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Biomedical subjects

K Hosoi

Publications and source records attributed to K Hosoi.

At least 73 records · Page 4Linked to original sources
PubMed1990

A new electrophoretic variant of alpha subunit of Na+/K(+)-ATPase from the submandibular gland of rats.

The alpha catalytic subunits of Na+/K(+)-ATPase were isolated from the kidney and brain of rats (alpha 1 and alpha 2, respectively). The antisera raised against these subunits were used as probes to analyze the isoform of catalytic subunits of Na+/K(+)-ATPase in various tissues of rats. Of 27 rat tissues examined, most had a catalytic subunit identical to alpha 1 but some, such as the nervous and muscle tissues, had both alpha 1 and alpha 2 isoforms as judged by their reactivities to antisera and their electrophoretic mobility. We found that the submandibular gland contained a new electrophoretic variant of immunoreactive alpha subunit (designated alpha(S) in this report) in addition to alpha 1 identical to those found in kidney and brain. The new variant, alpha(S), strongly cross-reacted with anti-alpha 1 antiserum, but to a lesser extent with anti-alpha 2 antiserum. The alpha(S) had a molecular mass which was found to be slightly less (approx. 90 kDa) than brain and kidney alpha 1. We examined whether or not the alpha(S) is formed by proteolytic cleavage of alpha subunits during preparation and concluded that this is not the case. The alpha(S) reacted with [gamma-32P]ATP, resulting in the formation of radioactive alpha subunit which was stabilized by 2 mM ouabain but which was labile in the presence of 70 mM potassium chloride. Since N-terminal amino acid sequence of alpha(S) protein [G()DKY()PAAVS] corresponds exactly and uniquely with the sequence of the alpha 1 chain between residues 1 and 11, it is very probable that alpha(S) protein originated from alpha 1 protein following the post-translational processing.

Amino Acid Sequence↗
PubMed1990

Preparation of asialofetuin-labeled liposomes with encapsulated human interferon-gamma and their uptake by isolated rat hepatocytes.

The selective delivery of human recombinant interferon (IFN)-gamma to isolated rat hepatocytes was studied with asialofetuin (AF)-labeled liposomes. AF-liposomes containing buffer solution were initially prepared by the detergent removal method, and IFN-gamma was subsequently encapsulated by the freeze-thawing method without loss of activity. Virtually no free [32P]IFN-gamma was internalized into isolated rat hepatocytes, whereas AF-liposomes containing [32P]IFN-gamma were taken up to a significant degree. Liposomal binding to the hepatocytes (estimated at 4 degrees C) was one-fifth of the uptake (estimated at 37 degrees C). Since the uptake was inhibited by the addition of free AF, AF-liposomes may be taken up by the action of galactose-binding protein on the hepatocytic cell surface. The liposome preparation method reported in this paper provides a useful means for the encapsulation of unstable macromolecules into AF-liposomes. AF-liposomes were found effectively to carry IFN-gamma into hepatocytes in vitro.

Animals↗
PubMed1990

Sodium and pH dependent carrier-mediated transport of antibiotic, fosfomycin, in the rat intestinal brush-border membrane.

The mechanism of intestinal absorption of an antimicrobial agent, fosfomycin (FOM), was investigated in rats using small intestinal brush-border membrane vesicles (BBMV). The uptake of [3H]FOM by BBMV was osmolarity- and temperature-sensitive and showed apparently saturable uptake kinetics consistent with the Michaelis-Menten equation, having Kt = 15.3 mM and Jmax = 7.78 nmol/30 s/mg protein at 37 degrees C. An overshoot uptake of FOM was observed in the presence of an inwardly direct Na+ gradient. The replacement of extravesicular Na+ with choline or mannitol significantly reduced the uptake. An addition of a protonophore, FCCP, significantly decreased the initial uptake of FOM in the absence of Na+ gradient but in the presence of a H+ gradient (pHin = 7.5, pHout = 6.0), whereas in the absence of a H+ gradient no significant difference was observed between the uptakes at an acidic pH (pHin = pHout = 6.0) and a neutral pH (pHin = pHout = 7.5). An inside negative potassium diffusion potential induced by valinomycin enhanced significantly the uptake of FOM. The uptake of FOM in the presence of both Na(+)- and H(+)-gradients was significantly inhibited by phosphate, arsenate and phosphonoformic acid (PFA), which are specific inhibitors of phosphate transport, but not by D-glucose. Based on these results, it is concluded that FOM transport in the small intestine is partially shared with the Na(+)-phosphate cotransport system and in part occurs via a H(+)-gradient dependent carrier-mediated system.

