Biomedical subjects

S Machida

Publications and source records attributed to S Machida.

At least 19 recordsLinked to original sources

PubMed2001

Constitutive "light" adaptation in rods from G90D rhodopsin: a mechanism for human congenital nightblindness without rod cell loss.

A dominant form of human congenital nightblindness is caused by a gly90-->asp (G90D) mutation in rhodopsin. G90D has been shown to activate the phototransduction cascade in the absence of light in vitro. Such constitutive activity of G90D rhodopsin in vivo would desensitize rod photoreceptors and lead to nightblindness. In contrast, other rhodopsin mutations typically give rise to nightblindness by causing rod cell death. Thus, the proposed desensitization without rod degeneration would be a novel mechanism for this disorder. To explore this possibility, we induced mice to express G90D opsin in their rods and then examined rod function and morphology, after first crossing the transgenic animals with rhodopsin knock-out mice to obtain appropriate levels of opsin expression. The G90D mouse opsin bound the chromophore and formed a bleachable visual pigment with lambda(max) of 492 nm that supported rod photoresponses. (G+/-, R+/-) retinas, heterozygous for both G90D and wild-type (WT) rhodopsin, possessed normal numbers of photoreceptors and had a normal rhodopsin complement but exhibited considerable loss of rod sensitivity as measured electroretinographically. The rod photoresponses were desensitized, and the response time to peak was faster than in (R+/-) animals. An equivalent desensitization resulted by exposing WT retinas to a background light producing 82 photoisomerizations rod(-1) sec(-1), suggesting that G90D rods in darkness act as if they are partially "light-adapted." Adding a second G90D allele gave (G+/+, R+/-) animals that exhibited a further increase of equivalent background light level but had no rod cell loss by 24 weeks of age. (G+/+, R-/-) retinas that express only the mutant rhodopsin develop normal rod outer segments and show minimal rod cell loss even at 1 year of age. We conclude that G90D is constitutively active in mouse rods in vivo but that it does not cause significant rod degeneration. Instead, G90D desensitizes rods by a process equivalent to light adaptation.

Adaptation, Ocular↗

PubMed2001

Introduction of tapasin gene restores surface expression of HLA class I molecules, but not antigen presentation of an HIV envelope peptide in a hepatoma cell line.

A hepatoma cell line, Hep G2, reveals the diminished HLA class I surface expression and the reduced expression of LMP2, LMP7, and tapasin transcripts, suggesting that the reduced expression of these transcripts may be associated with the low expression of HLA class I molecules. Introduction of tapasin gene dramatically up-regulates the surface expression of HLA class I molecules on Hep G2 cells, and unexpectedly, enhances the expression of LMP2 and LMP7 transcripts as well. Unlike Hep G2, these tapasin-transfected Hep G2 cells are recognized by allo-specific CTL. However, the transfectant is unable to endogenously present an HIV envelope peptide to an HIV-specific CTL clone, suggesting that a proteasome-independent antigen processing pathway exists and still remains defective in the transfectant. These data may provide significant evidence that the nonproteasomal antigen processing pathway as well as the proteasomal pathway may be impaired in tumor cells to escape immune surveillance performed by CTL.

Antigen Presentation↗

PubMed2001

Interaction of SMKT, a killer toxin produced by Pichia farinosa, with the yeast cell membranes.

SMKT (salt-mediated killer toxin), a killer toxin produced by the halotolerant yeast, Pichia farinosa, kills yeasts of several genera, including Saccharomyces cerevisiae. To elucidate the killing mechanism of SMKT, we examined the interaction of SMKT with membranes using liposomes. Leakage of calcein from calcein-entrapped liposomes was observed in the presence of SMKT. Destruction of liposomes was observed by dark-field microscopy. Comparison of intact S. cerevisiae cells with SMKT-treated cells by dark-field microscopy indicated that the spherical cell membrane is disrupted by SMKT. Using sodium carbonate extraction, we obtained direct evidence for the first time that SMKT is associated with the membrane of sensitive cells. Our results indicate that SMKT kills sensitive S. cerevisiae by interacting with the yeast cell membrane.

