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H Sohma

Publications and source records attributed to H Sohma.

At least 37 records · Page 2Linked to original sources
PubMed1999

Lazaroid U74389G ameliorates ischemia-reperfusion injury in the rat lung transplant model.

We investigated the effect of Lazaroid U74389G on ischemia-reperfusion injury in the rat orthotopic left lung transplantation model. Five groups of reperfused lungs were studied. In group I, donor lungs were transplanted after 12 hours of preservation in University of Wisconsin (UW) solution at 4C. In groups II, III, and IV, Lazaroid was intravenously administrated at a dose of 1 mg/kg, 8 mg/kg, and 15 mg/kg, respectively, to the donors 30 minutes before preservation and also to the recipients 30 minutes before reperfusion after 12 hours of storage in UW solution at 4C. In group V, Lazaroid was added to the UW solution (80 micromol/l), and also was administered intravenously (6 mg/kg) 30 minutes before reperfusion. After 1 hour of reperfusion, gas exchange function and tissue lipid peroxide levels were significantly improved in Lazaroid-treated groups III, and V compared with no treatment group I. Histologic damage was less severe in groups III, IV, and V than in group I. These findings suggest that Lazaroid U74389G ameliorates ischemia-reperfusion injury in the rat lung transplants by inhibiting lipid peroxidation, regardless of whether it is administrated intravenously or given as an additive to the preservation solution.

Analysis of Variance↗
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

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↗
PubMed1997

A novel type of binding specificity to phospholipids for rat mannose-binding proteins isolated from serum and liver.

Mannose-binding protein (MBP) belongs to the collectin subgroup of C-type lectins with specificity for mannose and N-acetylglucosamine sugars. We investigated whether rat MBPs isolated from serum (S-MBP) and liver (L-MBP) interact with phospholipids using antibody against each MBP. Both S- and L-MBPs bound to phosphatidylinositol coated onto microtiter wells in a concentration- and a Ca2+-dependent manner. L-MBP also bound to phosphatidylglycerol and weakly to phosphatidylserine. MBPs interacted with liposomes composed of these lipids. S- and L-MBPs bound to phosphatidylinositol 4-monophosphate. L-MBP also bound to cardiolipin. These results provide evidence for a novel type of ligand binding specificity for MBPs, and raise the possibility that phospholipids are ligands for collectins.

Acute-Phase Proteins↗
PubMed1997

The mannose-binding protein A region of glutamic acid185-alanine221 can functionally replace the surfactant protein A region of glutamic acid195-phenylalanine228 without loss of interaction with lipids and alveolar type II cells.

Pulmonary surfactant protein A (SP-A) is a C-type lectin that regulates the uptake and secretion of surfactant lipids by alveolar type II cells and binds dipalmitoylphosphatidylcholine (DPPC) and galactosylceramide (GalCer). We isolated mannose-binding protein A (MBP-A) from rat sera, which is structurally analogous to SP-A, and examined if it was functionally equivalent to SP-A. We found that MBP-A did not possess the ability to interact with lipids and type II cells. The purpose of this study was to investigate the SP-A region involved in binding lipids and interacting with type II cells by using chimeric proteins with MBP-A. Chimeras AM1, AM2, and AM3 were constructed with SP-A/MBP splice junctions at Cys218/Gln210, Lys203/Cys195, and Gly194/Glu185, respectively. All of the chimeras bound DPPC and GalCer with activity comparable to recombinant SP-A. The three chimeras retained the ability to induce phospholipid vesicle aggregation and augment lipid uptake by type II cells, albeit to a lesser extent than wild type SP-A. The chimeras inhibited lipid secretion from type II cells with an IC50 of 0.5 microg/mL and competed effectively for SP-A receptor binding. In addition all these chimeras contained the epitope for monoclonal antibody 1D6, which blocks specific SP-A function. From these results, we conclude that the MBP-A region of Glu185-Ala221 can functionally replace the homologous SP-A region of Glu195-Phe228 without loss of interaction with lipids and type II cells.

