Dyspnea: measurement and mechanism.
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Arm↗
Biomedical subjects
Y Honda
Publications and source records attributed to Y Honda.
Intrauterine high-resolution magnetic resonance imaging in fetal hydrocephalus and prenatal estimation of postnatal outcomes with "perspective classification".
OBJECT: It is possible to diagnose hydrocephalus prenatally based on the morphological appearance of the fetus on neurodiagnostic images; however, the prognosis of this disease shows wide variation. The authors previously proposed a classification system for the prediction of postnatal outcome based on progression of hydrocephalus and affected brain development, known as the "Perspective Classification of Congenital Hydrocephalus (PCCH)." In this study the authors have used their classification system to analyze long-term follow-up results obtained in each clinicoembryological stage of fetal hydrocephalus. METHODS: Sixty-one fetuses with hydrocephalus were examined to predict postnatal outcome by using this newly developed classification. The authors' recently developed method of using heavily T2-weighted imaging with a superconducting magnet clearly delineated the cerebrospinal fluid (CSF) space and the malformed brain and spinal cord. Imaging was achieved in less than 1 second per slice and required no sedation of the fetus. The technique appears to be simple and good at delineating intrauterine anatomy. Hydrocephalus was diagnosed in two fetuses at PCCH embryological Stage I (8-21 gestational weeks), in 28 fetuses at Stage II (22-31 weeks), and in 31 fetuses at Stage III (32-40 weeks). Among these 61 fetuses, clinicopathological typing showed that 19 had primary hydrocephalus (nine in Stage II and 10 in Stage III), 34 had dysgenetic hydrocephalus (two in Stage I, 16 in Stage II, and 16 in Stage III), and eight had secondary hydrocephalus (three in Stage II and five in Stage III). When the hydrocephalic state developed during PCCH Stage I or II, the prognosis was very poor, and only one of 18 fetuses with dysgenetic hydrocephalus and none of three fetuses with secondary hydrocephalus had an acceptable postnatal outcome. Even within the same category or subtype of fetal hydrocephalus, such as primary hydrocephalus in its simple form, or hydrocephalus with spina bifida aperta (myeloschisis), the postnatal outcomes differed depending on the time of onset of hydrocephalus. When the diagnosis of hydrocephalus was made during PCCH Stage II, the fetuses had a poorer postnatal outcome compared with those at Stage III (p < 0.05). CONCLUSIONS: It is emphasized that postnatal prognosis is not simply a function of the form of the diagnosis but is also dependent on the progression of hydrocephalus and the degree to which that process affects neuronal development. Early decompressive procedures, conventionally performed after but, hopefully, performed before birth, are indicated to obtain the optimal postnatal prognosis of fetuses with hydrocephalus diagnosed at PCCH Stage II.
Disease Progression↗
Ondine's curse and its inverse syndrome. Respiratory failure in autonomic vs. voluntary control.
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Autonomic Nervous System↗
Fluorophotometric detection of intravitreal peroxides after panretinal laser photocoagulation.
PURPOSE: To develop a highly sensitive method for in vivo quantitation of intravitreal peroxides by vitreous fluorophotometry with 2',7'-dichlorofluorescin (DCFH), a hydrogen peroxide (H2O2)-sensitive fluorescent dye, and to measure peroxides in the vitreous humor after panretinal laser photocoagulation (PRP). METHODS: In the presence of H2O2 and lipid hydroperoxides, nonfluorescent DCFH was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF; excitation, 495 nm; emission, 520 nm), which is detectable by vitreous fluorophotometry. Reactions of DCFH, including hematin with various concentrations of H2O2, were investigated in vivo. Fluorophotometry with DCFH was performed 1, 7, 14, and 28 days and 2 months after argon laser PRP. Untreated eyes served as the controls. RESULTS: Exogenously applied H2O2 oxidized DCFH to DCF in a dose-dependent manner, ranging from 6 x 10(-8) mol/l to 6 x 10(-5) mol/l in concentration in vivo. Intravitreal DCF concentration was 83.7 +/- 6.8 nmol/l in control eyes. A significant increase of DCF was detected 1 day after PRP (330.7 +/- 123.8 nmol/l, P < 0.002). The increase peaked on day 7 (352.4 +/- 239.5 nmol/l, P < 0.002) and remained elevated at 2 months after PRP (161.8 +/- 51.4 nmol/l, P < 0.01). CONCLUSIONS: This method allowed a highly sensitive quantitation of intravitreal peroxides in vivo. The authors' findings indicated that PRP induces increased production of peroxides in rabbit vitreous for 2 months. The data suggested that persistently high levels of peroxides in the vitreous humor affect the development of vitreous liquefaction after PRP.
