Cephalometric assessment of craniofacial morphology in Japanese male patients with obstructive sleep apnea-hypopnea syndrome

Sleep and Biological Rhythms - Craniofacial morphological anomalies can be divided into two principal categories: skeletal anomalies and soft tissue anomalies. This study examined the hypothesis...     

Morphological and functional characterization of a novel Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase-immunoreactive,follicle-like structure on the gill septum of Japanese banded houndshark, <Emphasis Type="Italic">Triakis scyllium</Emphasis>

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranch fish, however, the ionoregulatory function of the gills is still poorly understood. Although mitochondria-rich (MR) cells have also been found in elasmobranch fish, these cells are considered to function primarily in acid-base regulation. In this study, we found a novel aggregate structure made up of cells with basolaterally-expressed Na⁺/K⁺-ATPase (NKA), in addition to NKA-immunoreactive MR cells that have already been described in the gill filament and lamella. The cell aggregates, named follicularly-arranged NKA-rich cells (follicular NRCs), were found exclusively in the epithelial lining of the venous web in the cavernous region of the filament and the inter-filamental space of the gill septum. The follicular NRCs form a single-layered follicular structure with a large lumen leading to the external environment. The follicular NRCs were characterized by: (i) well-developed microvilli on the apical membrane, (ii) less prominent infoldings of the basolateral membrane and (iii) typical junction structures including deep tight junction between cells. In addition, large numbers of vesicles were observed in the cytoplasm and some of them were fused to the lateral membrane. The follicular NRCs expressed Na⁺/H⁺ exchanger 3 and Ca²⁺ transporter 1. The follicular NRCs thus have the characteristics of absorptive ionoregulatory cells and this suggests that the elasmobranch gill probably contributes more importantly to body fluid homeostasis than previously thought.     

Molecular assembly and dynamics of fluorescent protein-tagged single KCa1.1 channel in expression system and vascular smooth muscle cells

The large-conductance Ca(2+)-activated K(+) (K(Ca)1.1, BK) channel has pivotal roles in the regulation of vascular tone. To clarify the molecular dynamics of BK channels and their functionally coupled protein on the membrane surface, we examined single-molecule imaging of fluorescent-labeled BK subunits in the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. The dynamic mobility of yellow fluorescent protein (YFP)-tagged BKα subunit (BKα-YFP) expressed in human embryo kidney 293 (HEK) cells was detected in TIRF regions at the level of individual channels and their clusters on the plasma membrane with a diffusion coefficient of 6.7 × 10(3) nm(2)/s. When BKα-YFP was coexpressed with cyan fluorescent protein (CFP)-tagged BKβ1 subunit (BKβ1-CFP) in HEK cells, the mobility was reduced by ～50%. Fluorescent image analyses suggest that green fluorescent protein (GFP)-tagged BKα subunit (BKα-GFP) expressed in vascular smooth muscle cells (VSMCs), at low density, preferentially formed a heterotetrameric molecular assembly with native BKα subunits, rather than homotetrameric BKα-GFP. Movement of BKα-YFP in VSMCs (0.29 × 10(3) nm(2)/s) was far more restricted than BKα-YFP/BKβ1-CFP in HEK cells (2.5 × 10(3) nm(2)/s). Actin disruption by pretreatment with cytochalasin D in VSMCs appeared to increase the mobile behavior of BKα-YFP, which was then significantly reduced by addition of jasplakinolide. Most BKα-YFP colocalized with caveolin 1 (Cav1)-CFP in VSMCs, but unexpectedly not frequently in HEK cells. Fluorescence resonance energy transfer analyses showed the direct interaction between BKα-YFP and Cav1-CFP, particularly in VSMCs. These results, obtained by single molecule imaging in living cells, indicate that the dynamics of BKα molecules on the membrane surface are strongly restricted or regulated by its auxiliary β-subunit, cytoskeleton, and direct interaction with Cav1 in VSMCs.     

