Visualization of mitochondria with green fluorescent protein in cultured fibroblasts from patients with mitochondrial diseases

cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.     

Effects of short- and long-term dexamethasone treatment on growth and growth hormone (GH)-releasing hormone (GRH)-GH-insulin-like growth factor-I axis in conscious rats

Although the inhibitory effects of a chronic excess of glucocorticoids (GC) on body growth and GH secretion are well established, the mechanisms involved remain unclear. In this study, we examined the chronic effects of a high dose of dexamethasone (DEX) on spontaneous GH secretion and insulin-like growth factor (IGF)-I in conscious rats. The animals were given daily i.p. injections of DEX (200 microg/day) for either one or four weeks. Body growth assessed by tibia length and serum IGF-I levels was significantly inhibited 1 week after treatment. By contrast, spontaneous GH secretion was not altered 1 week after the treatment. Neither hypothalamic GRH and somtatostain mRNA levels nor GH responses to GRH from single somatotropes were affected 1 week after the treatment. Four weeks after DEX treatment, body growth of the rats was noticeably suppressed. Interestingly, spontaneous GH secretion, hypothalamic GRH mRNA levels and GH responses to GRH were all inhibited 4 weeks after treatment. Pituitary GRH receptor mRNA levels were not altered 1 week after treatment, but increased after 4 weeks. These results indicate that a high dose of DEX initially impairs IGF-I production and subsequently inhibits spontaneous GH secretion in rats. Inhibition of spontaneous GH secretion resulting from chronic GC excess is due, at least in part, to the impairment of hypothalamic GRH synthesis and pituitary GH responsiveness. An increase in the pituitary GRH receptor may be caused by decreased GRH secretion.     

Mechanical strengthening of Si cantilever by chemical KOH etching and its surface analysis by TEM and AFM

Mechanical strengthening of a Si cantilever by applying KOH wet etching was investigated. Two kinds of Si cantilever specimens having the different crystallographic orientations of the sidewall surfaces, i.e., Si{100} and Si{110}, were fabricated from the same SOI wafer by a Bosch process. The typical height and pitch of the scalloping formed on the sidewall were 248 and 917 nm, respectively. A 50 % KOH (40 °C) chemical wet etching was applied to increase the fracture stress of the Si cantilever. The fracture stress in the both of Si{100} and Si{110} cantilevers increased with the advance of the etching. The obtained maximum fracture stress in Si{100} and Si{110} were 4.2 and 3.7 GPa, respectively. Sidewall surface of the cantilever was analyzed to investigate the mechanical strengthening of Si cantilever by wet etching. The etched surface crystalline was analyzed by the transmission electron microscope (TEM), and confirmed that the thickness of the affected flow layer was less than 10 nm from the obtained TEM image. Then the change of the surface roughness by the KOH etching was analyzed by the atomic force microscope. The surface was smoothened with the advance of the KOH etching. The roughness value of Ra in Si{100} and Si{110} decreased to 12.1 and 37.7 nm, respectively.     

Experimental measurement on tangential momentum accommodation coefficient in a single microtube

The tangential momentum accommodation coefficient (TMAC) was investigated experimentally from the mass flow rate through a single microtube under the slip flow and the early part of the transition regime. The measurements were carried out by the constant-volume method under the mean Knudsen number smaller than 0.3, which is based on the mean pressure of the inlet and the outlet of the microtube, to apply the second-order slip boundary condition. To measure TMACs on various materials, quite large microtube was employed, which require the reduction in leakage. TMAC was obtained from the slip coefficient determined by the relation of the mass flow rate to the mean Knudsen number. The obtained mass flow rate was well explained by the theoretical equation. TMACs of deactivated-fused silica with argon, nitrogen, and oxygen were measured, showing the tangential momentum was not accommodated completely to the surface, and the values showed good agreement with previous studies. From the comparison between microtubes with different inner diameter, it is showed that TMAC is determined mainly by gas species and surface material.     

