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PubMed1996

Water-soluble Abeta (N-40, N-42) oligomers in normal and Alzheimer disease brains.

Ultracentrifugation and graded molecular sieving, as well as a sensitive sandwich enzyme-linked immunosorbent assay were used to isolate and quantitate the amounts of water-soluble oligomers of beta amyloid (Abeta) peptides N-40 and N-42 in cerebral cortex of normal and Alzheimer disease (AD) brains. AD brains contained 6-fold more water-soluble Abeta (wsAbeta) than control brains. The majority of water-soluble peptides in most AD cases was A beta N-42, representing 12 times the amount found in control brains. The wsAbeta was present in the form of monomers and oligomers ranging from less than 10 kDa to greater than 100 kDa. The amount of wsAbeta N-42 in AD brains is about 50 times greater than the level of soluble Abeta N-42 found in the CSF of AD patients. This disparity may be due to the rapid association of wsAbeta N-42 into fibrillar deposits and/or to the integrity of the anatomical barriers which separate the two extracellular spaces. In this paper, we consider soluble any form of Abeta which has not yet polymerized into its insoluble, filamentous form. This includes both the newly synthesized forms of Abeta and those peptides which may be loosely attached to insoluble filaments but which can, nevertheless, still be considered soluble. It has been previously shown that, once it has aggregated into its filamentous form, the Abeta peptides are resistant to disaggregation and degradation by a number of denaturing agents and aqueous buffers containing proteolytic enzymes. Therefore, it is likely that the water-soluble Abeta peptides we quantified are precursors to its insoluble, filamentous form. Consequently, reducing the levels of soluble Abeta in AD brains could have profound effects on AD pathophysiology.

Adult↗

PubMed2005

Increased soluble glycoprotein V concentration during the acute onset of unstable angina pectoris in association with chronic cigarette smoking.

Platelet hyperactivity is important in the pathobiology of acute coronary syndromes. Glycoprotein V (GPV) is an integral membrane protein of platelets in the function of the GPIb-V-IX receptor for vWf/shear-dependent platelet adhesion in arteries. Soluble GPV is a novel marker of platelet activation. The aim of this study is to assess circulating soluble GPV levels in unstable angina pectoris (UA). Twenty-one patients (15 men, six women, aged 52+/-7 years) with UA pectoris were studied. The inclusion criteria were angina at rest lasting >20 min during the preceding 6 h, with transient ST segment depression and/or T wave inversion and no evidence of myocardial infarction detected with the use of cardiac troponin-T. Coronary artery stenosis was angiographically confirmed in all patients. Twenty age- and sex-matched healthy adults (14 men, six women, aged 48+/-7 years) served as controls. There were no significant differences among the studied groups with respect to age, sex, obesity, smoking, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride and platelet counts. Plasma-soluble GPV concentrations were higher in the UA patient group (126+/-46 ng/ml) than those in the healthy controls (82+/-15 ng/ml) (P=0.001). There was a significant correlation only between plasma-soluble GPV levels and smoking (r=0.526, P=0.0001). Smoker UA patients had higher levels of soluble GPV than the non-smoker patients (139+/-40 vs. 113+/-50 ng/ml, respectively, P=0.02). However, soluble GPV levels were similar in smoker and non-smoker healthy controls (P=0.2). It is concluded that soluble GPV concentrations are significantly increased during the acute clinical course of unstable angina pectoris, indicating that soluble GPV may be useful marker of platelet activation in those patients. The level of the molecule is significantly affected from smoking in those patients.

Acute Disease↗

PubMed2001

The influence of complexing agents on the solubility and absorption of aluminium in rats exposed to aluminium in water.

The influence of citrate (0-31 mM), fluoride (0 or 2.6 mM) and silicate (0 or 2.6 mM) on the absorption of Al (0-18 mM) was studied in rats. We tested the hypothesis that the solubility and absorption of Al increases in the gastrointestinal (GI) tract in the presence of the complexing agents. Male rats were exposed for 6 or 7 weeks to soluble Al in acidic drinking water (pH 2.5-3.0) with or without the complexing agents. At the end of exposure Al was fractionated in the stomach content, in order to study if the solubility of Al was changed after ingestion. Al absorption was estimated by Al analysis of the right femur bone. Speciation calculations indicated that citrate and fluoride caused formation of soluble Al-citrate (97%) and -fluoride (> 60%) complexes in the water. Silicate did not affect the theoretical speciation. In all cases, a large fraction of soluble Al became insoluble in the stomach after ingestion. The concentration of soluble Al increased only in the presence of citrate or a mixture of fluoride and silicate, but citrate was the only complexing agent that influenced the absorption of Al in the rat. This indicates that the form of Al may be changed in the GI tract when soluble drinking-water Al is ingested, and that the solubility of Al in drinking water and GI tract may not be good predictors of the bioavailability of Al even when chelating agents are present.

