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PubMed2006

Elevated first trimester maternal levels of soluble fibrin polymer are associated with lower birthweight in twin gestation.

We assessed soluble fibrin polymer longitudinally in normal pregnancy, thrombophilic pregnancy and twin gestation Thirty-three thrombophilic pregnancies, 34 uncomplicated multiple gestations and 23 singleton normal pregnancies were studied. Maternal plasma samples were collected in the first (6-12 weeks), 2nd (13-25 weeks) and 3rd trimesters of pregnancy (26-40 weeks) and were stored at -70 degrees C until assay. Soluble fibrin polymer was assayed by enzyme-linked immunosorbent assay (ABS, Copiague, New York, USA). Statistical analysis were made by Spearman test and Levine's test for equality of variance (P < 0.05). First soluble fibrin polymer maternal levels in twin gestation were significantly higher than in normal pregnancy and thrombophilic pregnancy, (23.8 +/- 4.5 mug/ml versus 9.2 +/- 3.1 and 10.0 +/- 2.0 mug/ml respectively, P < 0.005). Second and third trimester maternal levels of SFP in twins were also significantly higher than in normal pregnancy. First trimester soluble fibrin polymer was highly correlated with birthweight in twin gestation (r = -1, P < 0.01). In the third trimester, maternal soluble fibrin polymer correlated with placental weight in twin gestation (r = 0.639, P < 0.01). Overall, soluble fibrin polymer was correlated with placental weight and birthweight in the three groups but this did not reach statistical significance. Elevated maternal plasma levels of soluble fibrin polymer in twin gestation may derive from an accelerated coagulation process due to extensive vascular remodelling. Current studies are underway to determine the utility of soluble fibrin polymer in assessing fetal growth abnormalities.

Adult↗

PubMed1992

Tissue distribution of constitutive and induced soluble peroxidase in rat. Purification and characterization from lacrimal gland.

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen-induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane-bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases. As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane-bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5-3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane-bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross-react with lactoperoxidase it did interact with lactoperoxidase on Western blot. The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane-bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue-specific regulation of gene expression at the molecular level.

Animals↗

PubMed2001

Maternal endothelial soluble cell adhesion molecules with isolated small for gestational age fetuses: comparison with pre-eclampsia.

OBJECTIVE: 1. To evaluate the activation profile of the endothelium in pregnancies complicated by small for gestational age fetuses compared with pre-eclampsia and normal pregnancy, by measuring the plasma levels of soluble adhesion molecules soluble E-selectin, intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. 2. To determine whether soluble adhesion molecules were related to the severity of small for gestational age fetuses and pre-eclampsia. DESIGN: Observational study. PARTICIPANTS: Sixteen women with small for gestational age fetuses; 15 women with pre-eclampsia and 15 healthy primigravidae were recruited as controls. METHODS: Plasma levels of soluble E-selectin, intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 were measured by ELISA. RESULTS: Compared with the healthy controls, soluble E-selectin was significantly increased in both small for gestational age fetuses and pre-eclampsia, whereas intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 were increased only in pre-eclampsia. In the small for gestational age fetuses group, soluble E-selectin correlated inversely with the ratio between birthweight and the expected normal birthweight (r = -0.4, P = 0.007). In the pre-eclampsia group, a significant correlation was observed between vascular cell adhesion molecule-1 and blood pressure (r = 0.54, P = 0.039). CONCLUSIONS: Endothelial activation, reflected by raised levels of soluble E-selectin, is a feature of small for gestational age fetuses and is correlated with the severity of the disease. Differences in the profile of soluble cell adhesion molecules suggest variations in the degrees of endothelial activation between pre-eclampsia and small for gestational age fetuses.

Adult↗

PubMed1992

Lack of correlation between soluble CD4-induced shedding of the human immunodeficiency virus type 1 exterior envelope glycoprotein and subsequent membrane fusion events.

The noncovalent association of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) is disrupted by soluble CD4 binding, resulting in shedding of the gp120 exterior envelope glycoprotein. This observation has led to the speculation that interaction of gp120 with the CD4 receptor triggers shedding of the exterior envelope glycoprotein, allowing exposure of gp41 domains necessary for membrane fusion steps involved in virus entry or syncytium formation. To test this hypothesis, a set of HIV-1 envelope glycoprotein mutants were used to examine the relationship of soluble CD4-induced shedding of the gp120 glycoprotein to envelope glycoprotein function in syncytium formation and virus entry. All mutants with a threefold or greater reduction in CD4-binding ability exhibited marked decreases in gp120 shedding in response to soluble CD4, even though several of these mutants exhibited significant levels of envelope glycoprotein function. Conversely, most fusion-defective mutants with wild-type gp120-CD4 binding affinity, including those with changes in the V3 loop, efficiently shed gp120 following soluble CD4 binding. Thus, soluble CD4-induced shedding of gp120 is not a generally useful marker for conformational changes in the HIV-1 envelope glycoproteins necessary for the virus entry or syncytium formation processes. Some gp120 mutants, despite being expressed on the cell surface and capable of efficiently binding soluble CD4, exhibited decreased gp120 shedding. These mutants were still sensitive to neutralization by soluble CD4, indicating that, for envelope glycoproteins exhibiting high affinity for soluble CD4, competitive inhibition may be more important than gp120 shedding for the antiviral effect.