Animals↗
Introduction of DNA into the rat and primate trabecular meshwork by fusogenic liposomes.
PURPOSE: To evaluate the feasibility of introducing exogenous genes and phosphorothioate oligonucleotides into the anterior chamber tissues of rats and monkeys using the authors' fusogenic liposomes. METHODS: Hemagglutinating virus of Japan liposomes containing LacZ DNA-high-mobility group 1 complexes or fluorescein isothiocyanate (FITC)-labeled phosphorothioate oligonucleotides were prepared and injected into the anterior chambers of rats (3 microliters) and rhesus monkeys (30 microliters). The expression of LacZ DNA was visualized histochemically by beta-Galactosidase assay and was followed for as long as 60 days in rats and 30 days in monkeys. FITC-labeled phosphorothioate oligonucleotides were observed by fluorescence microscopy for as long as 14 days in rats and 7 days in monkeys. RESULTS: Injection of LacZ DNA-high-mobility group 1 complexes encapsulated in hemagglutinating virus of Japan liposomes resulted in blue staining in the trabecular meshwork and iris-ciliary body of rats and selectively in the trabecular meshwork of monkeys at the concentrations used. This LacZ expression lasted for as long as 14 days after injection in both animals. Phosphorothioate oligonucleotides (3 microM) also were introduced into the rat trabecular meshwork and iris-ciliary body and into the primate trabecular meshwork when encapsulated in hemagglutinating virus of Japan liposomes, although the injection of naked FITC-labeled phosphorothioate oligonucleotides at the same concentration resulted in little fluorescence in any anterior chamber tissue. CONCLUSIONS: This study shows that the use of hemagglutinating virus of Japan liposomes can transfer LacZ DNA and phosphorothioate oligonucleotides to adult rat and primate trabecular meshwork. This system may enable progress in glaucoma research and in the development of nonviral somatic gene therapy of the trabecular meshwork to treat glaucoma.
Animals↗
[Idiopathic hypersomnia--review of literatures and clinical experiences on 16 Japanese cases].
The concept of idiopathic hypersomnia (IHS) was reviewed. The minimal criteria for IHS in the International Classification of Sleep Disorders (ICSD) are not clear enough to designate a clinical entity. We propose our criteria for IHS, which consist of 6 items. 1. Recurrent daytime sleep episodes with duration of longer than one hour that occur almost everyday for at least 6 months. 2. Absence of refreshed feeling after the daytime sleep episodes. 3. Length of nocturnal sleep is either normal or prolonged. 4. Total sleep time per day exceeds 10 hours. 5. Absence of cataplexy. 6. Does not meet criteria for other sleep disorders that account for the excessive daytime sleep episodes. Our clinical experiences with 16 Japanese IHS patients were described. Decrease in HLA Cw3 was found.
Adult↗
[The autopsy case of traumatic carotid artery dissection].
The results of an autopsy of a 78-year-old female patient with an occluded of the right internal carotid artery after a traffic accident are reported. She presented with consciousness disturbance, right conjugate deviation, left hemiparesis and left pathological reflex. Evidence of right skull and clavicular fractures seemed to suggest that severe hyperextension of her neck associated with contralateral lateral flexion had stretched her carotid artery. Although she was treated with antiplatelet therapy, her cerebral swelling due to right global infarction progressed and she died due to bilateral cerebral herniation three days after injury. The autopsy disclosed right dissecting carotid artery occlusion and subadventitial dissection was revealed histologically. When a hematoma dissect the media and adventitia of the carotid artery wall, the outer wall weakens and may dilate without narrowing the lumen of the carotid artery. In this case, the vessel occlusion was considered to be due to a primary intramural hematoma which developed and subsequently ruptured through the intima into the vessel lumen. Early diagnosis and treatment are necessary for improving the prognosis of this disease entity. A patient with poor colateral flow, such as in this case, will show a rapid progression of cerebral infarction. It is best to consider surgical treatment in this case if the other traumatic lesions are able to tolerate surgery.