Acute-Phase Proteins↗
PubMed1997

Immunoglobulin G is associated with surfactant protein A aggregate isolated from patients with pulmonary alveolar proteinosis.

We have previously shown that the purified preparation of surfactant protein A (SP-A) isolated from patients with pulmonary alveolar proteinosis (PAP) contains a very small amount of immunoglobulin G (IgG). We have recently found that there exists an abnormal multimerized form (alveolar proteinosis protein-I, APP-I) in SP-As isolated from patients with PAP in addition to normal-sized octadecameric APP-II. We examined which of the populations of APP that IgG is associated with. The APP was purified by mannose-affinity column followed by gelfiltration over Bio Gel A5m after the delipidation with 1-butanol. Analysis by gel filtration over Bio Gel A15m showed two elution peaks of APP-I and APP-II. When the fractions eluted from the Bio Gel A15m column were coated onto microtiter wells and reacted with HRP-labeled antihuman IgG, the elution peak of IgG was superimposed on that of APP-I but not on that of APP-II. The immunoblotting analysis also revealed that a very small amount of IgG, which could not be detected by staining with Coomassie blue or amido black, was associated with APP-I but not with APP-II or normal SP-A. APP-I bound to nonimmune IgG coated onto microtiter wells in a concentration-dependent manner, whereas APP-II, normal human SP-A, and rat SP-A exhibited almost no binding to IgG. The results indicate an unusual property of SP-A during the diseased state.

Chromatography, Gel↗
PubMed1996

Human surfactant protein A with two distinct oligomeric structures which exhibit different capacities to interact with alveolar type II cells.

The lung lavage fluids from patients with pulmonary alveolar proteinosis have been generally used as a source for human surfactant protein A (SP-A). We have recently found that a multimerized form of SP-A oligomer (alveolar proteinosis protein-I, APP-I) exists besides the normal-sized octadecamer (APP-II) in SP-As isolated from the patients. When analysed by Bio-Gel A15m column chromatography in 5 mM Tris buffer (pH 7.4), the apparent molecular masses of APP-I and APP-II were 1.65 MDa and 0.93 MDa, respectively. Gel-filtration analysis also revealed that APP-II is clearly separated from APP-I in the presence of 2 mM Ca2+ and 150 mM NaCI. We investigated the abilities of both SP-A oligomers to regulate phospholipid secretion and to bind to alveolar type II cells. Although APP-I inhibited lipid secretion, it was clearly a less effective inhibitor than APP-II. IC50 for inhibition of lipid secretion was apparently 0.23 +/- 0.08 microgram/ml (0.14 +/- 0.05 nM) and 0.055 +/- 0.019 microgram/ml (0.059 +/- 0.020 nM) for APP-I and APP-II, respectively. Both proteins bound to monolayers of type II cells in a concentration-dependent manner; however, APP-I clearly had a lower affinity to bind to type II cells. The apparent dissociation contants were, K(d) = 2.31 +/- 0.70 microgram/ml (1.40 +/- 0.43 nM) and 0.89 +/- 0.22 microgram/ml (0.95 +/- 0.24 nM) for APP-I and APP-II, respectively. Excess unlabelled rat SP-A replaced 45% of 125I-APP-I and 77% of 125I-APP-II for type II cell binding. Although 125I-APP-II competed with excess unlabelled APP-I or APP-II, 125I-APP-I failed to compete and instead its binding rather increased in the presence of unlabelled APPs. The biotinylated APP-I bound to APP-I and APP-II coated on to microtitre wells in a concentration-dependent manner, indicating that APP-I interacts with APPs. This study demonstrates that the multimerized form of human SP-A oligomer exhibits the following attributes: (1) the reduced capacity to regulate phospholipid secretion from type II cells, and (2) lower affinity to bind to type II cells, and that the integrity of a flower-bouquet-like octadecameric structure of SP-A oligomer is important for the expression of full activity of this protein, indicating the importance of the oligomeric structure of mammalian lectins with collagenous domains.