Blotting, Western↗

PubMed2001

Involvement of conserved hydrophobic residues in the CTLD of human lectin-like oxidized LDL receptor in ligand binding.

We previously identified the hydrophilic residues that are essential for ligand binding in the C-type lectin-like domain (CTLD) of human lectin-like oxidized LDL receptor (hLOX-1). To provide a more detailed understanding of ligand binding, we selected in the present study 13 conserved hydrophobic residues in the CTLD of hLOX-1 for mutagenesis analysis. The selected residues were replaced either by Ser (drastic mutation) or by size- and structure-based alternative hydrophobic residues (conserved mutation). Mutation targeted at F228, Y238, and G232 deprived hLOX-1 of ligand binding without alteration of protein expression and localization. In contrast, drastic mutation introduced into positions W203, W215, and W217 resulted in mislocalization, whereas conserved mutation at the same sites resulted in clones with similar cell surface localization and ligand binding to native hLOX-1. Our results indicate that F228, Y238, and G232 are essential for ligand binding, while W203, W215, W217, and L206 play a structural role.

Amino Acid Sequence↗

PubMed2001

Characterization of residues and sequences of the carbohydrate recognition domain required for cell surface localization and ligand binding of human lectin-like oxidized LDL receptor.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) has been cloned from human aortic endothelial cells, and has a sequence identical to that from human lung. Previous studies showed that human LOX-1 can recognize modified LDL, apoptotic cells and bacteria. To further explore the relationship between the structure and function of LOX-1, a mutagenesis study was carried out. Our results showed that the carbohydrate recognition domain (CRD) was the ligand-binding domain of human LOX-1. We also investigated the sequences and residues in CRD that were essential for protein cell surface localization and ligand binding. LOX-1s carrying a mutation on each of six Cys in CRD resulted in a variety of N-glycosylation and failed to be transported to the cell surface. This was strong evidence for the involvement of all six Cys in the intrachain disulfide bonds required for proper folding, processing and transport of LOX-1. The C-terminal sequence (KANLRAQ) was also essential for protein folding and transport, while the four final residues (LRAQ) were involved in maintaining receptor function. Both positive charged (R208, R209, H226, R229 and R231) and non-charged hydrophilic (Q193, S198, S199 and N210) residues were involved in ligand binding, suggesting that ligand recognition of LOX-1 is not merely dependent on the interaction of positively charged residues with negatively charged ligands.

Amino Acid Sequence↗

PubMed2001

Atypical membranoproliferative glomerulonephritis in a cat.

Membranoproliferative glomerulonephritis was observed in a 2-year-old male Japanese domestic cat with clinical renal failure. In the glomeruli, moderate mesangial hypercellularity with an increased mesangial matrix and thickening of the capillary walls were prominent. In addition, frequent duplication of the capillary walls, splitting, and spike formation were observed in the glomerular basement membrane. Granular cat IgG and complement component deposition were detected globally along the glomerular capillary walls and in the mesangium. Transmission electron microscopy revealed dense deposits in the subendothelial and subepithelial regions and the mesangium. Mesangial interposition was also observed. These glomerular lesions are also found in humans with membranoproliferative glomerulonephritis type III, which has not been reported in animals.

Animals↗

PubMed2001

Lens epithelium-derived growth factor promotes photoreceptor survival in light-damaged and RCS rats.

