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Biomedical subjects

Y Kuroki

Publications and source records attributed to Y Kuroki.

At least 91 records · Page 5Linked to original sources
PubMed1999

Purification and biochemical characterization of equine pulmonary surfactant protein D.

OBJECTIVE: To characterize surfactant protein D (SP-D) isolated from bronchoalveolar lavage fluid (BALF) of healthy horses. SAMPLE POPULATION: BALF from 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) without history or clinical signs of respiratory tract disease. PROCEDURE: BALF was obtained and centrifuged at 33,000 X g. The supernatant was applied to a mannose-Sepharose 6B affinity column in the presence of calcium, and the bound protein fraction was analyzed by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis; amino acid composition was determined and partial sequencing was done. Phospholipid binding and liposome aggregation assay were performed, using purified proteins. RESULTS: The protein isolated by use of mannose affinity matrices was SP-D. It bound carbohydrates and phosphatidylinositol, which are the characteristic features of SP-D isolated from other animal species. Amino acid analysis and partial primary sequence of the isolated protein indicated high homology with rat and human SP-D. Furthermore, immunoblot analysis indicated that equine SP-D reacted with human and rat SP-D-specific antibodies. CONCLUSION AND CLINICAL RELEVANCE: SP-D exists in equine lungs; its measurement may be useful in evaluating equine lung disease.

Amino Acids↗
PubMed1999

Low expression of alphaA-crystallins and rhodopsin kinase of photoreceptors in retinal dystrophy rat.

PURPOSE: The Royal College of Surgeons (RCS) rat has been extensively characterized as a model for inherited retinal dystrophy such as retinitis pigmentosa. In the present study, compositions of retinal proteins were compared between RCS (rdy-/-) and control (rdy+/+) rats during progression of the disease to understand the molecular pathologic course of the retinal degeneration. METHODS: Protein mapping was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional (2D)-PAGE using whole retinas or rod outer segments (ROS) obtained by a sucrose-density gradient centrifugation method from RCS or control rats at the age of 3 to 8 weeks. RESULTS: 2D-PAGE showed that retinal proteins of RCS rats were generally less abundant than those of the control animals and that the difference became more evident with aging. However, no significant difference was observed in the protein-mapping patterns in 2D-PAGE between RCS and control rats in any ages tested. Analysis by SDS-PAGE of ROS proteins and by western blot using antibodies against opsin, rhodopsin kinase (RK), recoverin, or arrestin demonstrated that a 20-kDa protein and RK were selectively less abundant in RCS than in control rats. Edman sequence analysis of the proteolytic peptides obtained by in-gel digestion of the corresponding protein band using endoproteinase Lys C identified the 20-kDa protein as alphaA-crystallin. Reverse transcription-polymerase chain reaction confirmed selective low levels of mRNA expressions of alphaA-crystallins and RK in RCS rats. CONCLUSIONS: This study demonstrates that decreased expression of alphaA-crystallins and RK in RCS rats, may have significant roles in the development of retinal dystrophy.

Amino Acid Sequence↗
PubMed1998

Surfactant proteins A and D: disease markers.

The abundant and restricted expression of surfactant proteins SP-A and SP-D within the lung makes these collectins specific markers for lung diseases. The measurement of SP-A and SP-D in amniotic fluids and tracheal aspirates reflects lung maturity and the production level of the lung surfactant in infants with respiratory distress syndrome (RDS). The SP-A concentrations in bronchoalveolar lavage (BAL) fluids are significantly decreased in patients with acute respiratory distress syndrome (ARDS) and also in patients at risk to develop ARDS. The prominent increase of these proteins in BAL fluids and sputum is diagnostic for pulmonary alveolar proteinosis (PAP). The concentrations of SP-A and SP-D in BAL fluids from patients with idiopathic pulmonary fibrosis (IPF) and interstitial pneumonia with collagen vascular diseases (IPCD) are rather lower than those in healthy controls and the SP-A/phospholipid ratio may be a useful marker of survival prediction. SP-A and SP-D appear in the circulation in specific lung diseases. Their serum concentrations significantly increase in patients with PAP, IPF and IPCD. The successive monitoring of serum levels of SP-A and SP-D may predict the disease activity. The serum SP-A levels increase in patients with ARDS. SP-A is also a marker for lung adenocarcinomas and can be used to differentiate lung adenocarcinomas from other types and metastatic cancers from other origins, and to detect metastasis of lung adenocarcinomas.

Enzyme-Linked Immunosorbent Assay↗
PubMed1998

Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells.

Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Mutational analysis of Arg197 of rat surfactant protein A. His197 creates specific lipid uptake defects.