Collagen type IV-specific tripeptides for selective adhesion of endothelial and smooth muscle cells

Controlling the balance of endothelial cells (ECs) and smooth muscle cells (SMCs) in blood vessels is critically important to minimize the risk associated with vascular implants. Extracellular matrix (ECM) plays a key role in controlling the cellular balance, suggesting a promising source of cell-selective peptides. To obtain EC- or SMC-selective peptides, we start by highlighting sequence differences found among ECM molecules as enriched targets for cell-selective peptides. We explored the EC- or SMC-selective performance of tripeptides that are specifically enriched only in collagen type IV, but not in types I, II, III, and V. Collagen type IV was chosen since it is the major ECM in the basement membrane of blood vessels, which separates ECs and SMCs. Among 114 collagen type IV-derived tripeptides pre-screened from in silico analysis, 22 peptides (19%) were found to promote cell-selective adhesion, as determined by peptide array. One of the best performing EC-selective peptides (Cys-Ala-Gly (CAG)) was mixed into an electrospun fine-fiber, a vascular graft material, for practical application. Compared to unmodified fiber, the CAG containing fiber surface was found to enhance adhesion of ECs (+190%) while limiting SMCs (-20%). These results are not only consistent with the hypothesis of ECM as a source of cell selective peptides, but also suggest a new genre of EC- or SMC-selective peptides for tissue engineering applications. Collectively, these findings favorably support the screening approach used to discover new peptides for these purposes.     

A sensitive cell-based method to screen for selective inhibitors of SMS1 or SMS2 using HPLC and a fluorescent substrate

Recent studies have revealed that sphingomyelin (SM) is involved in metabolic syndrome and is a new target of an anti-metabolic syndrome drug. Deficiencies in the enzyme SM synthase 1 (SMS1) result in severe abnormalities, whereas deficiencies in SMS2 do not. SMS1 and SMS2 synthesize SM under similar conditions, so their respective activities cannot be measured separately. We report here on a sensitive, high-throughput and reliable cell-based method to separately measure each SMS activity and to screen for SMS-specific inhibitors, using HPLC and fluorescent ceramide (Cer) analogs. We isolated SMS-null cells and stably transfected them with SMS1 or SMS2. Using these cells, individual SMS activities could be measured separately. Fluorescent Cer, SM, and glucosylceramide analogs could be separated within 4 min by HPLC using an NH₂ column. SMS activities of SMS1- or SMS2-expressing cells seeded in a single well of a 96-well plate could be measured using HPLC and fluorescent Cer analogs. This method clearly demonstrated that treatment of the cells with their respective siRNA or D609, an inhibitor of SMS, resulted in a significant decrease in each SMS activity. These results indicate that our newly developed method can be utilized for screening therapeutics against metabolic syndrome that target SMS2.     

Development and application of a method for identification of isothiocyanate-targeted molecules in colon cancer cells

In this study, we have developed a novel method to identify isothiocyanate (ITC)-targeted molecules using two well-studied ITCs: benzyl ITC (BITC) and phenethyl ITC (PEITC). The principle of this method is based on identifying a pattern of differences between BITC and PEITC given that they show similar chemical and biological behaviors. For method validation, dithiothreitol-reduced bovine insulin as a model molecule was incubated with either BITC or PEITC, and digested peptides were analyzed by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) and liquid chromatography quadrupole TOF-MS (LC-Q-TOF-MS). Three peptides-NYCN, FVNQHLCGSHLVE, and ALYLVCGE-were identified as being adducted with BITC or PEITC on their cysteine residues. Each set of peptides adducted with either BITC or PEITC showed retention times (RT(BITC)相似文献   

Induction of mitochondrial uncoupling enhances VEGF₁₂₀ but reduces MCP-1 release in mature 3T3-L1 adipocytes: possible regulatory mechanism through endogenous ER stress and AMPK-related pathways

Although white adipocytes contain a larger number of mitochondria per cytoplasmic volume, adipocyte mitochondrial uncoupling to reduce the efficiency of ATP production on cellular function including secretory regulation of bioactive molecules such as VEGF and MCP-1 remains to be elucidated. Here we induce mitochondrial uncoupling under hypoxia-independent conditions in mature 3T3-L1 adipocytes using a metabolic uncoupler, dinitrophenol (DNP). MCP-1 release was significantly decreased by 26% (p<0.01) in 24h DNP (30 μmol/L)-treated adipocytes compared to control cells. In contrast, secreted VEGF(120) lacking a heparin-binding domain was markedly increased 2.0-fold (p<0.01). CHOP content in these cells also were augmented (p<0.01), but no significant increase of endogenous oxidative stress was observed. Treatment with thapsigargin, which can induce exogenous endoplasmic reticulum (ER) stress, clearly attenuated MCP-1 release (p<0.01), but exhibited no effects on VEGF(120) secretion. On the other hand, exogenous H(2)O(2) amplified both MCP-1 and VEGF(120) secretion (p<0.05). In addition, under chronic activation of AMPK by AICAR, MCP-1 release was significantly diminished (p<0.05) but VEGF(120) secretion was increased (p<0.01). JNK phosphorylation in mature adipocytes was decreased by treatment with either DNP or AICAR (p<0.01). Enhanced VEGF(120) secretion with either DNP or AICAR was markedly suppressed by PI3K inhibitor LY294002 (p<0.01). Thus, induced mitochondrial uncoupling in adipocytes can reduce MCP-1 release through induction of endogenous ER stress and by reduced JNK activities via chronic activation of AMPK. Under this condition, VEGF(120) secretion was increased through PI3K-dependent pathways, which were chronically activated by AMPK, and not through ER stress. Because the decrease of MCP-1 secretion under mitochondrial uncoupling might attenuate chronic low-grade inflammation by suppressing macrophages recruitment to adipose tissue, clarification of the mechanism might reveal novel therapeutic targets for ameliorating obesity-associated insulin resistance in metabolic syndrome and type 2 diabetes.     