Animals↗
PubMed1990

A human-mouse hybrid cell line expressing both human leukocyte and histocompatibility-2 antigens.

Clone-1d, a sub-line of mouse L cells, was transfected with E. coli neo gene cloned in pSV2 vector (pSV2 neo) to obtain C1-1d neo cells. These cells are able to survive in the presence of geneticin (G418) but are killed by the medium containing hypoxanthine, aminopterin, and thymidine (HAT) because of the deficiency of thymidine kinase (TK) gene. By using these cells, it became possible to produce stable hybrid cells between these neo cells and any other cells since the hybrid cells are selected in the culture medium containing both G418 and HAT. We produced such hybrid cells by fusing C1-1d neo and A431 human epidermoidal carcinoma cells and studied the expression of human leucocyte antigens (HLA) and histocompatibility-2 antigens (H-2) in three hybrid cell lines. We found that one out of three hybrid cell lines expresses both HLA and human beta 2 microglobulin besides H-2 antigens, whereas the other two express only H-2 antigens; this indicated better stability of mouse genes than of human genes.

Animals↗
PubMed1990

[Genesis and clinical significance of an apical diastolic rumble in patients with mitral Björk-Shiley valve: a Doppler echocardiographic study].

The genesis and clinical significance of an apical diastolic rumble after replacement with mitral Björk-Shiley prosthesis were studied in 20 patients by left ventricular (LV) inflow dynamics using Doppler echocardiography. 1. The rumble was audible in 17 of the 20 patients (85%). In all but one patient, its intensity was less than Levine 2/6. Duration of the rumble ranged from 248 to 544 (mean 377) msec. There was no case of prosthetic valve dysfunction. Duration of the slow filling wave (SF interval) of the apexcardiogram in 19 cases ranged from 150 to 541 (mean 317) msec, and it correlated significantly with the duration of the rumble (r = 0.95) in 15 cases. 2. In 20 patients, peak velocity of the LV inflow obtained from continuous wave Doppler echocardiograms ranged from 1.17 to 1.89 (mean 1.56) m/sec, and pressure half time (PHT) ranged from 82 to 233 (mean 134) msec. In 19 patients, the SF interval correlated significantly with PHT (r = 0.87), but not with peak inflow velocity. 3. Peak inflow velocity was significantly slower in cases without a rumble than in cases with a rumble. PHT and the SF interval tended to be shorter in the former than in the latter. 4. There was no significant difference in the SF interval, peak inflow velocity and PHT between eight patients with smaller valve sizes (less than or equal to 25 mm) and 12 with larger valve sizes (greater than or equal to 27 mm). 5. There was no obvious relation between the direction of the main LV inflow jet as assessed by Doppler color flow mapping and the presence of the rumble in 19 patients. 6. In 19 patients, the SF interval correlated significantly with left atrial dimension (r = 0.47), but it did not correlate with LV end-diastolic dimension, fractional shortening of the LV, and the peak lengthening rate of LV internal dimension. 7. Three patients with both the rumble and SF lasting 500 msec had prolonged PHT longer than 190 msec. One of these three had congestive heart failure and suspected prosthetic mitral valve stenosis. From these results, we concluded that an apical diastolic rumble after replacement with mitral Björk-Shiley valve is mainly due to the increase in resistance and velocity of the LV inflow, and that both durations of the rumble and the SF intervals as well as PHT of the Doppler echocardiogram are useful in detecting stenosis of prosthetic mitral valves.