Aged↗
Alterations of retinal microcirculation in response to scatter photocoagulation.
PURPOSE: To perform acridine orange digital fluorography on rats after scatter photocoagulation to investigate alterations of retinal microcirculation at the capillary level, the authors used leukocyte dynamics as a parameter. METHODS: Twenty-five pigmented rats (Long-Evans) were studied. Argon laser photocoagulation, extending 6 disc diameters from the optic disc, was delivered to one half of the retina, and the other half was untreated. The total number of burns was 200 +/- 10. Leukocyte hemodynamics in retinal microcirculation were evaluated 4, 7, 14, and 28 days after photocoagulation by acridine orange digital fluorography. The fundus image was obtained by using an argon laser in a scanning laser ophthalmoscope and was recorded on magnetic tapes at a video rate. The images were analyzed by a personal computer-based image analysis system. RESULTS: Leukocyte velocities in the retinal capillaries were significantly decreased immediately after photocoagulation. In the laser-treated area, mean capillary leukocyte velocities were 0.73, 0.92, 1, and 1.3 mm/second on days 4, 7, 14, and 28, respectively (velocity in normal control animals 1.4 mm/second). In addition, leukocyte hemodynamics were compromised in the untreated retina: Mean capillary leukocyte velocities were 0.88, 1.1, 1.2, and 1.3 mm/second on days 4, 7, 14, and 28, respectively. Twenty-eight days after photocoagulation, the velocities recovered to normal values in the treated and the untreated areas of the retina. CONCLUSIONS: Retinal capillary hemodynamics were impaired after scatter photocoagulation, and the hemodynamics in the untreated retina were also affected. Photocoagulation to the retina may influence capillary hemodynamics by diffusible chemical substances and by direct tissue injury.
Acridine Orange↗
In vivo evaluation of leukocyte dynamics in retinal ischemia reperfusion injury.
PURPOSE: To evaluate quantitatively leukocyte dynamics in vivo in the rat retinal microcirculation during ischemia reperfusion injury with the use of acridine orange digital fluorography. METHODS: Retinal ischemia was induced in anesthetized pigmented rats by a temporary ligation of the optic nerve. After 60 minutes of ischemia, leukocyte behavior in the retinal microcirculation was evaluated, with acridine orange digital fluorography--consisting of a scanning laser ophthalmoscope and the fluorescent nuclear dye, acridine orange--during reperfusion at 1, 2, 4, 6, 12, 24, 48, 96, and 168 hours. The obtained images were recorded on videotape and analyzed with a computer-assisted image analysis system. RESULTS: Rolling leukocytes along the major retinal veins were observed in treated rats during the reperfusion period; no rolling leukocytes were observed in the control rats. The number of rolling leukocytes gradually increased and peaked at 102 +/- 40 cells/minute 12 hours after reperfusion; few rolling leukocytes were observed at 96 hours. The velocity of rolling leukocytes at 12 hours (19.1 +/- 3.5 microns/second; P < 0.05) was significantly lower than that at the other three times. No rolling leukocytes were observed along the arterial walls throughout the experiments. The number of accumulated leukocytes increased as time elapsed, peaked at 931 +/- 187 cells/mm2 24 hours after reperfusion, and decreased thereafter. CONCLUSIONS: Leukocyte dynamics in the retinal microcirculation can be quantitatively evaluated during ischemia reperfusion injury.
Acridine Orange↗
Upregulation of transforming growth factor-beta after panretinal photocoagulation.