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-Pro-Gly units

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X-Y-Gly)(n) repeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)-hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)-hydroxyproline (hyp) at the X-position destabilizes the helix as demonstrated that the triple helix of (hyp-Pro-Gly)(15) is less stable than that of (Pro-Pro-Gly)(15) and that a shorter peptide (hyp-Pro-Gly)(10) does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro-Hyp-Gly)(4) -(hyp-Pro-Gly)(2) -(Pro-Hyp-Gly)(4) and analyzed its structure by X-ray crystallography and CD spectroscopy. In the crystal, the main-chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra-molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac-hyp-NMe(2) by quantum mechanical calculations.     

Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

Alternative substrates for cryopreservation at −20 °C have been little explored for basidiomycetes and could bring new possibilities of lower cost cryopreservation. Nevertheless, freezing temperatures between −15 and −60 °C are very challenging because they frequently result in cryoinjuries. The objective of this study was to evaluate substrates associated to cryoprotective agents for Pleurotus ostreatus cryopreservation at −20 or −70 °C in order to develop alternative techniques for basidiomycete cryopreservation. P. ostreatus was grown on potato dextrose agar or whole grains of oat, wheat, rice or millet and transferred to cryovials with cryoprotective solution with 1 % dimethyl sulfoxide, 5 % glycerol, 10 % saccharose, 4 % glucose, 6 % polyethylene glycol-6000 or 5 % malt extract. The mycelium in the cryovials were cryopreserved at −20 or −70 °C and recovered for evaluation of the mycelial growth viability after 1 and 3 years. Both substrates and cryoprotectants affect the viability of the mycelial growth cryopreserved at −20 or −70 °C; wheat grains combined with cryoprotectants such as saccharose or glucose are effective for keeping mycelium viable after cryopreservation at −20 °C for 1 or 3 years; for cryopreservation at −70 °C after 1 or 3 years, any substrate combined with any cryoprotectant is effective for preserving the mycelium viable, except for millet grains with polyethylene glycol after 3 years; semi-permeable cryoprotective agents such as saccharose and glucose are the most effective for cryopreservation at −20 or −70 °C for at least 3 years.     

Additional rDNA ITS sequences and its phylogenetic consequences for the genus Leveillula with emphasis on conidium morphology

Taxonomy of the genus Leveillula has long been considered as a challenge in powdery mildew systematics. The rDNA diversity has recently been used for phylogenetic analysis of several specimens of the genus Leveillula. In the present study, additional rDNA ITS sequences are provided and a new phylogenetic analysis is carried out aiming at a better understanding of the genetic diversity in the genus Leveillula. New analyses confirmed that L. taurica is unique in the genus, as it exhibits an intraspecific gene sequence diversity considerably higher than in other species. In several cases L. taurica s. lat. on a certain host plant species has a sequence different from L. taurica on other host plants. Moreover, DNA data indicated different lineages among L. taurica specimens which were hardly distinguishable by morphology. More than one genotype occurring on a single host is sometimes possible. According to these results, several races such as Leveillula on Artemisia, Acroptilon, Onobrychis, are molecularly well characterized. While there is enough molecular evidence to delimit such races as independent taxa, clear morphological delimitations between these new and already published taxa are very difficult or even impossible. However, ecological features, and above all, host specificity for biotrophic fungi such as powdery mildew, would be a good criterion to discriminate cryptic taxa along with rDNA sequences. In fact, many collections of Leveillula strains on different hosts show their own type of conidial morphology, which is usually consistent for a strain on a single host species. Hence, we have proposed to describe new species for Leveillula on some host plants such as Artemisia, Acroptilon, Echinops and Onobrychis.