PURPOSE: To investigate possible protective effects of lens epithelium-derived growth factor (LEDGF) against photoreceptor death in light-damaged, Royal College of Surgeons (RCS) and P23H rhodopsin transgenic rats. METHODS: Twelve-week-old Sprague-Dawley (SD), 6-week-old RCS, and 10-day-old P23H (line 1, heterozygote) rats received an intravitreal injection of LEDGF fused with glutathione-S-transferase (GST-LEDGF). Fellow eyes received vehicle and served as control specimens. Two days after the injections, the SD rats were exposed to light of 2000 lux for 48 hours. Corneal Ganzfeld ERGs were recorded 10 days after light damage, at 10 weeks of age in RCS rats, and at 4 weeks of age in P23H rats. The eyes were then processed for histologic analysis. Heat shock protein (hsp) content in the sensory retina was analyzed quantitatively by protein immunoblot. RESULTS: In light-damaged rats, the ERG indicated retinal protection in GST-LEDGF-injected eyes, with b-wave and STR thresholds being 1.14 +/- 0.50 (mean +/- SD) and 0.60 +/- 0.26 log candela (cd)/m2 lower, respectively, than in vehicle-injected eyes (P < 0.01). The GST-LEDGF-treated eyes had maximum b-wave amplitudes that were significantly larger (P < 0.0005), had more than twice as many remaining photoreceptors, and had better organized outer segments than the control eyes. In RCS rats, the treated eyes had 2.76 +/- 0.73 and 0.83 +/- 0.09 log cd/m(2) lower thresholds for the b-wave and STR, respectively (P < 0.005), and had significantly larger maximum b-wave amplitude (P < 0.0005). GST-LEDGF-treated eyes of RCS rats also had more photoreceptors remaining (P < 0.005) and a thinner debris layer than control eyes. In P23H rats, GST-LEDGF treatment did not protect either retinal function or structure. The retinas from GST-LEDGF-treated eyes of SD and RCS rats had higher levels of hsp25 and alphaB-crystallin than vehicle-injected eyes. CONCLUSIONS: GST-LEDGF protects photoreceptor structure and function in both light-damaged and RCS rats. The increased expression of hsp25 and alphaB-crystallin may play a role in this protection. The absence of rescue in P23H raises the possibility that some forms of inherited retinal degeneration may not be amenable to treatment by intraocular injection of LEDGF.

Animals↗

PubMed2000

Cycloamylose as an efficient artificial chaperone for protein refolding.

High molecular weight cyclic alpha-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form.

Animals↗

PubMed2000

Complete sequence and characterization of chick ventricular myosin heavy chain in the developing atria.

We isolated five complementary DNA (cDNA) clones, encoding the chick ventricular myosin heavy chain (MyHC) by reverse transcription polymerase chain reaction (RT-PCR). The entire cDNA consists of 5995 nucleotides with the 52 bp 5'-untranslated region and the 129 bp 3'-untranslated region. The complete cDNA encodes 1937 amino acids. Expression of the chick ventricular MyHC gene was also studied by Northern blot analysis. This gene continued to be strongly expressed in the ventricle during cardiac development. On the other hand, its expression was moderate in the early embryonic atria, and was down-regulated during development. In the adult atria, this gene was expressed at very low levels. To determine the localization of the ventricular MyHC protein, an immunohistochemical study was performed. The ventricular MyHC was present in early embryonic atrial myocytes. During development, the expression of this protein in the atrial myocytes was down-regulated, but continued to be present in the atrial conduction system. Our results indicate that the ventricular MyHC appears in the primary atrial myocardium and is then localized in the conduction cells of the atria.

Animals↗

PubMed2000

Dual quantum nondemolition measurements via successive soliton collisions

Quantum mechanics allows quantum nondemolition (QND) variables to be measured without being changed. This requires QND variables to be initially in an eigenstate and measurement backaction noise to be directed into conjugate variables. Experimental demonstrations thus require two measurements: the first to collapse variables toward an eigenstate and the second to show results identical to the first. Here, we report results from two successive soliton-collision QND measurements that optical correlation measurements show to be nearly identical.

Journal Article↗

PubMed2000

Evidence for the expression of neonatal skeletal myosin heavy chain in primary myocardium and cardiac conduction tissue in the developing chick heart.

