Biomedical subjects
K Fukuda
Publications and source records attributed to K Fukuda.
Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva.
PURPOSE: Amniotic membrane transplantation has been reported to be an effective surgical procedure for the reconstruction of the anterior segment of the eye. To understand better the function of transplanted amniotic membrane, we compared the distributions of subchains of type IV collagen and laminin in the amniotic membrane to those in the cornea and conjunctiva. METHODS: Five human corneas with conjunctivas and three human amniotic membranes were frozen in OCT compound. Cryosections were cut with a cryostat and stained by an indirect immunofluorescent microscopy. We used antibodies of the collagen alpha2(IV) and alpha5(IV) subchains, laminin-1, laminin-5, fibronectin, and type VII collagen. RESULTS: In the conjunctival basement membrane and the amniotic membrane, fluorescence was evident for collagen alpha2(IV) but not for collagen alpha5(IV). By contrast, in the corneal basement membrane, fluorescence was apparent for the collagen alpha5(IV) subchain but not for the collagen alpha2(IV) subchain. Laminin-1, laminin-5, fibronectin, and type VII collagen were present in all the basement membranes examined. CONCLUSION: The distribution of alpha subchains of type IV collagen in the amniotic membrane was identical to that in the conjunctiva but different from that in the cornea. No difference in the distribution pattern of other basement membrane components was observed. These results demonstrate that the basement membrane of the amniotic membrane and the conjunctiva might share the same components; therefore, the amniotic membrane might be useful as a replacement for the basement membrane of the conjunctiva.
Monolayer Behavior of Tetraphenylporphyrin Derivatives Containing Four Fluorocarbon Chains and Optical Properties of Their Monolayer Assemblies.
For tetraphenylporphyrin derivatives substituted with four fluorocarbon chains (TFPP), monolayers on the water surface were studied with the Brewster angle microscope together with pressure-area curves. It was found that heterogeneous aggregated structures of the monolayers were dependent upon the chain length of fluorocarbons, the metal-free or Mn-TFPP, and the periods after spreading. Polarized UV-visible spectra for the monolayer assemblies indicated that the porphyrin rings in the films were tilted on the average, but had a tendency to adopt an orientation more parallel to the water surface as the chain length of the fluorocarbon increased. The third-order nonlinear optical susceptibility for the monolayer assembly of TFPP with the shorter chains was found to be 7.0 x 10(-13) esu, and those of Mn-TFPP were enlarged. Copyright 1998 Academic Press.
A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies.
When the two-dimensional crystal of bacteriorhodopsin (bR), purple membrane, is incubated at high temperature (32 degreesC) with a small amount of the neutral detergent octylthioglucoside in the presence of the precipitant ammonium sulfate, a large fraction of the membrane fragments is converted into spherical vesicles with a diameter of 50 nm, which are able to assemble into optically isotropic hexagonal crystals when the precipitant concentration is increased. The vesicularization of purple membrane takes place under such a condition that the miscibility of the detergent to the aqueous phase becomes very low, and we suggest that it is initiated by insertion of the detergent molecules into the membrane. At low temperature, the transformation into the vesicular structure is inhibited and no large crystal is produced directly from membrane/detergent/precipitant mixtures. When a suspension of the spherical vesicles produced at the high temperature is cooled and concentrated below 15 degreesC, however, a birefringent hexagonal crystal is produced that diffracts X-rays beyond 2.5 A resolution. This new crystal belongs to the space group P622 with unit cell dimensions of a=b=104.7 A and c=114.1 A, and it is shown to be made up of stacked planar membranes, in each of which the bR trimers are arranged on a honeycomb lattice and the space among the proteins is filled with the detergent molecules and native lipids. These stacked membranes are suggested to be produced by successive fusion of the spherical vesicles. This implies that the crystallization is achieved without any step for complete solubilization of the protein. The present result offers a unique crystallization method that may be applicable to such membrane proteins that are liable to denature in the presence of an excess amount of detergent.
Morphology and function of human benign tumors and normal thyroid tissues maintained in severe combined immunodeficient mice.