Adult↗
PubMed1990

[Relationship between regurgitant flow dynamics and cardiac physical in tricuspid regurgitation: a phono-, mechano- and Doppler echocardiographic study].

To evaluate the relationships between regurgitant flow dynamics of tricuspid regurgitation (TR) and cardiac physical signs, and to clarify the role of atrial function on central venous flow, we investigated physical signs by cardiac auscultation and palpation of the liver. In addition, phonocardiography, jugular venous and hepatic pulse tracings and Doppler echocardiographic recordings were performed. The subjects, 109 patients with Doppler-detected TR, were categorized as an SR group of 42 with sinus rhythm, an Af group of 63 with atrial fibrillation and four with sinus arrest. Thirty-five patients underwent open heart surgery before six months or more. Results were as follows: 1. In the Af group, the maximum systolic flow velocity data in the superior vena cava (SVC) and hepatic vein (HV) correlated well with the maximum tricuspid regurgitant signal area on the color Doppler echocardiogram, and systolic backward flow from the heart was more evident in the HV than in the SVC. In the SR group, however, no correlation was observed between the maximum systolic flow velocity and the TR signal area, and systolic backward flow was not evident even in cases with severe TR. 2. After open heart surgery, systolic flow velocities in the SVC and HV were significantly decreased in the SR group compared to the Af group. 3. There was close correlation between the presence of hepatomegaly and systolic backward flow towards the liver. Hepatomegaly was more marked in the Af group than in the SR group. 4. Jugular venous and hepatic pulse data correlated well with the flow velocity data in the SVC and HV and with the TR signal area. 5. Intensity of the tricuspid regurgitant murmur as estimated by the Levine's classification correlated relatively well with the systolic pressure gradient between the right ventricle and right atrium as calculated by the modified Bernoulli equation, but did not correlate with the TR signal area. From these results, we conclude that the intensity of the tricuspid regurgitant murmur and the jugular venous or hepatic pulse patterns are useful for evaluating tricuspid regurgitant dynamics, when they are applied clinically with precise recognition of their significance and limitations, and that sinus rhythm or atrial fibrillation is also an important factor.

Atrial Fibrillation↗
PubMed1990

[Effects of L-ascorbic acid and bone metabolism factors on alkaline phosphatase activity of and 45Ca2+ incorporation by ROS 17/2.8 cells].

Among several bioactive substances known as coupling factors, transforming growth factor-beta (TGF-beta), interleukin-1 (IL-1), and prostaglandin (PG) E1 and E2 increased not only the activity of alkaline phosphatase but also the rate of incorporation of 45Ca2+ into ROS 17/2.8 during a 3-day culture: the former two factors are known to be formed at the site where bone is resorbed, while PG's are known as one of the factors involved in bone resorption. Parathyroid hormone, another hormone that affects bone metabolism, elevated the incorporation of 45Ca2+ by and decreased the alkaline phosphatase activity of the cells. The facts indicate the possibility that the osteoblastic cells are involved in the transport of calcium ions when bones are being resorbed. On the other hand, when these osteosarcoma cells were cultured in DMEM containing ascorbate and beta-glycerophosphate, followed by staining with silver nitrate by the procedure of von Kossa, there appeared many groups of cells that were positively stained as dark brown spots. Cells were then cultured under the same conditions in the presence of radioactive calcium, and the radioactivity accumulated was measured. The result showed that the presence of both ascorbate and beta-glycerophosphate in the culture medium dramatically increased the accumulation of 45Ca2+. It appears from these facts that ROS 17/2.8 cells are capable of incorporating and/or accumulating calcium ion if they are cultured under appropriate conditions. These cells will probably be able to produce a calcified matrix in vitro.

Alkaline Phosphatase↗
PubMed1990

Reversal of relative proteinase F activity and onset of androgen-dependent proteinases in the submandibular gland of postnatal mice.