We isolated a neonatal skeletal myosin heavy chain (MHC) cDNA clone, CV11E1, from a cDNA library of embryonic chick ventricle. At early cardiogenesis, diffuse expression of neonatal skeletal MHC mRNA was first detected in the heart tube at stage 10. During subsequent embryonic stages, the expression of the mRNA in the atrium was upregulated until shortly after birth. It then diminished, dramatically, and disappeared in the adult. On the other hand, in the ventricle, only a trace of the expression was detected throughout embryonic life and in the adult. However, transient expression of mRNA in the ventricle was observed, post-hatching. At the protein level, during the embryonic stage, the atrial myocardium was stained diffusely with monoclonal antibody 2E9, specific for chick neonatal skeletal MHC, whereas the ventricles showed weak reactivity with 2E9. At the late embryonic and newly hatched stages, 2E9-positive cells were located clearly in the subendocardial layer, and around the blood vessels of the atrial and ventricular myocardium. These results provide the first evidence that the neonatal skeletal MHC gene is expressed in developing chick hearts. This MHC appears during early cardiogenesis and is then localized in cardiac conduction cells. Dev Dyn 2000;217:37-49.

Amino Acid Sequence↗

PubMed2000

Intravascular ultrasound imaging of the pulmonary arteries in primary pulmonary hypertension.

OBJECTIVE: Intravascular ultrasound has the unique ability to provide cross-sectional images of the arterial wall. This study examined intravascular ultrasound (IVUS) images of the proximal pulmonary arteries in primary pulmonary hypertension (PPH). METHODOLOGY: Study 1: Specimens from four patients who had died of PPH (in vitro PPH group) were compared with those of three patients who had died of subarachnoid haemorrhage but had no evidence of cardiopulmonary disease (in vitro control group). Three-centimetre segments of the following levels were examined by IVUS: pulmonary trunk, eight secondary branch arteries of the upper, middle, and lower lobes of both lungs, and the thoracic descending aorta. Study 2: Four patients with PPH (in vivo PPH group) and five patients without pulmonary hypertension and no evidence of cardiopulmonary disease (in vivo control group) were examined. The IVUS images of the apical segmental artery of the right upper lobe and the descending branch of the right pulmonary artery were studied. RESULTS: Echographic examination of formalin-fixed preparations of secondary branch sections of the pulmonary artery failed to show a clear three-layer structure in the in vitro control group (24 preparations), but a distinct three-layer structure and increased vessel wall thickness were observed in the in vitro PPH group (32 preparations). Similar findings were obtained in the in vivo study. The mean echo density of the proximal pulmonary arterial wall correlated well with the mean pulmonary arterial pressure (mPA) in the in vitro PPH, and also correlated with the mPA in the in vivo study (r = 0.960, P < 0.0001). The echo intensity of secondary branch sections of the pulmonary artery was higher in the in vitro PPH group than in the in vitro control group (180.5 +/- 27.0 vs 132.5 +/- 26.7 counts, P < 0.001); similar results were obtained in the in vivo study (144.7 +/- 23.4 vs 85.0 +/- 14.3 counts, P < 0.01). CONCLUSIONS: These results suggest that the histological changes detected in the pulmonary artery walls in the PPH group were responsible for the increased echo intensity.

Adult↗

PubMed2000

Computed tomographic fluoroscopy-guided transthoracic needle biopsy for diagnosis of pulmonary nodules.

BACKGROUND: The purpose of this study was to evaluate the usefulness of computed tomographic (CT) fluoroscopy-guided transthoracic needle biopsy (TTNB) with an 18-gauge automatic biopsy gun for the diagnosis of pulmonary nodules. METHODS: Between March 1996 and January 1998, 50 patients in whom pulmonary lesions could not be diagnosed cytopathologically with fiberoptic bronchoscopy or were not clearly visualized with fluoroscopy underwent CT fluoroscopy-guided TTNB. RESULTS: Final pathological diagnoses were 23 lung carcinomas, five pulmonary metastases and 22 benign lesions. Sufficient tissue for analysis was obtained from 48 of the 50 lesions (96%). The overall diagnostic yield of CT fluoroscopy-guided TTNB was 90%. The sensitivity, specificity and accuracy for malignancy were 89%, 100% and 94%, respectively. In 20 of the 22 cases (91%) of benign lesions, histological analysis yielded correct and specific diagnoses. Complications occurred in 22 of the 50 cases (44%). The most common complication was pneumothorax, which occurred in 21 of the 50 cases (42%). Chest tube insertion was required in 6 (12%). CONCLUSIONS: Although CT fluoroscopy could not decrease the complication rate, CT fluoroscopy-guided TTNB with an automatic biopsy gun appears to be a promising technique for diagnosing pulmonary lesions, particularly benign lesions.