In the improved SCID (severe combined immunodeficient) mice, various human benign tumors of the head and neck region were well maintained morphologically and functionally for 3 years until the experiments were terminated, e.g. transplanted parathyroid adenoma secreted parathyroid hormone (PTH) in the SCID mice for more than 1 year. Normal human thyroid tissue was also well maintained in the SCID mice for 3 years. Rapid and high uptake of radioiodine into the transplanted human thyroid tissue was observed. Furthermore, transplanted human thyroid tissue secreted thyroid hormone (T3) and T3 secretion was stimulated by the injection of human thyroid stimulating hormone (TSH). These findings suggest that the improved SCID mice will provide an invaluable experimental system for investigating the function of normal human tissues and the influence of endogenous and exogenous factors on human tissues.
The epidemiology of chronic fatigue in San Francisco.
Despite considerable research on chronic fatigue syndrome (CFS) and conditions associated with unexplained chronic fatigue (CF), little is known about their prevalence and demographic distribution in the population. The present study describes the epidemiology and characteristics of self-reported CF and related conditions in a diverse urban community. The study used a cross-sectional telephone screening survey of households in San Francisco, followed by interviews with fatigued and nonfatigued residents. Respondents who appeared to meet case definition criteria for CFS, based on self-reported fatigue characteristics, symptoms, and medical history, were classified as CFS-like cases. Subjects who reported idiopathic chronic fatigue (ICF) that did not meet CFS criteria were classified as ICF-like cases. Screening interviews were completed for 8,004 households, providing fatigue and demographic information for 16,970 residents. Unexplained CF was extremely rare among household residents <18 years of age, but was reported by 2% of adult respondents. A total of 33 adults (0.2% of the study population) were classified as CFS-like cases and 259 (1.8%) as ICF-like cases. Neither condition clustered within households. CFS- and ICF-like illnesses were most prevalent among women and persons with annual household incomes below $40,000, and least prevalent among Asians. The prevalence of CFS-like illness was elevated among African Americans, Native Americans, and persons engaged in clerical occupations. Although CFS-like cases were more severely ill than those with ICF-like illness, a similar symptom pattern was observed in both groups. In conclusion, conditions associated with unexplained CF occur in all sociodemographic groups but appear to be most prevalent among women, persons with lower income, and some racial minorities.
Chronic multisymptom illness affecting Air Force veterans of the Gulf War.
CONTEXT: Gulf War (GW) veterans report nonspecific symptoms significantly more often than their nondeployed peers. However, no specific disorder has been identified, and the etiologic basis and clinical significance of their symptoms remain unclear. OBJECTIVES: To organize symptoms reported by US Air Force GW veterans into a case definition, to characterize clinical features, and to evaluate risk factors. DESIGN: Cross-sectional population survey of individual characteristics and symptoms and clinical evaluation (including a structured interview, the Medical Outcomes Study Short Form 36, psychiatric screening, physical examination, clinical laboratory tests, and serologic assays for antibodies against viruses, rickettsia, parasites, and bacteria) conducted in 1995. PARTICIPANTS AND SETTING: The cross-sectional questionnaire survey included 3723 currently active volunteers, irrespective of health status or GW participation, from 4 air force populations. The cross-sectional clinical evaluation included 158 GW veterans from one unit, irrespective of health status. MAIN OUTCOME MEASURES: Symptom-based case definition; case prevalence rate for GW veterans and nondeployed personnel; clinical and laboratory findings among veterans who met the case definition. RESULTS: We defined a case as having 1 or more chronic symptoms from at least 2 of 3 categories (fatigue, mood-cognition, and musculoskeletal). The prevalence of mild-to-moderate and severe cases was 39% and 6%, respectively, among 1155 GW veterans compared with 14% and 0.7% among 2520 nondeployed personnel. Illness was not associated with time or place of deployment or with duties during the war. Fifty-nine clinically evaluated GW veterans (37%) were noncases, 86 (54%) mild-to-moderate cases, and 13 (8%) severe cases. Although no physical examination, laboratory, or serologic findings identified cases, veterans who met the case definition had significantly diminished functioning and well-being. CONCLUSIONS: Among currently active members of 4 Air Force populations, a chronic multisymptom condition was significantly associated with deployment to the GW. The condition was not associated with specific GW exposures and also affected nondeployed personnel.
Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. I. Molecular determination.
Interactions of G-protein alpha (Galpha) and beta gamma subunits (Gbeta gamma) with N- (alpha1B) and P/Q-type (alpha1A) Ca2+ channels were investigated using the Xenopus oocyte expression system. Gi3alpha was found to inhibit both N- and P/Q-type channels by receptor agonists, whereas Gbeta1 gamma2 was responsible for prepulse facilitation of N-type channels. L-type channels (alpha1C) were not regulated by Galpha or Gbeta gamma. For N-type, prepulse facilitation mediated via Gbeta gamma was impaired when the cytoplasmic I-II loop (loop 1) was deleted or replaced with the alpha1C loop 1. Galpha-mediated inhibitions were also impaired by substitution of the alpha1C loop 1, but only when the C terminus was deleted. For P/Q-type, by contrast, deletion of the C terminus alone diminished Galpha-mediated inhibition. Moreover, a chimera of L-type with the alpha1B loop 1 gained Gbeta gamma-dependent facilitation, whereas an L-type chimera with the N- or P/Q-type C terminus gained Galpha-mediated inhibition. These findings provide evidence that loop 1 of N-type channels is a regulatory site for Gbeta gamma and the C termini of P/Q- and N-types for Galpha.
Transforming growth factor-beta1-induced degradation of activated Src tyrosine kinase in rat fibroblasts.
Transforming growth factors-beta (TGF-betas) play pivotal roles in the regulation of cell growth and differentiation, although little is known regarding the regulation of cytoplasmic components by TGF-betas. Src tyrosine kinase is required for signal transduction of various cytokine receptors, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and G-protein coupled receptors. Moreover, Src tyrosine kinase is important in signal cross-talk between these receptors. To determine whether Src kinase is also involved in TGF-beta signaling, we examined the effects of TGF-beta1 on Src in the rat fibroblast cell line 3Y1 and in v-Src-transformed 3Y0 (SR-3Y1). TGF-beta1 inhibited mitogen-activated protein kinase activity and inhibited growth in SR-3Y1 cells, while it did not affect the growth of 3Y1 cells. TGF-beta1 significantly decreased v-Src kinase activity and protein abundance in SR-3Y1 cells, mainly by accelerating the degradation of v-Src. In contrast, in 3Y1 cells, TGF-beta1 did not affect c-Src abundance or kinase activity. However, upon activation of c-Src in 3Y1 cells by PDGF, TGF-beta1 decreased Src abundance. Additionally, in 3Y1 cells transfected with an activated c-Src mutant which lacks the SH3 domain, its level was decreased by TGF-beta1 treatment. These findings suggest that TGF-beta1 specifically induces degradation of activated Src kinase. This may be a novel mechanism for cross-talk between growth factors and TGF-beta1.
Nociceptin receptor-mediated Ca2+ channel inhibition and its desensitization in NG108-15 cells.
It has been shown that the membrane of hybrid NG108-15 neuroblastoma x glioma cells contains a high-affinity binding site for nociceptin. In the present study, we first demonstrated the expression of nociceptin receptor mRNA in NG108-15 cells. Application of nociceptin to NG108-15 cells produced a concentration-dependent (EC50 = 29 nM) inhibition of Ca2+ channel currents in a pertussis toxin-sensitive fashion. This nociceptin-induced inhibition of Ca2+ channel currents was prevented in the presence of omega-conotoxin GVIA, a blocker of the N-type Ca2+ channel, and had both voltage-dependent and -independent components. Prolonged application of nociceptin elicited homologous desensitization of the inhibition with a time constant of 5.3 min. These results indicate that the nociceptin receptor is coupled to the N-type Ca2+ channel via pertussis toxin-sensitive G proteins in NG108-15 cells and that this coupling is associated with rapid and homologous desensitization.
Treatment of neurotrophic keratopathy with substance-P-derived peptide (FGLM) and insulin-like growth factor I.
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High incidence of esophageal cancer in esophageal achalasia by the oral administration of N-amyl-N-methylnitrosamine and its prevention by nicardipine hydrochloride in mice.