By isoelectric focusing, we separated trypsin-like proteinases of the mouse submandibular gland (ICR strain) into isozymes with pI values of 4.6 (proteinase F), 5.6 (protease D), 5.8 (protease A), 7.1 and 9.9 (P-esterase). During postnatal development, proteinase F appeared earliest (on the 15th day after birth) and increased in both sexes; however, its percentage ratio to total activity decreased markedly with time because of the rapid increase of other proteinases. On the 22nd day of life, proteinases A and D appeared, and the increase of a proteinase with pI-7.1 followed thereafter. P-esterase was the last isozyme to appear, becoming detectable around 29-45 days. After maturation, the activities of protease A plus D, P-esterase, and the isozyme with a pI value of 7.1 were higher in males than in females, whereas the relative level of proteinase F was reversed. We conclude that proteinase F is appreciably different from the other four proteinases in its development pattern as well as in its responsiveness to sexual hormones.

Androgens↗
PubMed1990

[On the mechanisms of mitral regurgitation in rheumatic mitral valve disease: with special reference to the role of mitral valve prolapse].

To assess the role of mitral valve prolapse (MVP) in the pathogenesis of mitral regurgitation (MR) in rheumatic mitral valve disease (RMD), we performed phonocardiography (PCG), transthoracic and transesophageal two-dimensional and color Doppler (CD) echocardiography in 22 patients with RMD including three with pure mitral stenosis (MS), 11 with predominant MS, six with predominant MR and two with pure MR. Results were as follows: 1. Prolapse of the mitral valve (MV) was differentiated from systolic ballooning of the whole MV by the findings that the anterior leaflet's tip (rough zone) protruded into the left atrium with an acute angle between the body (clear zone) and rough zones of the anterior MV and that the posterior leaflet protruded markedly above the level of the mitral ring. 2. MR was detected in six patients (slight MR) by only the CD method and in 13 (mild, moderate or greater MR) by both the PCG and CD methods. 3. MR was absent or slight in five patients (three of pure MS and two of predominant MS) without valve thickening and with systolic ballooning of the whole valve due to commissural fusion. 4. Mitral valve abnormalities related to significant (mild, moderate or severe) MR were dependent on valve thickening (five patients), prolapse of the leaflet's tip toward the left atrium (four), or both (four). 5. An apical systolic click was found in only one of the nine patients with systolic ballooning, but in four of 11 with MVP. 6. The MR murmur in six of the nine patients with valve thickening showed the decrescendo or flat contour, but that in four of the eight patients with MVP showed a crescendo contour. From these results, we concluded that mitral valve prolapse should be considered as one of the important causes of mitral regurgitation in rheumatic mitral valve disease.

Adult↗
PubMed1989

Establishment of strongly neutralizing monoclonal antibody to human interleukin-6 and its epitope analysis.

Three monoclonal antibodies against human interleukin-6 were established and characterized. One antibody was shown to strongly neutralize both the Ig-inducing and hybridoma/plasmacytoma growth activity of interleukin-6. The results of its epitope analysis using protease treated interleukin-6 and immobilized antibody indicated that this neutralizing antibody binds to a peptide corresponding to Leu151-Lys171 of interleukin-6 molecule. Further analysis using synthetic peptides showed that a shorter peptide corresponding to Ala153-Thr162 can also inhibit the binding of the antibody to interleukin-6. These results suggest that this carboxyl-terminal region plays a crucial role in interleukin-6 functions.

Amino Acid Sequence↗
PubMed1989

Characterization of four different mammalian-cell-derived recombinant human interferon-beta 1s. Identical polypeptides and non-identical carbohydrate moieties compared to natural ones.

In order to evaluate the structural identification of various recombinant human interferon-beta 1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian-cell-derived recombinant human interferon-beta 1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast-derived natural human interferon-beta 1. Specific activities, amino acid compositions, amino-terminal sequences, peptide maps on C18 reversed-phase high-performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones. On the other hand, the patterns of isoelectric focusing were different between mammalian-cell-derived recombinant human interferon-beta 1s and natural human interferon-beta 1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8- and C127-derived recombinant proteins by N-glycanase. These results indicate that mammalian-cell-derived recombinant human interferon-beta 1s have identical polypeptides to those of natural human interferon-beta 1 but their carbohydrate moieties, including unusual N-linked oligosaccharides, are individually different from natural ones and depend on the host cell.