PURPOSE: To determine whether upregulation of transforming growth factor-beta (TGF-beta) gene expression occurs after panretinal photocoagulation (PRP). To quantitate two TGF-beta isoforms in the aqueous and vitreous humors and to localize TGF-beta 2-like immunoreactivities (TGF-beta 2-LI) and TGF-beta 2 mRNA in the retina after PRP. METHODS: PRP was performed on Brown Norway rats by using an argon-green laser. Sensory retina, aqueous and vitreous humors were collected from the rats on days 1, 3, and 7 after PRP, and semiquantitative polymerase chain reaction analyses were carried out. TGF-beta 2 in the aqueous and vitreous humors was quantitated by enzyme-linked immunosorbent assay. Localization of TGF-beta 2-LI was demonstrated by immunohistochemistry, and that of TGF-beta 2 mRNA by in situ reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Gene expression of both TGF-beta 1 and -beta 2 was upregulated by PRP. Expression of TGF-beta 2 was greater than that of beta 1 (12.8-fold increase versus 3.24-fold increase, respectively, compared with control level on day 3). TGF-beta 2 concentration was increased in the vitreous humor (4.37-fold increase, P < 0.01) but not in the aqueous humor (the same level compared with control) on day 1. TGF-beta 2-LI and TGF-beta 2 mRNA were detected in cells of the outer retinal layer and in retinal pigment epithelial cells adjacent to the laser burns. CONCLUSIONS: PRP upregulates expression of TGF-beta 2 in the retina, and increased TGF-beta 2 concentrations are found in the retina and the vitreous humor. Retinal pigment epithelial cells and sensory retina around the laser burn appear to play a major role in the upregulation.
Animals↗
Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells.
PURPOSE: Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture. METHODS: Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation. RESULTS: VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS: These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.
Animals↗
Contrast enhanced magnetic resonance imaging for femoral neck fracture.
Femoral head perfusion was evaluated in 29 patients after acute femoral neck fracture using contrast enhanced fat saturation magnetic resonance imaging. The patients were followed up with T1 and T2 weighted spin echo magnetic resonance imaging without fat saturation, which is suitable in detecting avascular necrosis. The mean interval from injury to the initial contrast enhanced fat saturation magnetic resonance imaging was 24.5 hours. The mean age at the time of injury was 69 years, and the mean followup was 26.9 months. Three distinct patterns of femoral head enhancement were recognized in the acute phase after fracture. When the whole femoral head was well enhanced (Type 3, n = 11), no avascular necrosis developed. In contrast, when the head showed no enhancement (Type 1, n = 6), avascular necrosis developed in all patients. In patients with partial enhancement (Type 2, n = 12), avascular necrosis developed in five patients. These data showed the current method provided an accurate prediction of the development of avascular necrosis in the patients with Type 1 and Type 3 enhancement. However, overall predictive value was 59% (17 of 29 patients) because of the uncertainty in the patients with partial enhancement (Type 2), and additional study is needed for this method to become routine in clinical use.
Adolescent↗
Protective effects of FK506 against glutamate-induced neurotoxicity in retinal cell culture.
PURPOSE: To examine the effects of FK506 on glutamate neurotoxicity in cultured retinal neurons. METHODS: Experiments were performed with primary retinal cultures obtained from 17- to 19-day-old rat fetuses. To assess the effects of FK506 and other drugs on glutamate neurotoxicity, cultures were treated with a drug beginning 10 minutes before application of glutamate and continuing during the subsequent 10 minutes of glutamate exposure. The treated cells were then incubated for 1 hour in a drug-free and glutamate-free medium. After a 1-hour incubation, cell viability was quantitatively measured by the trypan blue exclusion method. RESULTS: Brief exposure to glutamate markedly decreased cell viability. FK506 protected against glutamate neurotoxicity in a dose-dependent manner. Rapamycin is a competitive inhibitor of FK506 that binds FK506 binding protein. Simultaneous application of rapamycin and FK506 negated the protective effects of FK506. Cyclosporin A, which binds and inhibits calcineurin, mimicked the protective effects of FK506. Treatment with FK506 did not affect the intracellular maximum Ca2+ concentration induced by glutamate application. Although FK506 exhibited protective action against Ca2+ ionophore-induced neurotoxicity, it had no effect on nitric oxide-induced neurotoxicity. Treatment with FK506 reduced the activity of nitric oxide synthase (NOS). CONCLUSION: FK506 protected against glutamate neurotoxicity by inhibiting NOS activity in cultured retinal neurons.
Animals↗
Contribution of E-selectin to cellular infiltration during endotoxin-induced uveitis.