Esophageal achalasia (EA) is a rare disease in man and animals and there are many discussions on its higher risk of esophageal cancer. N-Amyl-N-methylnitrosamine (AMN) which specifically induces esophageal tumors in mice and rats was given to three mutant mouse strains, i.e. 101/N, STX/Le and BXH-8, which develop a high incidence of EA. The incidence of EA in 101/N, STX/Le, BXH-8 and normal C57BL/6J mice was 38.5% (110/286), 30.1% (43/143), 91.8% (190/207) and 0% (0/167), respectively. The average numbers of AMN-induced esophageal tumors in EA(+) were significantly higher than those of EA(-) in all of the 101/N, STX/Le and BXH-8 mice. Furthermore, significantly larger size tumors and invasive squamous cell carcinomas were found in EA(+) mice than in EA(-) mice. These results indicate the higher sensitivity of EA for both tumor induction and promotion, possibly due to the longer retention of AMN. In fact, relaxation of the lower esophagus by a smooth muscle relaxing calcium-channel blocker, nicardipine hydrochloride, significantly prevented the induction of esophageal tumors.
Adaptations to chronic agonist exposure of mu-opioid receptor-expressing Chinese hamster ovary cells.
To investigate cellular adaptation responses induced by chronic agonist treatment of the mu-opioid receptor, Chinese hamster ovary (CHO) cells were stably transfected with the rat mu-opioid receptor cDNA. Chronic treatment with agonists selective for the mu-opioid receptor, [D-Ala2, N-MePhe4, Gy-ol5]enkephalin (DAMGO), morphine and fentanyl, time- and dose-dependently induced down-regulation of the mu-opioid receptor. The down-regulation was not significantly affected by pretreatment with pertussis toxin, but was completely blocked by treatment with hypertonic sucrose, suggesting that receptor internalization mediated by clathrin-coated vesicles is an essential step in the mu-opioid receptor down-regulation. On the other hand, forskolin-stimulated cyclic AMP formation was increased by chronic DAMGO treatment, which was inhibited by pertussis toxin pretreatment. These results indicate that two adaptation responses induced by chronic agonist treatment of the mu-opioid receptor-expressing CHO cells, down-regulation of the mu-opioid receptor and supersensitization of adenylate cyclase, are mediated by distinct mechanisms.
Biphasic activation of the JAK/STAT pathway by angiotensin II in rat cardiomyocytes.
This study was designed to demonstrate the characteristic pattern of angiotensin II-induced JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) activation in cultured rat cardiomyocytes by comparing it with leukemia inhibitory factor (LIF)-induced activation. Angiotensin II (10(-7) mol/L) induced rapid phosphorylation of JAK2 and Tyk2, but not JAK1, and phosphorylated STAT1 and STAT2, but not STAT3, in the early stage up to 30 minutes. The time course of JAK/STAT activation by angiotensin II was apparently slower than that by LIF. Interestingly, angiotensin II phosphorylated STAT3 and rephosphorylated STAT1 in the late stage at 120 minutes. We also found that angiotensin II induced the formation of interferon-stimulating gene factor (ISGF) complexes biphasically, in the early stage at 15 to 30 minutes and in the late stage at 120 minutes, and that angiotensin II induced delayed activation of the sis-inducing factor (SIF) complex at 120 minutes. Formation of ISGF and SIF complexes in response to angiotensin II paralleled the phosphorylation pattern of STAT1 and STAT3 and was quite different from those obtained in response to LIF. The phosphorylation of STAT1 was suppressed by pretreatment with the angiotensin II type-1 (AT1) receptor antagonist CV11974, but the delayed addition of CV11974 failed to suppress phosphorylation of STAT3 at 120 minutes. In conclusion, angiotensin II-induced JAK/STAT activation in rat cardiomyocytes is biphasic and entirely different from LIF-induced activation.
Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.