Adenocarcinoma↗
PubMed1989

Exogenous ATP and other nucleoside phosphates modulate epidermal growth factor receptors of A-431 epidermoid carcinoma cells.

The binding of epidermal growth factor (EGF) by A-431 human epidermoid carcinoma cells was reduced after exposure of the cells to low concentrations (0.01-1 mM) of ATP and other nucleoside 5'-triphosphates at 37 degrees C, but not at 0 degree C. This was due to loss of high-affinity EGF binding sites. The modulation was associated with transient increases in inositol phosphate synthesis and intracellular Ca2+ and with phosphorylation of the EGF receptor on serine and threonine. There was no evidence for entry of labeled ATP into the cells. ATP appeared to bind to specific cell surface receptors. Such binding was demonstrated directly with the nonmetabolizable ATP analogue adenosine 5'-[beta,gamma-imido]triphosphate.

Adenosine Triphosphate↗
PubMed1989

Highly sensitive enzyme immunoassay of human interferon-beta 1.

A highly sensitive sandwich enzyme immunoassay for human interferon-beta 1 (HuIFN-beta 1) was developed. HuIFN-beta 1-containing samples and horseradish peroxidase (HRP)-labeled mouse anti-HuIFN-beta 1 monoclonal antibody (Fab') were incubated overnight at 2-10 degrees C in the wells of a 96-well microtiter plate, onto which affinity-purified rabbit anti-HuIFN-beta 1 polyclonal antibody was coated. The EIA was able to detect 0.5 IU/ml of HuIFN-beta 1, thus showing higher sensitivity than bioassay. The values obtained by the EIA closely paralleled those obtained by bioassay in the concentration which bioassay can detect. In order to detect the concentration below 0.5 IU/ml of HuIFN-beta 1, the avidin/biotin-amplified EIA was also developed. The use of biotinylated mouse anti-HuIFN-beta 1 monoclonal antibody (F(ab')2) and HRP-avidin in the EIA made it possible to detect 0.1 IU/ml of HuIFN-beta 1. These EIAs were applied for the studies such as process control of HuIFN-beta 1 production, pharmacokinetics of HuIFN-beta 1, and determination of serum level of HuIFN-beta 1 in healthy subjects.

Antibodies, Monoclonal↗
PubMed1989

Postnatal changes in an alpha subunit isoform, alpha(S), of Na+,K(+)-ATPase in the submandibular gland of rats.

The alpha and alpha(+) isoforms of Na+,K(+)-ATPase were isolated from the kidney and brain of rats and purified. Their antisera were raised to analyze the alpha isoforms in rat tissues. We found that the submandibular gland (SMG) contains a new immunoreactive alpha subunit isoform, designated alpha(S) in this report, in addition to alpha identical with those found in the kidney or brain. The new alpha(S) strongly reacted with anti-alpha-antiserum but to a much lesser extent with anti-alpha(+)-antiserum. The alpha(S) had a slightly lower molecular weight (approximately 90,000) than the brain and kidney alpha isoforms. Various fractions of SMG tissues were added to the SMG microsomes and incubated in order to test whether or not the alpha(S) is formed artificially; no increase of alpha(S) was observed by these treatments, suggesting that the alpha(S) was not the product formed from alpha during the preparation of microsome sample, but was rather a protein originally present in the SMG. The alpha(S) protein was not detected in the SMG of 2- or 5-week-old rats, but it gradually increased in rats older than 8 weeks, reaching the maximum in 30-week-old animals. The Na+,K(+)-ATPase activity in the SMG increased concomitantly with the increase of alpha(S), indicating that Na+,K(+)-ATPase comprising alpha(S) also shows enzyme activity; it is speculated that alpha(S) may have some unique and unknown function(s) in older rats.

Animals↗
PubMed1989

A simple and rapid purification procedure for the preparation of tetramethylrhodamine-labeled antibodies.