Animals↗

PubMed2004

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression.

BACKGROUND: In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. RESULTS: Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. CONCLUSIONS: By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.

Amino Acid Sequence↗

PubMed2000

Elevated levels of soluble adhesion molecules in sera and BAL fluid of individuals infected with human T-cell lymphotropic virus type 1.

STUDY OBJECTIVE: T-lymphocytic alveolitis and increased levels of interleukin-2 receptor-alpha (CD25)-bearing T cells in the BAL fluid (BALF) of human T-cell lymphotropic virus type 1 (HTLV-1) carriers have been reported. Several chemokines and adhesion molecules may contribute to the accumulation of T lymphocytes in the lungs of HTLV-1 carriers. To clarify the correlation between adhesion molecules and HTLV-1-associated pulmonary disorders, we compared the distribution of T-lymphocyte subsets and soluble adhesion molecules, including soluble intercellular adhesion molecule (sICAM)-1, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble L-selectin (sL-selectin), soluble E-selectin (sE-selectin), and soluble P-selectin (sP-selectin), in BALF and peripheral blood, between HTLV-1 carriers and noninfected healthy subjects. DESIGN: Flow cytometric analysis with monoclonal antibodies to cell-surface antigens was used to identify T-lymphocyte subsets in BALF samples from HTLV-1 carriers (n = 13) and noninfected healthy control subjects (n = 10). The levels of various soluble adhesion molecules in serum and in BALF were estimated by enzyme-linked immunosorbent assay. RESULTS: Higher percentages of CD3+ cells, CD3-expressing human leukocyte antigen-DR antigen, and CD3+CD25+ cells were detected in the BALF of HTLV-1 carriers than in that of noninfected control subjects. The concentrations of sICAM-1, sVCAM-1, sL-selectin, SE- selectin, and sP-selectin in the sera of patients were significantly higher than those in noninfected healthy control subjects. The concentration of sICAM-1 in the BALF of patients was significantly higher than that in noninfected healthy control subjects, and the concentration of sICAM-1 correlated well with the percentage of CD3+CD25+ cells. CONCLUSION: The concentrations of adhesion molecules in the sera of and sICAM-1 in the BALF of HTLV-1 carriers were significantly higher than those in noninfected individuals, and the concentration of sICAM-1 correlated well with the percentage of CD3+CD25+ cells in BALF. Our results suggest a possible interaction between activated T cells bearing CD25 and soluble adhesion molecules, especially sICAM-1, which may contribute to the pulmonary involvement in HTLV-1 carriers.

Adult↗

PubMed2003

Transcardiac release of soluble adhesion molecules during coronary artery bypass grafting: effects of crystalloid and blood cardioplegia.

BACKGROUND: Dysfunction of myocardium as a result of ischemia/reperfusion during coronary artery bypass grafting (CABG) is currently one of the biggest problems in cardiovascular surgery. In previous studies, it has been well established that activated leukocytes and coronary vascular endothelial cells play an important role in the development of cardiac tissue damage during ischemia followed by reperfusion. Interactions between both of these cell types require the expression of adhesion molecules on their surface. In certain conditions, on cell activation, the adhesion proteins may be released from activated cells in soluble form into circulation. The purpose of our study was to establish whether the use of blood cardioplegia modifies plasma levels of soluble intracellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), and soluble L-selectin (sL-selectin) in comparison with crystalloid cardioplegia in patients undergoing CABG. METHODS: Patients undergoing CABG were classified into two groups to receive cold crystalloid cardioplegia (St. Thomas' Hospital) or cold blood cardioplegia (method of Buckberg), followed by a "warm-shot" of the solution. Coronary sinus and arterial blood samples were obtained from 50 patients (42 men and 8 women; age range, 34 to 73 years) before aortic cross-clamping, at the beginning of reperfusion, and after 30 min of reperfusion. Plasma levels of soluble adhesion molecules were measured using sensitive enzyme-linked immunosorbent assays. RESULTS: The transcardiac release of sICAM-1 and sVCAM-1 following myocardial ischemia/reperfusion during CABG was evident in both groups of patients. However, the increase of soluble forms of both of these adhesion proteins was more significant in the group of patients receiving crystalloid cardioplegia. Crystalloid cardioplegia resulted in decreased plasma level of sE-selectin in the coronary sinus blood sample after 30 min of reperfusion. No significant changes in plasma levels of sL-selectin in either group were observed. CONCLUSION: Cardioplegia may affect the release of soluble forms of adhesion molecules from ischemic myocardium and modify endothelium activation in patients undergoing CABG.