An avian H5N1 influenza A virus (A/Hong Kong/156/97) was isolated from a tracheal aspirate obtained from a 3-year-old child in Hong Kong with a fatal illness consistent with influenza. Serologic analysis indicated the presence of an H5 hemagglutinin. All eight RNA segments were derived from an avian influenza A virus. The hemagglutinin contained multiple basic amino acids adjacent to the cleavage site, a feature characteristic of highly pathogenic avian influenza A viruses. The virus caused 87.5 to 100 percent mortality in experimentally inoculated White Plymouth Rock and White Leghorn chickens. These results may have implications for global influenza surveillance and planning for pandemic influenza.
Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier.
The induction of the stress protein heme oxygenase-1 (HO-1) was studied in the rat brain after intracarotid administration of hyperosmolar mannitol. HO-1 was immunolocalized in fixed sections of brain 24 h to 7 days after injection. Immunoglobulin G (IgG) was immunolocalized in adjacent sections to demonstrate areas of breakdown of the blood-brain barrier. Induction of HO-1 was also evaluated by Western immunoblots, performed at 24 h after the insult. Immunofluorescent double labelling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor was used to determine if glia/macrophages expressed HO-1. There was pronounced, widespread induction of HO-1 in the ipsilateral hemisphere and cerebellum by 24 h both by immunocytochemistry and by Western blots. This induction was markedly attenuated at later times. HO-1 was induced in astrocytes and microglia/macrophages in the ipsilateral hemisphere. In addition, the protein was induced in Bergmann glia and scattered microglia/macrophages in the cerebellum. The mechanism of induction of HO-1 in glia after opening of the blood-brain barrier could include exposure to heme proteins, denatured proteins and other plasma constituents known to induce HO-1. This glial induction may reflect a protective response of these cells.
Beta-catenin is frequently mutated and demonstrates altered cellular location in azoxymethane-induced rat colon tumors.
Beta-Catenin is a key regulator of the cadherin-mediated cell-cell adhesion system and an important element in the Wnt signal transduction pathway. Stabilization and accumulation of cytoplasmic beta-catenin, which result from mutations in either the adenomatous polyposis coli or beta-catenin genes, are causatively associated with colon carcinogenesis. In the present study, we examined the expression of beta-catenin in rat colon tumors induced by azoxymethane in comparison with adjacent normal colon mucosa by immunostaining and immunoblotting. Cytoplasmic and nuclear immunostaining was pronounced in all colon adenoma and carcinoma tissues, whereas antibody binding was limited to membranes at the intercellular borders in normal colon epithelial cells. Increase of the free beta-catenin fraction in tumor cells was also indicated by immunoblot analysis of fractionated tissue lysates. Investigation of mutations in the glycogen synthase kinase-3beta phosphorylation consensus motif of the beta-catenin gene by PCR-single strand conformation polymorphism methods and direct sequencing revealed eight mutations in six of the eight colon carcinomas, and seven of these were shown to be G:C to A:T transitions, with five being CTGGA to CTGAA. Such frequent mutations of the beta-catenin gene in azoxymethane-induced rat colon tumors suggest that consequent alterations in the stability and localization of the protein may play an important role in this colon carcinogenesis model.
Characteristic difference in gastrointestinal excretion of clarithromycin and roxithromycin.
Excretion characteristics of two new macrolides; clarithromycin and roxithromycin, into the intestinal and the gastric lumens was studied by in situ single-pass perfusion and loop methods in rats. Roxithromycin maintained higher serum levels than clarithromycin after their intravenous administrations at a dose of 5 mg kg(-1) each. Radioactivities of clarithromycin and roxithromycin exsorbed into the intestinal lumen were 8.6 and 18.9% of dose in 2 h, respectively, whereas clarithromycin and roxithromycin excreted into the bile were 28.4 and 5.9%, respectively. These results suggest that roxithromycin is transported mainly by exsorption across the intestinal membrane, whereas clarithromycin mainly by excretion through the biliary tract. On the other hand, radioactivities of clarithromycin and roxithromycin exsorbed into the gastric lumen were much less then those into the intestinal lumen and were 0.72 and 1.34% of dose in 4 h, respectively. Thus, the exsorption into the gastric lumen seems to be a minor route for the elimination of both macrolides. Consequently, the transport into the intestinal lumen via the intestinal membrane and/or the bile tract may play a significant role in the overall elimination of both macrolides.