Adenocarcinoma↗

PubMed2000

Design of a novel membrane-destabilizing peptide selectively acting on acidic liposomes.

The design of amphipathic peptides resulted in a novel peptide with a selective ability to destabilize lipid bilayers of acidic liposomes. The newly synthesized peptide, termed mast 21, is a 21-residue long amino acid chain and can only act effectively on acidic liposomes lacking cholesterol. Moreover, mast 21 killed gram-positive and gram-negative bacteria, and it had no hemolytic activity. The antimicrobial and hemolytic activities paralleled the results of membrane destabilizing activity using liposomes. Circular dichroism and Trp-fluorescence emission spectra showed changes in the peptide conformation and circumstances around the peptide during interaction with liposomes. These changes were consistent with an increased alpha-helical content and a less polar environment for the tryptophan residue of the peptide. Mast 21 was observed under dark-field microscopy in real time attacking liposomes. Acidic liposomes were attacked, which resulted in peeling of the lipid bilayer with its subsequent destruction.

Amino Acid Sequence↗

PubMed2000

Lack of effect of running training at two intensities on cardiac myosin isozyme composition in rats.

Little information is available regarding the influence of the intensity of endurance training over biochemical profiles in cardiac muscle. We assessed the effect of running training at two different intensities on cardiac myosin isozyme composition in rats. Male Sprague-Dawley rats (4 weeks old) were divided into four groups: sedentary control (SC), trained at 20 m/min (T20), trained at 40 m/min (T40), and weight-matched sedentary control (WMSC) groups. The T20 and T40 group rats were trained by treadmill running for 60 min/d, 5 d/week at 20 or 40 m/min, respectively, for 11 to 12 weeks. In both groups the left ventricle was significantly heavier than in WMSC animals. The ratio of left ventricle weight to body weight was significantly greater in T40 rats than in either the untrained (SC and WMSC) or trained T20 rats. Thus the extent of exercise-induced cardiac hypertrophy appears to be influenced by the intensity of running training. However, neither of the training programs (1) induced a change in cardiac myosin isozyme composition or (2) had any effect on myocardial succinate dehydrogenase or citrate synthase activity. These results suggest that although the intensity of running training may play an important role in cardiac morphological adaptation, it does not modulate the cardiac biochemical adaptation to running training.

Animals↗

PubMed2000

The effect of calcium channel blocker diltiazem on photoreceptor degeneration in the rhodopsin Pro213His rat.

PURPOSE: To determine whether the calcium channel blocker D-cis-diltiazem promotes photoreceptor survival in rats with the Pro23His rhodopsin mutation. METHODS: Heterozygous Pro23His rhodopsin line 1 rats (n = 11) were treated daily, according to a protocol applied successfully in rd mice, with D-cis-diltiazem hydrochloride increased incrementally from 21 to 54 mg/kg in a divided dose (8 AM and 8 PM) administered by intraperitoneal (i.p.) injection for 21 days, beginning on days of age 10 through 12. Saline-treated line 1 rats (n = 6) received i.p. injections of an equal volume of 0.9% saline. Analysis on day 35 of age included dark-adapted corneal electroretinogram (ERG) b- and a-waves recorded from threshold to 0.63 log candela-seconds [cd-s]/m2, saturated a-waves elicited with a 2.1 log cd-s/m2 flash, and morphometry of the outer nuclear layer (ONL) and rod outer segments (ROS). RESULTS: ONL width and cell counts of diltiazem-treated and saline-treated animals at 35 days were reduced to 64%-68% of 15-day-old untreated P23H line 1 retinas. No photoreceptor rescue was found by measuring ONL width (P = 0.84), cell count (P = 0.42), or ROS length (P = 0.85). Functional assays by ERG b-wave threshold (P = 0.57), b-wave maximum amplitude (P = 0.46), and saturated a-wave amplitude (P = 0.59) also showed no rescue. CONCLUSIONS: D-cis-Diltiazem did not rescue photoreceptors of Pro23His rhodopsin mutation line 1 rats treated according to the protocol used in rd mouse.