Adult↗

PubMed2002

Soluble cytokine receptors in biological therapy.

Due to their fundamental involvement in the pathogenesis of many diseases, cytokines constitute key targets for biotherapeutic approaches. The discovery that soluble forms of cytokine receptors are involved in the endogenous regulation of cytokine activity has prompted substantial interest in their potential application as immunotherapeutic agents. As such, soluble cytokine receptors have many advantages, including specificity, low immunogenicity and high affinity. Potential disadvantages, such as low avidity and short in vivo half-lifes, have been addressed by the use of genetically-designed receptors, hybrid proteins or chemical modifications. The ability of many soluble cytokine receptors to inhibit the binding and biological activity of their ligands makes them very specific cytokine antagonists. Several pharmaceutical companies have generated a number of therapeutic agents based on soluble cytokine receptors and many of them are undergoing clinical trials. The most advanced in terms of clinical development is etanercept (Enbrel, Immunex), a fusion protein between soluble TNF receptor Type II and the Fc region of human IgG1. This TNF-alpha; antagonist was the first soluble cytokine receptor to receive approval for use in humans. In general, most agents based on soluble cytokine receptors have been safe, well-tolerated and have shown only minor side effects in the majority of patients. Soluble cytokine receptors constitute a new generation of therapeutic agents with tremendous potential for applications in a wide variety of human diseases. Two current areas of research are the identification of their most promising applications and characterisation of their long-term effects.

Asthma↗

PubMed1991

Increased soluble interleukin 2 receptor levels in autoimmune thyroid disease.

We measured soluble interleukin 2 receptor, a part of the Tac protein (p55), in peripheral blood to study the immunological condition of the T cell in autoimmune thyroid disease. In 26 patients with untreated Graves' disease and 7 hyperthyroid patients with Hashimoto's thyroiditis, the mean levels of soluble IL-2 receptor were both significantly higher than in normal controls (1497 +/- 649 (mean +/- SD), 641 +/- 137 vs 221 +/- 63 10(3) U/l, p less than 0.001). There was good correlation between soluble IL-2 receptor levels and blood thyroxine levels (r = 0.684, p less than 0.001) in patients with untreated Graves' disease, but no correlation of soluble IL-2 receptor with TSH-inhibitory immunoglobulins, TS-ab, thyroidal autoantibodies to thyroglobulin and thyroidal microsomal antigen was found. We thought that the level of soluble IL-2 receptor is not dependent only on immunological conditions, but also on thyroid hormone status. When T3 was administered to subjects in remission from Graves' disease and in normal controls, the soluble IL-2 receptor levels significantly increased. Moreover, the mean level of soluble IL-2 receptor in patients with toxic multinodular goitre was also significantly higher than in normal controls (411 +/- 148 vs 221 +/- 63 10(3)U/l, p less than 0.05). We conclude that the soluble IL-2 receptor levels are higher in sera of subjects with elevated levels of thyroid hormone.

Adult↗

PubMed1992

Relationship between absorption of radiolabeled soluble insulin, subcutaneous blood flow, and anthropometry.

OBJECTIVE: To evaluate the interrelationships between the rate of absorption of soluble insulin, SCBF, and anthropometry in normal subjects. RESEARCH DESIGN AND METHODS: In 12 normal men (age range 23-30 yr, BMI 18.2-41.3 kg/m2), simultaneous assessment of the absorption of 125I-labeled soluble insulin and SCBF (99mTc clearance) was performed, on separate study days, for the anterior abdominal wall, anterior midthigh, and the upper arm sites. Each site was examined in a randomized order on two separate occasions. Absorption of 125I-soluble insulin was determined by external monitoring of residual radioactivity levels at the injection site for 6 h postinjection. Residual radioactivity level-time curves, including the characteristic early phase of slow absorption of soluble insulin (the lag phase), were described using two- and three-parameter biexponential models. Anthropometric measurements included BMI, ultrasonic measurement of the subcutaneous adipose tissue layer, and caliper skin fold thickness at the anterior abdominal wall, biceps, triceps, anterior midthigh, and subscapular sites. RESULTS: A highly significant positive relationship was observed between the rate of absorption of 125I-soluble insulin and SCBF (rS = 0.44-0.52; P < 0.01-0.001). The duration of the lag phase was inversely correlated with SCBF (rS = 0.34 - 0.51; P < 0.01-0.001). Inverse relationships also were observed for the subjects' degree of adiposity with the rate of soluble insulin absorption (rS = -0.43(-)-0.71; P < 0.001) and SCBF (rS = -0.27(-)-0.62; P < 0.05-0.001). Significantly shorter lag phase was observed for the abdominal site compared with thigh and arm injection sites (P < 0.05-0.01). CONCLUSIONS: The rate of absorption of soluble insulin, including during the lag phase, is positively correlated with SCBF. Increasing adiposity prolongs the duration of the early lag phase and reduces the rate of absorption of soluble insulin and SCBF.

