to exhibit its biological functions, and an unidentified matrix metalloproteinase is involved in the release of Gal-9. Our recent studies also showed that Gal-9 has other functions, such as cell differentiation, aggregation, adhesion, and death. Now, we and other groups are on the way of investigating the regulation and function of Gal-9 in a variety of physiological and pathological conditions. In this article, we will show the possible role of Gal-9 in physiological and pathological conditions by using our recent findings. Published in 2004.     

Glucocorticoid enhances Na(+)/K(+) ATPase mRNA expression in rat olfactory mucosa during regeneration: a possible mechanism for recovery from olfactory disturbance.

Systemic or topical application of glucocorticoid is the treatment of choice for olfactory disturbance. Recently, Na(+)/K(+) ATPase and glucocorticoid receptor immunoreactivity in the olfactory mucosa was reported. To elucidate a glucocorticoid action on Na(+)/K(+) ATPase production, an animal model was produced by an intra-nasal application of 5% ZnSO(4) solution to Wistar rats. Dexamethasone was injected i.p. (0.01 mg/100 g) for 14 days after the insult. Histologically, the regeneration process was completed on day 14 in both dexamethasone- and saline-injected control rats. We used a quantitative polymerase chain reaction (PCR) method to evaluate mRNA production of Na(+)/K(+) ATPase and glucocorticoid receptor. In dexamethasone-injected rats, up-regulation of glucocorticoid receptor mRNA (95% more than control rats, P = 0.00068, unpaired t-test) and of Na(+)/K(+) ATPase mRNA expression (76% more than control rats, P = 0.0042) was observed on day 14. The increased Na(+)/K(+) ATPase expression in the regenerated olfactory mucosa is thought to be beneficial for an active uptake of K(+), which is released during excitation, around olfactory neurons and for the transepithelial absorption of Na(+) from olfactory mucus. Dexamethasone may thus contribute to the recovery of function after the morphological regeneration in part, at least, through its receptor by regulation of the ionic concentration in the olfactory mucosal microenvironment.