Aluminum Compounds↗

PubMed1994

Differential trafficking of soluble and integral membrane secretory granule-associated proteins.

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.

Amino Acid Sequence↗

PubMed1997

Long-term effect of soluble-fiber foods on postprandial fat metabolism in dyslipidemic subjects with apo E3 and apo E4 genotypes.

To determine the long-term effect of soluble fiber on postprandial fat metabolism, we studied 33 dyslipidemic subjects, 16 with apolipoprotein (apo) E3/3 (E3) and 17 with E3/4 or E4/4 (E4) genotypes. They ate preweighed low-fat (20% of energy), high-fiber (> 5.7 g/MJ) diets for two 4-mo periods separated by a 2-mo washout period according to a randomized, crossover design. One diet contained foods rich in insoluble fiber and the other foods rich in soluble fiber. On 1 d during the last 2 wk of each diet, subjects ingested a standard, fiber-free, fatty liquid meal containing retinyl palmitate as a marker of intestinally derived lipoproteins. Plasma samples were obtained at hourly intervals for 10 h. Compared with the insoluble-fiber diet, soluble fiber reduced fasting plasma total cholesterol in both E3 (6.6 +/- 2.1%, P = 0.007)and E4 subjects (5.6 +/- 2.1%, P = 0.017). Soluble fiber increased fecal total bile acid output in both E3 (76 +/- 18%, P < 0.001) and E4 subjects (85 +/- 19%, P < 0.001). The incremental area under the chylomicron triacylglycerol response curve was significantly greater after soluble fiber than after insoluble fiber in E3 (3.56 +/- 0.56 compared with 2.87 +/- 0.38 mmol x h/L, respectively, P = 0.046) but not in E4 subjects (5.19 +/- 0.78 compared with 4.92 +/- 0.81 mmol x h/L). Kinetic analysis suggested an increase in retinyl palmitate absorption in E3 subjects after soluble fiber, but no difference in E4 subjects. These results suggest that a long-term increase in dietary soluble fiber has no effect on postprandial fat metabolism in subjects with an apo E3/4 or E4/4 genotype. However, soluble fiber enhances apparent fat absorption in E3 subjects, which could be due to an increased bile acid pool and increased micelle formation.

Apolipoprotein E3↗

PubMed2004

Experimental and numerical determination of odorant solubility in nasal and olfactory mucosa.

Odorant deposition in the nasal and olfactory mucosas is dependent on a number of factors including local air/odorant flow distribution patterns, odorant mucosal solubility and odorant diffusive transport in the mucosa. Although many of these factors are difficult to measure, mucosal solubility in the bullfrog mucus has been experimentally determined for a few odorants. In the present study an experimental procedure was combined with computational fluid dynamic (CFD) techniques to further describe some of the factors that govern odorant mucosal deposition. The fraction of odorant absorbed by the nasal mucosa (eta) was experimentally determined for a number of odorants by measuring the concentration drop between odorant 'blown' into one nostril and that exiting the contralateral nostril while the subject performed a velopharyngeal closure. Odorant concentrations were measured with a photoionization detector. Odorants were delivered to the nostrils at flow rates of 3.33 and 10 l/min. The velopharyngeal closure nasal air/odorant flows were then simulated using CFD techniques in a 3-D anatomically accurate human nose modeland the mucosal odorant uptake was numerically calculated. The comparison between the numerical simulations and the experimental results lead to an estimation of the human mucosal odorant solubility and the mucosal effective diffusive transport resistance. The results of the study suggest that the increase in diffusive resistance of the mucosal layer over that of a thin layer of water seemed to be general and non-odorant-specific; however, the mucosa solubility was odorant specific and usually followed the trend that odorants with lower water solubility were more soluble in the mucosa than would be predicted from water solubility alone. The ability of this approach to model odorant movement in the nasal cavity was evaluated by comparison of the model output with known values of odorant mucosa solubility.

Adult↗

PubMed1995

Purification and functional properties of soluble forms of membrane cofactor protein (CD46) of complement: identification of forms increased in cancer patients' sera.