In previous studies, tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids. We substituted Ala, Lys, His, Asp, and Asn mutations for Arg to evaluate the role of a basic amino acid at position 197 in SP-A action. Unexpectedly, Ala197 retained complete activity in the SP-A functions of carbohydrate binding, type II cell binding, inhibition of surfactant secretion, lipid binding, lipid aggregation, and lipid uptake by type II cells. The results unambiguously demonstrate that Arg197 is not mechanistically essential for SP-A function. The Lys197 mutation displayed all functions of the wild type protein but exhibited a 2-fold increase in lipid uptake activity. The His197 mutation displayed all SP-A functions studied except for lipid uptake. The results obtained with the His197 mutation clearly demonstrate that lipid aggregation alone by SP-A is insufficient to promote lipid uptake by type II cells. These findings indicate that specific interactions between type II cells and SP-A are involved in the phospholipid uptake processes.

Animals↗
PubMed1998

Analysis of chimeric proteins identifies the regions in the carbohydrate recognition domains of rat lung collectins that are essential for interactions with phospholipids, glycolipids, and alveolar type II cells.

Pulmonary surfactant proteins A (SP-A) and D (SP-D) are collectins in the C-type lectin superfamily. SP-A binds to dipalmitoylphosphatidylcholine and galactosylceramide, and it regulates the uptake and secretion of surfactant lipids by alveolar type II cells. In contrast, SP-D binds to phosphatidylinositol (PI) and glucosylceramide (GlcCer). We investigated the functional region in the carbohydrate recognition domain of rat SP-A and SP-D that is involved in binding lipids and interacting with alveolar type II cells by using chimeric proteins. Chimeras ad3, ad4, and ad5 were constructed with SP-A/SP-D splice junctions at Gly194/Glu321, Gln173/Thr300, and Met134/Cys261, respectively. All three chimeras lost SP-A-specific functions. Chimeras ad3, ad4, and ad5 bound to PI with increasing activity. In contrast, chimeras ad3 and ad4 did not bind to GlcCer, whereas ad5 avidly bound this lipid. From these results, we conclude that 1) the SP-A region of Glu195-Phe228 is required for lipid and type II cell interactions, 2) the SP-D region of Cys261-Phe355 is required for optimal lipid interactions, and 3) the structural requirement for the binding of SP-D to PI is different from that for GlcCer.

Animals↗
PubMed1998

BAL surfactant protein A and Clara cell 10-kDa protein levels in healthy subjects.

Lung surfactant protein A (SP-A) and Clara cell 10-kDa protein (CC10) are the most abundant proteins produced locally in the lower respiratory tract, as assessed in bronchoalveolar lavage (BAL) analysis. However, it is not known what factors influence SP-A and CC10 levels in BAL fluids, and the relationship between SP-A and CC10 levels in BAL fluids has been unclear. We measured SP-A and CC10 concentrations in BAL fluids from 11 healthy nonsmokers and 12 healthy smokers by enzyme-linked immunosorbent assays using specific antibodies. Mean SP-A and CC10 levels in BAL fluids of healthy smokers were significantly lower than those of healthy nonsmokers. SP-A values correlated significantly with CC10 and phospholipid values in BAL fluids. On BAL examinations using three 50-ml aliquots, the mean SP-A level in the second lavage was 2.0-fold and 2.4-fold, respectively, of that in the first and third lavages, and the mean CC10 level in the first lavage was 5.0-fold and 5.6-fold, respectively, of that in the second and third lavages. We conclude that BAL fluid SP-A and CC10 levels are influenced by the BAL methods and by cigarette smoking. There is a significant positive correlation between SP-A and CC10 values in BAL fluids of healthy subjects.

Adult↗
PubMed1998

Clinical characteristics of children with hypoparathyroidism due to 22q11.2 microdeletion.

UNLABELLED: The phenotypes of chromosomal 22q11.2 microdeletion are quite variable among individuals and hypoparathyroidism (HP) constitutes a definite portion of the clinical spectrum. For the correct diagnosis and pertinent follow up of the HP children due to del22q11.2, we tried to delineate the clinical characteristics of such patients. By employing fluorescence in situ hybridization (FISH) to all the patients diagnosed as HP in our clinic, ten possessed the 22q11.2 microdeletion. Among them, the incidence of cardiac defect (5/10), recurrent infection (1/10) and cleft palate (1/10) was modest. Additionally, seven of them had been diagnosed as HP during the infantile period, when their facial abnormality and intellectual problem had not become evident. Notably, two patients were complicated by Graves disease, while the association of idiopathic thrombocytopenic purpura was also observed in two girls. CONCLUSION: HP due to del22q11.2 may be misdiagnosed as idiopathic, especially in an infant who lacks apparent complications like cardiac anomaly. They should be closely followed up for auto-immune complications.