Methylcyclohexane dehydrogenation for hydrogen production via a bimodal catalytic membrane reactor

The dehydrogenation of methylcyclohexane (MCH) to toluene (TOL) for hydrogen production was theoretically and experimentally investigated in a bimodal catalytic membrane reactor (CMR), that combined Pt/Al₂O₃ catalysts with a hydrogen‐selective organosilica membrane prepared via sol‐gel processing using bis(triethoxysilyl) ethane (BTESE). Effects of operating conditions on the membrane reactor performance were systematically investigated, and the experimental results were in good agreement with those calculated by a simulation model with a fitted catalyst loading. With H₂ extraction from the reaction stream to the permeate stream, MCH conversion at 250°C was significantly increased beyond the equilibrium conversion of 0.44–0.86. Because of the high H₂ selectivity and permeance of BTESE‐derived membranes, a H₂ flow with purity higher than 99.8% was obtained in the permeate stream, and the H₂ recovery ratio reached 0.99 in a pressurized reactor. A system that combined the CMR with a fixed‐bed prereactor was proposed for MCH dehydrogenation. © 2015 American Institute of Chemical Engineers AIChE J, 61: 1628–1638, 2015     

Characterization of alternative plasticizers in poly(vinyl chloride) sheets for blood containers

This study aimed to optimize the ratio of dioctyl 4‐cyclohexene‐1,2‐dicarboxylate (DOTH) and di‐isononyl‐cyclohexane‐1,2‐dicarboxylate (DINCH^®) for use as plasticizers in poly(vinyl chloride) (PVC) sheets. We also evaluated the biological safety of DOTH for its potential to be part of a safe PVC‐based blood container. The suppression of hemolysis in mannitol‐adenine‐phosphate / red cell concentrates (MAP/RCC) with DOTH/(DINCH^®‐PVC) sheets and the elution of plasticizers from the sheets increased with higher DOTH compositions. The properties of the PVC sheet containing DOTH and DINCH^® in the ratio of 25:33 parts against PVC 100 parts as a weight were almost identical to the PVC sheet made of di(2‐ethylhexyl) phthalate. From a subchronic toxicity test, DOTH did not show any adverse effects on all organs, including the testes, epididymis, liver, and kidneys. The no‐observed‐adverse‐effect level was 300 mg/kg body weight/day in a rat. These results suggest that DOTH/DINCH^® (25:33) is a promising candidate for the replacement of di(2‐ethylhexyl) phthalate in blood containers. J. VINYL ADDIT. TECHNOL., 22:520–528, 2016. © 2015 Society of Plastics Engineers     

Single-molecular AFM probing of specific DNA sequencing using RecA-promoted homologous pairing and strand exchange

The specific sequence in a linearlized double-stranded DNA target has been identified at a single-molecular level by atomic force microscopy (AFM). This was accomplished using RecA-coated, single-stranded DNA probes which were paired with a specific complementary DNA sequence in a linear double-stranded DNA target by strand-exchange reaction at a homologous sequence site with target DNA. The sites of interaction between the nucleoprotein filaments and the double-stranded DNA targets were directly visualized by AFM in solution containing 4 mM magnesium acetate. Measurements of the position of RecA-coated probes paired to individual target DNA showed that DNA probes specifically paired at their corresponding homologous target sequences. Strand exchange promoted by RecA and the visualization by AFM provided a rapid and efficient way to identify homologous sequence on a single-molecule target DNA.     

Is Trypanoplasma Salmositica Present in Lake Ontario Salmonids?

During the period June-October 1981, fish, including lake trout (Salvelinus namaycush), brown trout (Salmo trutta), rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch), were collected from four locations on Lake Ontario. In addition, juvenile coho salmon reared from Lake Ontario fish eggs and maintained in a hatchery were collected. Subsequently, air-dried blood smears were stained with Leishman-Giesma and examined under oil emersion for Trypanoplasma salmositica. In this study of 131 fish, no T. salmositica were found. From these findings it may be assumed that T. salmositica are either rare or not present in Lake Ontario salmonids.