Normal human sera contained 10-60 ng/ml of soluble membrane cofactor protein (MCP, CD46) whereas sera of > 50% of the cancer patients contained > 60 ng/ml. MCP purified by immunoaffinity chromatography from both normal and cancer patients' sera consisted of three bands of 56, 47 and 29 kDa on SDS-PAGE/immunoblotting. The upper two components were increased in cancer patient sera. The 56 and 47 kDa soluble forms served as a cofactor for factor I-mediated cleavage of C3b. MCP expressed on Chinese hamster ovary (CHO) cells protects host cells from human C3 deposition and complement-mediated cytolysis, especially by activation of the alternative pathway. In this same assay system, exogenously added soluble MCP also protected untransfected CHO cells; however, its potency was much less than that of the endogenous membrane form. For example, 8 micrograms/ml of soluble MCP was equal to 10(4) copies/cell of the expressed MCP. Recombinant soluble forms possessed similar activity to the naturally occurring soluble forms and high doses (> 150 micrograms) blocked Arthus-like reaction induced in guinea-pigs by anti-Forssman antibody. These data establish that soluble forms of MCP are present in human sera that possess cofactor activity and their concentrations, especially the 56 and 47 kDa forms, are increased in sera of cancer patients. High doses of the recombinant soluble forms may be therapeutically useful for suppressing inflammatory responses.

Animals↗

PubMed1989

A detailed analysis of the potential of water-soluble classical class I MHC molecules for the suppression of kidney allograft rejection and in vitro cytotoxic T cell responses.

Water-soluble classical (RT1-A) class I MHC molecules were purified from aqueous extracts of DA strain liver. Following monoclonal antibody affinity, lentil lectin affinity, and gel filtration chromatography, 600 micrograms of soluble RT1-A class I molecules with antigen activity equivalent to 1.3 x 10(11) nucleated DA spleen cells (greater than 500 DA spleens) was obtained. Both PVG and LEW strain recipients of DA kidney allografts were pretreated with intravenous injections of the DA soluble class I molecules, in doses with antigen activity equivalent to 10(8) nucleated DA spleen cells. Three protocols of pretreatment were used: twice-weekly injections for 4-5 weeks, with grafting 3 or 4 days after the last injection; a single injection 7 days pregraft; or a single injection 1 day pregraft. The PVG and LEW rats received the soluble class I pretreatment either alone or in combination with suboptimal doses (2 mg/kg/day) of cyclosporine after grafting, making a total of 12 experimental groups treated with soluble class I antigen. In no case did treatment with soluble class I antigen elicit an antibody response in prospective graft recipients; influence kidney graft survival in any way; or enhance or suppress the antibody response to the kidney graft. The soluble DA class I MHC molecules were tested in vitro for their effect on the generation and effector function of allospecific PVG and LEW anti DA RT1-A class I cytotoxic T cells and TNP specific, self RT1-Aa restricted cytotoxic T cells. Concentrations up to 5 micrograms/ml (10(-7) M), equivalent to 10(9) nucleated DA spleen cells/ml, were without any effect. We conclude that monomeric forms of water-soluble classical class I molecules are poor immunogens--and, at doses conventionally used for active enhancement, do not influence cytotoxic T cell responses and have little potential for donor-specific immunosuppression.

Animals↗

PubMed2002

Soluble invertase expression is an early target of drought stress during the critical, abortion-sensitive phase of young ovary development in maize.

To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development.

Abscisic Acid↗

PubMed2001

Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins.

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.

ATP-Binding Cassette Transporters↗

PubMed1992

Release of soluble Fc gamma RII/CD32 molecules by human Langerhans cells: a subtle balance between shedding and secretion?