Child↗
PubMed1998

Neuroblastoma in an adult with a high serum level of carbohydrate antigen, CA125: report of a case.

We report herein the case of a 56-year-old woman with a neuroblastoma associated with a high serum level of carbohydrate antigen, CA125. The patient presented with massive ascites and a firm mass in her upper abdomen for which a laparotomy was performed. However, a recurrent tumor was found 6 months later and she died of the disease within 1 year of surgery despite several courses of adjuvant chemotherapy. Neuroblastoma rarely occurs in adults, and the features of 58 adult cases described in the world literature is summarized following the presentation of the clinical data on this case. The distribution of primary sites in adults is dispersed compared to that seen in pediatric cases, while the natural history of the disease in adults may be longer and less sensitive to chemotherapy than in children. The survival rate of adults with this disease is poor. We conclude that aggressive surgical intervention combined with appropriate chemotherapy protocols as applied in children should be performed in an attempt to achieve complete remission and improve the survival rate of adults with neuroblastoma.

Abdominal Neoplasms↗
PubMed1998

Wear of bipolar hip prostheses.

A method was developed to take radiographs showing the inner articulation of bipolar hip prostheses. By this method, wear was measured in 68 hips whose inner head diameter was 22 mm. Average annual wear rate was 0.17 mm. Osteolysis was observed in 25 hips (37%) and there was no difference between the annual wear rate of hips with and without osteolysis. Studying 19 retrieved prostheses, abrasion of the rim was deeper in hips with osteolysis than those without it. Wear rate of the inner articulation in bipolar hip prosthesis is much larger than that in Charnley's prosthesis, as linear penetration into the articulation surface reduces the motion range of the inner articulation and this increases impingement and advances rim abrasion.

Acetabulum↗
PubMed1998

Polymerase chain reaction analysis of the Y chromosome long arm in azoospermic patients: lack of the Y chromosome recognition motif (YRRM1) gene.

We analyzed DNA from a patient with azoospermia whose Y chromosome was cytogenetically normal. A total of 16 loci on the Y chromosome long arm were examined: 15 loci between DYS7E and DYZ1, and the Y chromosome RNA recognition motif (YRRM1) locus, a candidate gene for the azoospermic factor AZF. We did not detect the YRRM1 gene in this patient. This finding supports the theory that YRRM1 is an essential gene for spermatogenesis.

Adult↗
PubMed1998

Further evidence for the involvement of human chromosome 6p24 in the aetiology of orofacial clefting.

Chromosomal translocations affecting the 6p24 region have been associated with orofacial clefting. Here we present a female patient with cleft palate, severe growth retardation, developmental delay, frontal bossing, hypertelorism, antimongoloid slant, bilateral ptosis, flat nasal bridge, hypoplastic nasal alae, protruding upper lip, microretrognathia, bilateral, low set, and posteriorly rotated ears, bilateral microtia, narrow ear canals, short neck, and a karyotype of 46,XX,t(6;9)(p24;p23). The translocation chromosomes were analysed in detail by FISH and the 6p24 breakpoint was mapped within 50-500 kb of other breakpoints associated with orofacial clefting, in agreement with the assignment of such a locus in 6p24. The chromosome 9 translocation breakpoint was identified to be between D9S156 and D9S157 in 9p23-p22, a region implicated in the 9p deletion syndrome.

Chromosome Mapping↗
PubMed1998

A 50-kDa variant form of human surfactant protein D.

The dominant form of human surfactant protein D (SP-D) is a multimeric collagenous glycoprotein composed of monomeric subunits that have a molecular mass of 43 kDa under reducing conditions. However, in evaluating monoclonal antibodies to human SP-D, an additional monomeric subunit was identified with a reduced molecular mass of 50 kDa. This 50-kDa variant was detected in approximately half of the samples evaluated and was found in lavage fluid from normal subjects, patients with alveolar proteinosis or idiopathic pulmonary fibrosis and in amniotic fluid. This 50-kDa variant had the same amino-terminal sequence, amino acid composition and apparent size of the carboxy-terminal collagenase-resistant fragment (20 kDa) as the 43-kDa subunit. The major difference was in the amino-terminal portion of the molecule and was due to altered glycosylation, as determined by carbohydrate staining, chemical deglycosylation, treatment with N-glycanase and neuraminidase and reduced signals for threonine at positions 5, 9 and 10 during amino-terminal sequencing. After gel filtration chromatography, the 50-kDa form was not present in the high molecular weight fraction, which is commonly used in purification of SP-D, but was found only in the smaller molecular weight fraction of monomers and trimers of SP-D. In conclusion, the 50 kDa-form of surfactant protein D is produced by post-translational glycosylation and does not form higher ordered oligomers, but its precise physiological function remains to be determined.

Amino Acids↗