A conjugate preparation of antibody (Fab) and tetramethylrhodamine (TMR) generally adsorbs free TMR which is very difficult to remove because of its strong hydrophobic binding. On the basis of criteria such as sodium dodecyl sulfate (SDS) gel electrophoresis and staining of the plasma membrane of live cells, we found that simple extraction with n-butyl alcohol or iso-amyl alcohol could remove the contaminating free dye. The procedure is especially useful when one needs to prepare conjugates with low nonspecific binding for the study of lateral diffusion of cell membrane-associated antigens.

1-Butanol↗
PubMed1989

[Effects of various factors involved in bone metabolism on 45Ca2+ incorporation and alkaline phosphatase activity of ROS 17/2.8 cells].

The effects of interleukin 1, transforming growth factor-beta (coupling factors), prostaglandin E1, and prostaglandin E2 on incorporation of 45Ca2+ and on alkaline phosphatase activity were studied using cultured ROS 17/2.8 cells, one of cell lines derived from rat osteosarcoma. We found that all these factors stimulate both the incorporation of 45Ca2+ and alkaline phosphatase activity of these cells. On the other hand, one of the bone resorption hormones, parathyroid hormone (PTH), suppressed the proliferation of cells and decreased the alkaline phosphatase activity at considerably low concentrations (1 X 10(-12)-1 X 10(-11) M). However, the hormone stimulated the incorporation of 45Ca2+ by these cells in a dose-dependent manner; the maximum stimulation on day 3 was observed at 1 X 10(-7) M and it was approximately 3 times the control value. The data suggest therefore, that the osteoblasts incorporated calcium ions and transported them while bone resorption was occurring. Thus the ROS 17/2.8 cell line appears to be an advantageous experimental system for the study of calcium metabolism of osteoblasts in vitro.

Alkaline Phosphatase↗
PubMed1989

[Assessment of prolapsing pattern of the anterior mitral valve in mitral valve prolapse: new echocardiographic diagnostic criteria].

In the present study, the ballooning pattern of the anterior mitral valve (AMV) in mitral valve prolapse (MVP) was investigated, and new diagnostic criteria for MVP were established using two-dimensional and Doppler echocardiography. The study population consisted of 164 patients with prolapse of the AMV alone, including 86 patients with idiopathic MVP, 52 associated with atrial septal defect (ASD), 17 having chordal rupture and nine associated with connective tissue disorders. There were 60 normal controls. The results were as follows: 1. The AMV was divided into two zones, clear and rough (CZ and RZ), according to the point of insertion of the strut chordae based on two-dimensional long-axis echocardiograms of the left ventricle. The severity of AMV prolapse was determined by an angle between the posterior aortic wall (PAO)-CZ and the CZ-RZ. a) Type A: Apparently there was a transitional point between CZ and RZ, despite normal PAO-CZ and CZ-RZ angles. The RZ showed mild ballooning or prolapse into the left atrium. b) Type B: Although the PAO-CZ angle was normal, the CZ-RZ angle was markedly narrowed. Therefore, prolapse of the RZ was more severe compared with that of type A. c) Type C: An overall zone of the AMV showed ballooning or prolapse into the left atrium due to a narrowed PAO-CZ angle. 2. Type B prolapse was frequently observed in idiopathic MVP (58%), the ASD group (71%) and the chordal rupture group (71%), and type C prolapse in MVP of connective tissue disorders (89%). 3. All of 18 patients (100%) with type A, 48 of 99 (48%) with type B, and 10 of 47 (21%) with type C could not be diagnosed as MVP using Gilbert's criterion. 4. Doppler mitral regurgitation (MR) was detected in 40 of the 47 patients (85%) with type C in 56 of the 99 (59%) with type B, and in seven of the 18 (39%) with type A. These results suggested that classification of the two-dimensional echocardiography of the AMV into two zones, clear and rough (CZ and RZ), could contribute to determining not only the severity of AMV prolapse, but also to the extent of myxomatous changes of the AMV and to evaluating the correlations between the degree of MVP and the incidence of MR.

Adolescent↗