Animals↗
PubMed1996

The differences in the expressions of visual pigments and transducin in photoreceptor cell differentiation.

The distribution and accumulation of visual pigments, i.e., rod pigment, rhodopsin and red sensitive cone pigment, iodopsin, and transducin in the retina of chicken and chicken embryo were investigated immunohistochemically using their specific antibodies. The immunoreactivities of these proteins appeared at the early stage of photoreceptor differentiation (embryonal day 15) and increased in the photoreceptor cells appeared to reach maximum at the end of the embryonal period (embryonal day 20). On the other hand, although the immunoreactivity of beta gamma subunit of transducin (T beta gamma) was detected at embryonal day 15, the expression level of T beta gamma still remained in low level during the embryonal period. These observations suggest that both T beta gamma and visual pigments are expressed during the embryonic period in chicken photoreceptor cells, but their accumulations in the cells are different.

Animals↗
PubMed1995

Ca(2+)-dependent binding of annexin IV to surfactant protein A and lamellar bodies in alveolar type II cells.

Surfactant protein A (SP-A), a lung-specific glycoprotein in pulmonary surfactant, is synthesized and secreted from the alveolar type II cells. It has been shown that SP-A is a Ca(2+)-binding protein with several binding sites and that the high-affinity site(s) is located in the C-terminal region of SP-A. In the present study we isolated the proteins from bovine lung soluble fraction that bind to SP-A in a Ca(2+)-dependent manner using DEAE-Sephacel and SP-A-conjugated Sepharose 4B. At least three different protein bands with molecular masses of 24.5, 32, and 33 kDa were observed on SDS/PAGE. The main protein, with molecular mass of 32 kDa, was identified as annexin IV by the partial-amino-acid-sequence analyses and an immunoblot analysis with anti-(annexin IV) antiserum. We also found from the immunoblot analysis that the cytosolic fraction of isolated rat alveolar type II cells contains annexin IV. In addition, when rat lung cytosol was loaded on to the lung lamellar body-conjugated Sepharose 4B in the presence of Ca2+, two proteins, with molecular masses of 32 and 60 kDa on SDS/PAGE respectively, were eluted with EGTA. The 32 kDa protein was shown to be annexin IV by an immunoblot analysis with the antiserum against annexin IV. The lung annexin IV augmented the Ca(2+)-induced aggregation of the lung lamellar bodies from rats. However, the augmentation of aggregation of the lung lamellar bodies by annexin IV was attenuated when the lamellar bodies were preincubated with polyclonal anti-SP-A antibodies. SP-A bound to annexin IV under conditions where contaminated lipid was removed. These results suggest that SP-A bound to annexin IV based on protein-protein interaction, though both proteins are phospholipid-binding proteins. All these findings suggest that the interaction between SP-A and annexin IV may have some role in alveolar type II cells.

Amino Acid Sequence↗
PubMed1995

Epitope mapping for monoclonal antibody against human surfactant protein A (SP-A) that alters receptor binding of SP-A and the SP-A-dependent regulation of phospholipid secretion by alveolar type II cells.

Surfactant protein A (SP-A) is a lung-specific glycoprotein in pulmonary surfactant and has a collagen like sequence on its N-terminal. SP-A has been shown to function as an inhibitor of phospholipid secretion by primary culture of alveolar type II cells via cell surface receptor(s) for SP-A. In a previous report, we showed that the C-terminal non-collagen like domain of human SP-A possessed the biological activities, and that a monoclonal antibody against human SP-A, PE10, abolished the biological activity of SP-A (Murata et al. (1993) Biochem. J. 291, 71-76). In the present study, we investigated an epitope of SP-A for PE10. Western blot analysis with fragmented peptides of human SP-A generated by both lysyl endopeptidase and BrCN showed that PE10 reacted with the peptide corresponding with Glu202 to the C-terminal but that it lacked the ability to bind to the peptide corresponding with Tyr208 to the C-terminal. The antibodies against a synthetic peptide (P1) corresponding with Glu202 to Asn217 of human SP-A inhibited the binding of PE10 to SP-A, suggesting that a similar site was recognized by both PE10 and anti-P1 antibodies. Anti-P1 antibodies as well as PE10 suppressed the biological activity of SP-A. A direct interaction between P1 and rat lung membranes, or between P1 and alveolar type II cell membranes was shown from the measurement of the fluorescence emission spectra of dansyl-labeled P1. These results suggest that an area contiguous to or near the region from Glu202 to Met207 of SP-A is important for expressing the biological activities.