Freshly isolated human Langerhans cells (LC) express two forms of Fc gamma RII: a membrane-associated form detected by monoclonal antibody (MoAb) anti-CD32, which recognize an extracytoplasmic epitope of the molecule, and a soluble secreted form, whose existence is suggested by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. Indeed, RT-PCR performed on total LC RNA reveals the presence of two Fc gamma RIIA mRNA, one encoding the FC gamma RIIA with a transmembrane region (membranous form) and the other without this region (soluble form). Densitometry studies performed on the two PCR products reveal that the ratio between the membranous form and the soluble secreted form is about 1.5. LC maintained in culture for 24-48 h lose the major part of their membrane Fc gamma RII expression (shown by flow cytometry) and release soluble Fc gamma RII molecules (revealed by dot-blot assay), but maintain the same ratio of the two Fc gamma RIIA mRNA. The disappearance of the membrane-associated Fc gamma RII may be explained either by modification of its recycling pathway or by proteolytic cleavage of the receptor at the cell surface. Thus, soluble Fc gamma RII molecules generated during LC culture may result from proteolytic cleavage of the cell-surface receptor and/or secretion of a soluble form derived from the translation of an alternate spliced mRNA. Interestingly, addition of TNF-alpha (10 ng/ml) to the culture medium i) maintains the expression of the membranous form, which can be detected on the LC surface at the same level as on freshly isolated LC, and ii) reverses the ratio (to 0.6) of the two Fc gamma RII mRNA, the mRNA encoding the soluble form becoming predominant. Thus, TNF-alpha seems to modify the expression of the Fc gamma RII at the mRNA level, favoring the secretion of soluble Fc gamma RII molecules, and changes the fate of the membranous Fc gamma RII.

Humans↗

PubMed1994

Soluble CD8 and CD25 in serum of patients after heart transplantation.

To evaluate the diagnostic value of serum cytokine levels and cytokine receptor levels in the diagnosis of acute rejection after heart transplantation, we measured soluble CD8 and soluble CD25 in the serum of heart transplant recipients. The results were compared with endomyocardial biopsy (EMB) histopathology, lymphocyte activation by morphologic inspection of peripheral blood cells (cytoimmunologic monitoring), clinically manifested infections, and the maintenance immunosuppressive therapy. Significantly increased levels were observed in cases of lymphocyte activation in cytoimmunologic monitoring indicative of either rejection or infection. In clinically documented cytomegalovirus (CMV), bacterial, and Pneumocystis carinii infections, increased levels of soluble CD25 were observed. Soluble CD8 was only increased in a single case of P. carinii infection. A statistically significant correlation was calculated between the levels of soluble CD8 and whole blood cyclosporin A level. Considering chemotherapy, the levels of soluble CD8 showed an inverse correlation with the daily dosage of azathioprine. In conclusion, the levels of soluble CD8 and CD25 are associated with lymphocyte activation in peripheral blood, but do not differentiate between lymphocyte activation indicative of rejection or infection. No relationship was observed between levels of soluble CD8 and CD25, and EMB histopathology. Therefore, the assessment of these two cell products has no diagnostic potential for monitoring acute rejection after heart transplantation.

CD8 Antigens↗

PubMed1990

Rapidly absorbed solid oral formulations of ibuprofen using water-soluble gelatin.

Rapidly absorbed oral dosage forms of ibuprofen using water-soluble gelatin (hydrolysate of common gelatin: mean mol. wt: 6000) have been studied and compared with tablets prepared with common gelatin (mean mol. wt: 100,000) and commercial tablets. Spray-dried and speed-kneaded powders, two types of granules and tablets were prepared with water-soluble gelatin. The in-vitro dissolution rates of water-soluble gelatin preparations were significantly faster than those of commercial tablets, whereas the tablets prepared using common gelatin had slower dissolution rates than commercial tablets. Water-soluble gelatin enhanced the dissolution rate of ibuprofen by improving the wettability of the drug particle surface by water, without any interaction in solution and the solid state. The absorption behaviour of various preparations was evaluated in four beagle dogs. The peak concentration time (tmax) of the water-soluble gelatin preparations was significantly shorter than that of tablets prepared with common gelatin and commercial tablets. The maximum concentration (cmax) and the area under the serum concentration-time curve (AUCo-10 h) were similar in all cases. The serum concentration profiles of water-soluble gelatin solid preparations were almost the same as those of the solutions. On the other hand, the profiles of the common gelatin tablets were similar to those of the commercial tablets. The mean absorption time (MAT) from water-soluble gelatin preparations was about 0.7 h, while the MAT from commercial tablets and common gelatin tablets was about 1.2 h. The differences in the MAT of water-soluble gelatin preparations and commercial tablets or common gelatin tablets were the same as the differences in mean dissolution time (MDT) in gastrointestinal fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene.

Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis. Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE. The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Bath also produces a soluble MMO.

Base Sequence↗

PubMed1992

Characterization of novel, phenol-soluble polypeptides which confer rigidity to the sheath of Methanospirillum hungatei GP1.