PURPOSE: The selectin family is a group of early-reactive adhesion molecules that plays a role in the rolling phase of leukocytes in cellular infiltration. It has been reported that P-selectin is expressed on vascular endothelium in the iris-ciliary body 15 minutes after lipopolysaccharide (LPS) treatment in endotoxin-induced uveitis (EIU) and may contribute to the initial phase of ocular inflammation. The objective of the present study was to identify the expression pattern of E-selectin, another member of the selectin family, and to investigate the role of E-selectin during the course of EIU. METHODS: Endotoxin-induced uveitis was induced in male Lewis rats by a footpad injection of 200 microg LPS. The time-dependent expressions of E-selectin in EIU in the iris- ciliary body and the retina were studied by immunohistochemistry using wholemounts and paraffin-embedded sections and by monitoring the level of E-selectin mRNA expression. A monoclonal antibody to E-selectin and a control antibody were each injected intravenously to evaluate the effects of E-selectin inhibition on ocular inflammation at the time of maximum uveitis. In the anterior uvea, the effect was evaluated by the number of infiltrated cells and by the protein concentration in the aqueous humor 24 hours after LPS treatment; in the retina, the myeloperoxidase (MPO) activity was measured 48 hours after LPS treatment. The effect of the combined injection of anti-P-selectin and anti-E-selectin antibodies was also studied. It was then determined whether the delayed inhibition of E-selectin (6, 12, or 24 hours after LPS injection) could contribute to the early resolution of the uveitis. RESULTS: E-selectin immunoreactivity was observed on the vessel walls of the iris and retina 7 hours after LPS treatment in wholemounts and paraffin-embedded sections and remained positive for 24 hours after LPS treatment. The expression of E-selectin messenger RNA gene peaked at 6 hours and again at 18 hours after LPS treatment in the iris- ciliary body and retina. The expression returned to the basal level 24 hours after LPS treatment in the iris- ciliary body and 48 hours after LPS treatment in the retina. The selective inhibition of E-selectin significantly blocked the cellular infiltration into the aqueous humor (P < 0.005) but had a milder effect on the protein concentration in the aqueous humor (P=0.0536). The inhibition of E-selectin and P-selectin almost abrogated cellular infiltration (P < 0.001). Myeloperoxidase activity in the retina 48 hours after LPS treatment was again significantly decreased by the inhibition of E-selectin alone and by the inhibition of E-selectin and P-selectin (P < 0.0001). A single injection of anti-E-selectin antibody 6, 12, or 24 hours after LPS injection effectively blocked cellular infiltration in the aqueous humor 24 and 48 hours after LPS treatment. CONCLUSIONS: In EIU, E-selectin may be expressed on the vascular endothelium and remain after the period of expression of P-selectin and until approximately the time of maximum uveitis. The present results suggest that, in contrast to the role of P-selectin as an initiator of cellular infiltration, E-selectin may contribute to continuing cellular infiltration into the inflammatory site during inflammation, and its inhibition may contribute to the early resolution of the uveitis.
Animals↗
Estrogen receptor expression in bovine and rat retinas.
PURPOSE: To investigate the expression and distribution of estrogen receptor protein and mRNA in bovine and rat retinas. METHODS: Western blot analysis with an antiestrogen receptor monoclonal antibody (mAb) was used to detect estrogen receptor protein in the bovine retina. Immunohistochemistry with an antiestrogen receptor mAb and in situ hybridization with an oligodeoxynucleotide sequence coding for estrogen receptor were applied to study the cellular distribution of estrogen receptor protein and its mRNA in male bovine retina and rat retina of both sexes. RESULTS: Estrogen receptor protein was detected in bovine retina by Western blot analysis. Immunohistochemical staining with the antiestrogen receptor mAb was widespread throughout the neural retina. Specific staining showed extensive distribution localizing in the nerve fiber layer, the ganglion cell layer, the inner nuclear layer, and the outer plexiform layer. Retinal pigment epithelium and choroid were also stained with the antiestrogen receptor mAb. By in situ hybridization, the expression of estrogen receptor mRNA was predominantly observed in ganglion cell layer, the inner nuclear layer, and outer portion of the outer nuclear layer. No significant difference was found between male and female rats in the immunostaining of retinas with the antiestrogen receptor mAb. CONCLUSIONS: This study provides the first evidence for the presence of estrogen receptor in bovine and rat retinas. Expression of estrogen receptor throughout the retina suggests that estrogen may have important functions in the retina.