Amino Acid Sequence↗
PubMed1993

Different functional forms of G-protein beta gamma-subunits, beta gamma-I and beta gamma-II, in bovine brain.

Heterotrimeric GTP binding regulatory proteins (G proteins) are involved in the signal transduction process in cells. We have previously demonstrated that G protein (Gi/o) in bovine brain contains two subspecies of the beta gamma-subunit, beta gamma-I and beta gamma-II, with distinct gamma subunits, i.e., gamma-I and gamma-II, but identical beta-subunit. We found that gamma-I agreed with the gamma-subunit reported elsewhere, while gamma-II was a novel gamma-subunit. In the present study we separated the fractions containing G-protein isoforms, Go*, Gi1, Go and Gi2, with a Mono Q column and they were subjected to 15% polyacrylamide gel electrophoresis. All isoforms were shown to possess both gamma subunits, gamma-I and gamma-II. The molar ratio of the two gamma-subunit isoforms was one to one, based on the relative intensity of Coomassie blue-stained bands. Differences in biological activity between G proteins composed of beta gamma-I and beta gamma-II were investigated. The amount of GTP gamma S bound to the alpha-subunit was larger in alpha beta gamma-I than in alpha beta gamma-II. The pertussis toxin-catalyzed ADP-ribosylation on alpha-subunit was enhanced by either beta gamma-subunit subspecies, but the effect was larger with beta gamma-I than with beta gamma-II. From gel filtration with a Sephacryl S 300, it appeared that all alpha- and either beta gamma-subunit, i.e., beta gamma-I or beta gamma-II, formed a trimer complex. These findings suggest the possible existence of two different functional forms in each G-protein isoform depending on the beta gamma-subunit subspecies.

Animals↗
PubMed1993

Purification and characterization of three MEKA-like proteins in liver: association of a 94 kDa protein with beta gamma subunits of G-proteins.

Retinal 32 kDa MEKA protein (rMEKA) exists in the photoreceptor cells and forms a complex with beta gamma subunit of transducin. Bovine liver contained three MEKA-like proteins (94 kDa, 35 kDa-a, 35 kDa-b) which reacted with a rMEKA antibody. Each protein was purified as a single band on a SDS-PAGE and used for a reconstitution experiment with alpha and beta gamma subunits of cerebral G-proteins (Go/i). The 94 kDa protein inhibited GTP-binding ability of G alpha by forming a complex with beta gamma subunit.

Animals↗
PubMed1993

Identification of a novel gamma-subunit from bovine brain GTP binding regulatory proteins (Gi/o).

Heterogeneity of the gamma-subunit of G proteins has been demonstrated by cDNA cloning and by partial sequence analyses. We have isolated two intact beta gamma-subunit isoforms from bovine brain Gi/o mixture, in which only gamma subunits are distinct (Sohma, H., et al. (1992) Biochem. Biophys. Res. Commun. 184, 175-182). In this study, we isolated the gamma-subunit isoforms, gamma-I and gamma-II, and examined their amino acid sequences. Both gamma-I and gamma-II had blocked N-terminal amino acid residues, and the terminal amino acids of both were able to be truncated by an acylamino-acid-releasing enzyme. Gamma-I seemed to be identical with the gamma-subunit reported elsewhere, while the gamma-II appeared to be a novel protein. Antibodies to synthetic peptides based on the part of the amino acid sequences of gamma-I and gamma-II reacted specifically to gamma-I and gamma-II, respectively.