Animals↗

PubMed2000

P23H rhodopsin transgenic rat: correlation of retinal function with histopathology.

PURPOSE: To correlate retinal functional changes with structural changes in P23H rhodopsin transgenic rats as a model of autosomal dominant retinitis pigmentosa. METHODS: P23H heterozygote (lines 1 and 3) and Sprague-Dawley control rats were studied at 4 to 29 weeks by retinal histology, electroretinogram (ERG), and a-wave transduction modeling. RESULTS: Both line 1 (faster degeneration) and line 3 (slower degeneration) showed progressive rod outer segment (ROS) shortening and outer nuclear layer (ONL) cell loss with age. ERG b-wave maximum amplitude (Vb(max)) decreased with age, but b-wave threshold remained constant within each line despite progressive ONL thinning and ROS shortening. The only exception was in line 1 at 29 weeks, which showed a slight threshold change relative to earlier ages. Va(max) and a-wave threshold changed more rapidly and were more sensitive than the b-wave in reflecting histologic degeneration. Va(max) was linearly proportional to the product of (ROS x ONL) across a two log unit range of data combined from both lines. The photopic b-wave was normal for both lines until the ONL thinned beyond 50%. Phototransduction sensitivity was normal for both lines, and dark-adaptation recovery after bleaching rhodopsin was normal. CONCLUSIONS: The P23H transgenic rat has a slow rod degeneration with initially normal cone function, consistent with clinical findings of P23H patients. However, the normal bleach recovery and the normal phototransduction sensitivity in this rat model are different from human P23H disease. a-Wave measures were more sensitive than the b-wave for tracking changes. b-Wave threshold was inexplicably poor for tracking degeneration. Although line 1 degenerated faster than line 3, the functional-structural correlates were the same. The tight linear relationship between saturated a-wave amplitude and the product of (ROS x ONL) indicates that the density of cGMP-gated channels per unit ROS plasma membrane area remains constant over a wide range of degenerations.

Animals↗

PubMed1999

An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis.

Although the amino acid homology in the catalytic domain of FXYN xylanase from Streptomyces olivaceoviridis E-86 and Cex xylanase from Cellulomonas fimi is only 50%, an active chimeric enzyme was obtained by replacing module 10 in FXYN with module 10 from Cex. In the family F/10 xylanases, module 10 is an important region as it includes an acid/base catalyst and a substrate binding residue. In FXYN, module 10 consists of 15 amino acid residues, while in Cex it consists of 14 amino acid residues. The Km and kcat values of the chimeric xylanase FCF-C10 for PNP-xylobioside (PNP-X2) were 10-fold less than those for FXYN. CD spectral data indicated that the structure of the chimeric enzyme was similar to that of FXYN. Based on the comparison of the amino acid sequences of FXYN and Cex in module 10, we constructed four mutants of FXYN. When D133 or S135 of FXYN was deleted, the kinetic properties were not changed from those of FXYN. By deletion of both D133 and S135, the Km value for PNP-X2 decreased from the 2.0 mM of FXYN to 0.6 mM and the kcat value decreased from the 20 s(-1) of FXYN to 8.7 s(-1). Insertion of Q140 into the doubly deleted mutant further reduced the Km value to 0.3 mM and the kcat value to 3.8 s(-1). These values are close to those for the chimeric enzyme FCF-C10. These results indicate that module 10 itself is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function. Since changes of the spatial position of these amino acids would be expected to result in enzyme inactivation, module 10 must have some flexibility in its tertiary structure. The structure of module 10 itself also affects the substrate specificity of the enzyme.

Amino Acid Sequence↗