Animals↗
Phylogenetic analysis of the human papillomavirus type 2 (HPV-2), HPV-27, and HPV-57 group, which is associated with common warts.
Human papillomavirus types 2 (HPV-2), HPV-27, and HPV-57, are three closely related viruses within the phylogenetic supergroup formed by the remotely related genital papillomaviruses. In contrast to this phylogenetic association, these three viruses are most often found in common warts at nongenital sites, but also occasionally in genital warts and mucosal lesions of the nasopharyngeal cavity. We studied the genomic diversity of HPV sequences in skin warts presumably caused by these viruses. These biopsies were sampled from 75 patients living in Germany, Japan, or Singapore. Among 27 warts with HPV-2, we found seven new genomic variants and among 32 with HPV-57, eight new variants. In both cases, we did not detect the original prototype genomes. In contrast, 13 of 16 warts with HPV-27 contained the prototype genome, and only one new variant was found in three patients. We did not find variants clearly intermediate between any two types, although HPV-2 and HPV-27 are among the most closely related of the extent HPV types. We also did not detect novel HPV types, although the samples were examined with polymerase chain reaction protocols that would have detected remotely related HPVs. So we propose that the phylogenetic group formed by HPV-2, HPV-27, and HPV-57 has no or only very are additional members. One of the HPV-57 variants found, HPV-57-G44, was most likely identical to the subtype HPV-57b, previously proposed to be associated with nasal neoplasia, but found here frequently in common skin warts. Our publication establishes a foundation for pathological and phylogenetic comparisons of HPV types in skin warts.
Base Sequence↗
Enzyme-linked immunosorbent assay using F(ab')2 fragment for the detection of human pulmonary surfactant protein D in sera.
We developed an enzyme-linked immunosorbent assay (ELISA) for detection of SP-D in serum using recombinant SP-D as a standard and horseradish peroxidase conjugated F(ab')2 fragment of mouse monoclonal antibody IgG to avoid the interaction of serum factors including rheumatoid factor. The use of F(ab')2 fragment dramatically decreased the value of serum SP-D concentration in rheumatoid arthritis patients without pulmonary complication to the close level of healthy volunteer. In contrast, the patients with collagen disease having interstitial pulmonary pneumonia exhibited consistently elevated levels of serum SP-D. The use of new ELISA with recombinant SP-D and F(ab')2 fragment of anti-SP-D monoclonal antibody gives a greater advantage for the accurate detection of SP-D in sera from patients with idiopathic pulmonary fibrosis, interstitial pneumonia with collagen disease and pulmonary alveolar proteinosis without interference of rheumatoid factor.
Adult↗
Kinetic analysis of barley chitinase.
The endochitinase from barley is the archetypal enzyme for a large class of plant-derived antifungal chitinases. The X-ray structure was solved previously in our laboratory and a mechanism of action proposed based on structural considerations. In this manuscript we report the use of a defined soluble substrate, 4-methylumbelliferyl beta-N,N',N"-triacetylchitotrioside, to characterize kinetic parameters of the enzyme. The pH profile shows that activity is controlled by a base with a pKa of 3.9 (Glu 89) and an acid with a pKa of 6.9 (Glu 67). The Km using the synthetic substrate is 33 microM, and the k(cat) is 0.33 min(-1), while the Km for (GlcNAc)4 is 3 microM and k(cat) is 35 min(-1). Binding constants were measured for beta-linked oligomers of N-acetylglucosamine. The monomer does not bind and dissociation constants for the dimer, trimer, and tetramer are 43, 19, and 6 microM, respectively. Analysis of kinetic and dissociation constants proves the mechanism of barley chitinase is consistent with a Bi-Bi kinetic model for hydrolysis, with (GlcNAc)4 and water as substrates and (GlcNAc)2 as products. Substrate cleavage patterns show that (GlcNAc)6 is cleaved in half to (GlcNAc)3 as well as into (GlcNAc)4 and (GlcNAc)2 with almost equal efficiency. NMR analysis of cleavage products confirms that the enzyme proceeds with anomeric inversion of products.
Acetylglucosamine↗