Amino Acid Sequence↗
PubMed1993

Calcium and dithiothreitol dependent conformational changes in beta-sheet structure of collagenase resistant fragment of human surfactant protein A.

Ca2+ dependent conformational change of collagenase resistant fragment (CRF) of human surfactant protein A (SP-A) was studied by measurements of the far UV circular dichroism spectrum. The spectrum was altered by Ca2+ and DTT. The beta-sheet content was decreased by the addition of Ca2+ from 28.1 to 26.6%. On the other hand, the beta-sheet content was increased in the presence of dithiothreitol from 28.1 to 36.0%, and decreased by the addition of Ca2+ from 36.0 to 30.5%. The total Ca2+ concentration required for half maximal change of the ellipticity at 220 nm was estimated to be 30 microM both in the presence and absence of dithiothreitol. One of the functions of SP-A, enhancement of phospholipid uptake by alveolar type II cells, was abolished by the addition of 2-mercaptoethanol. These results strongly indicate a relationship between the conformation of CRF and SP-A functions.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

[Light-induced dephosphorylation of phosphoproteins in rod outer segments in bovine and frog retina].

Several lines of evidence have suggested that protein phosphorylation and dephosphorylation may play an important role in the regulation of metabolism and signal transduction processes. In our present study, bovine and frog retinas were incubated in Krebs' solution containing [32P] H3PO4 for the labelling of all phosphoproteins. Then, photoreceptor outer segments were isolated from each retina, and further incubated under dark or light conditions. In such conditions, several phosphoproteins were dark- or light-dependently dephosphorylated. Interestingly, the light-dependent dephosphorylated 39 kDa protein as well as the 35-36 kDa protein was commonly observed in both bovine and frog retinas. The 35-36 kDa protein is considered to be the same as the 33 kDa protein that has previously been shown to be phosphorylated light-dependently, whereas 39 kDa protein is thought to be a novel protein that undergoes light-dependent dephosphorylation in retinal photoreceptor outer segments. Thus, these proteins were thought to have significant roles in the visual transduction processes.

Animals↗
PubMed1992

Calcium dependent conformational changes of surfactant protein A (SP-A) and its collagenase resistant fragment with or without dithiothreitol.

Calcium-dependent conformational changes of surfactant protein A (SP-A) and the collagenase resistant fragment (CRF) of SP-A were studied by measuring fluorescence spectra. The emission peaks of both SP-A and CRF in the absence of Ca2+ appeared at 343 nm when they were excited at 280 nm. In the presence of Ca2+, the peaks appeared at 340 nm and were accompanied by an increase in the fluorescence intensity. The magnitude of the fluorescence intensity change induced by Ca2+ was amplified by the addition of dithiothreitol (DTT) in both SP-A and CRF. The Ca2+ binding of CRF was measured by a flow dialysis method with 45CaCl2 in the Ca2+ concentration range where the Ca(2+)-induced fluorescence changes occurred. The maximum binding number of Ca2+ to CRF was about 2 mol per mol of CRF, and the value was independent of the presence of DTT.

Calcium↗
PubMed1992

Two gamma-subunits, gamma-I and gamma-II, complex with the same beta-subunits in bovine brain G-proteins (Gi/o).

When a mixture of bovine brain G-proteins (Gi/o) was loaded onto an octyl sepharose column in the presence of AlF4-, alpha-subunits of molecular weights 39 kDa and the 41 kDa were eluted separately, followed by the appearance of two distinct peaks containing beta gamma-subunits (beta gamma-I, beta gamma-II). Both beta gamma-I and beta gamma-II possessed identical beta-subunits but different gamma-subunits. The molecular weights of the two gamma-subunits determined by SDS-polyacrylamide gel electrophoresis both in the presence and absence of urea were 4.5 kDa (gamma-I) and 5.0 kDa (gamma-II). Tests indicated that the two isolated gamma-subunits are intact and have not undergone proteolysis. The amino acid composition of gamma-I appeared to be distinct from that of gamma-II. Therefore, this method is a simple procedure for isolating beta gamma-I and beta gamma-II.

Amino Acids↗