Treatment of the Methanospirillum hungatei GP1 sheath with 90% (wt/vol) phenol resulted in the solubilization of a novel phenol-soluble group of polypeptides. These polypeptides were purified by the removal of insoluble material by ultracentrifugation and represented approximately 19% of the mass of the sheath. The phenol-insoluble material resembled untreated sheath but had lost its rigidity and cylindrical form. Recombination of phenol-soluble and phenol-insoluble fractions by dialysis to remove phenol resulted in cylindrical reassembly products. Although bona fide sheath (complete with the 2.8-nm lattice) was not produced, a role for the phenol-soluble polypeptides in the maintenance of sheath rigidity is implied. The phenol-soluble polypeptides have limited surface exposure as detected by antibodies on intact sheath; therefore, they are not responsible for the 2.8-nm repeat occurring on the outer face of the sheath. However, longitudinal and transverse linear labeling by protein A-colloidal gold on the outer and inner faces, respectively, occurred with monoclonal antibodies specific to the phenol-soluble polypeptides. Restricted surface exposure of phenol-soluble polypeptides on the sheath highlighted molecular defects in sheath architecture. These lattice faults may indicate sites of sheath growth to accommodate cell growth or division (longitudinal immunogold label) and filament division (transverse immunogold label). The identification of a second group of polypeptides within the infrastructure of the sheath suggests that the sheath is a trilaminar structure in which phenol-soluble polypeptides are sandwiched between sodium dodecyl sulfate-beta-mercaptoethanol-EDTA-soluble polypeptides (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) (phenol-insoluble material).

Antibodies, Monoclonal↗

PubMed2005

Inhibition of coxsackie B virus infection by soluble forms of its receptors: binding affinities, altered particle formation, and competition with cellular receptors.

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus B3 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Animals↗

PubMed1992

Ontogeny of membrane and soluble amino-oligopeptidases in rat intestine.

Intestinal amino-oligopeptidase (AOP) plays an essential role in protein digestion. To characterize its postnatal development, we measured AOP activity in intestinal membrane and cytosolic fractions in suckling and weaned rats, compared the subunit structures of the membrane and soluble enzymes, and assessed the biochemical relationship of these peptidases. At weaning, jejunal membrane AOP activity doubled while soluble AOP activity in the ileum fell abruptly. The maturational increase in the molecular mass of ileal membrane AOP was due to alterations in the N-linked glycosylation of this protein. Ileal membrane and soluble AOP exhibited similar substrate affinities, pH optima, inhibition characteristics, and antigenic epitopes. However, soluble AOP was 25-35 kDa smaller than the membrane enzyme. Peak incorporation of [35S]methionine into ileal brush-border AOP preceded maximal radioactivity in soluble AOP, suggesting that the membrane peptidase is a precursor of the soluble enzyme. We conclude that membrane and soluble AOP are closely related proteins with distinct developmental profiles and that the soluble peptidase may be derived from endocytosis of the membrane enzyme.

Aminopeptidases↗

PubMed2003

Vascular localization of soluble epoxide hydrolase in the human kidney.

Epoxyeicosatrienoic acids are cytochrome P-450 metabolites of arachidonic acid with multiple biological functions, including the regulation of vascular tone, renal tubular transport, cellular proliferation, and inflammation. Epoxyeicosatrienoic acids are converted by soluble epoxide hydrolase into the corresponding dihydroxyeicosatrienoic acids, and epoxyeicosatrienoic acid hydration is regarded as one mechanism whereby their biological effects are eliminated. Previous animal studies indicate that soluble epoxide hydrolase plays an important role in the regulation of renal eicosanoid levels and systemic blood pressure. To begin to elucidate the mechanism of these effects, we determined the cellular localization of soluble epoxide hydrolase in human kidney by examining biopsies taken from patients with a variety of non-end-stage renal diseases, as well as those without known renal disease. Immunohistochemical staining of acetone-fixed kidney biopsy samples revealed that soluble epoxide hydrolase was preferentially expressed in the renal vasculature with relatively low levels in the surrounding tubules. Expression of soluble epoxide hydrolase was evident in renal arteries of varying diameter and was localized mostly in the smooth muscle layers of the arterial wall. Western blot analysis and functional assays confirmed the expression of soluble epoxide hydrolase in the human kidney. There were no obvious differences in soluble epoxide hydrolase expression between normal and diseased human kidney tissue in the samples examined. Our results indicate that soluble epoxide hydrolase is present in the human kidney, being preferentially expressed in the renal vasculature, and support an essential role for this enzyme in renal hemodynamic regulation and its potential utility as a target for therapeutic intervention.

Arterioles↗