Absorption↗

PubMed1995

Transferrin reduces the production of soluble transferrin receptor.

The effect of homologous diferric transferrin from which contaminating transferrin receptor has been removed by monoclonal antibody affinity chromatography on soluble transferrin receptor concentrations was studied in K562 cells and HL60 cells in culture. Diferric transferrin in K562 cells caused a dose-dependent decrease in cellular receptor expression, a dose-dependent increase in cellular ferritin content, and a reduction in soluble receptor concentration which was of greater proportional magnitude than the reduction in cell receptor content. In HL60 cells, while there was a dose-dependent increase in cellular ferritin, cellular receptor content was relatively unaffected, while there was a consistent reduction in soluble receptor concentration. In both cells, the inhibitory effect of diferric transferrin on soluble receptor concentration was evident as early as 3 hr into the incubation. Apotransferrin, by contrast, did not reduce soluble receptor concentration. While elemental iron was capable of producing similar changes in cellular receptor and ferritin content, it had no inhibitory effect on proportional soluble receptor content. Studies employing other proteins, including human and bovine serum albumin, human lactoferrin, and rat ferritin, had no inhibitory effect on soluble receptors concentration, thus confirming the specificity of the findings. Control studies excluded an assay artifact as the explanation for the current findings. Prior contrary reports appear completely explained by the combination of soluble transferrin receptor contaminating the transferrin employed for study and a systematic difference in the assays employed between free and transferrin-bound receptor.

Ferritins↗

PubMed1975

Triggering mechanism of B lymphocytes. I. Effect of anti-immunoglobulin and enhancing soluble factor on differentiation and proliferation of B cells.

Stimulation of rabbit mesenteric lymphocytes with anti-immunoglobulin antibody (anti-Ig) for 24 hr (first stage of culture) followed by the addition of enhancing soluble factor (second stage of culture) induced the formation of IgG. The presence of mitotic inhibitors, such as cytosine arabinoside (Ara-C), hydroxyurea (HU), with anti-Ig for the first 24 hr of incubation did not inhibit the production of IgG induced by anti-Ig and soluble factor. The addition of cytochalasin-B in the first stage of culture with anti-Ig did not inhibit, but rather enhanced, the formation of IgG. On the other hand, the presence of Ara-C, HU, or cytochalasin-B with soluble factor in the second stage of culture completely inhibited the production of IgG. Soluble factor preferentially induced the uptake of thymidine of anti-Ig-stimulated cells. Lipopolysaccharide (LPS) also induced the proliferation of rabbit lymphocytes, but did not induce the IgG production of anti-Ig-stimulated cells. However, LPS-stimulated cells formed a significant amounf of IgG when they were cultured with soluble factor. Anti-Ig-stimulated cells absorbed the enhancing activity of soluble factor, but non-stimulated cells did not absorb the activity. These results showed that cross-linkage of Ig receptors or LPS receptors with anti-Ig or LPS induced the formation of acceptor sites for soluble factor without the requirement of cell division and these activated cells started to proliferate and maturate to antibody-forming cells under the influence of soluble factor.

Animals↗

PubMed2000

Comparison of the pharmacokinetic profiles of soluble aspirin and solid paracetamol tablets in fed and fasted volunteers.

The aim of this study was to investigate the absorption of popular preparations of two common analgesics--soluble aspirin and solid paracetamol tablets. An open, randomised, crossover study design was used to compare the pharmacokinetic parameters of soluble aspirin and solid paracetamol tablets in 16 healthy, male volunteers from the University of the Witwatersrand, South Africa, in both fed and fasted states. Plasma concentrations of paracetamol, aspirin and salicylic acid were measured. It was found that the rate of absorption was significantly faster for soluble aspirin than for solid paracetamol, regardless of fed or fasting state, considering time to maximum concentration (p < 0.01), time to first quantifiable concentrations (p < 0.05) and absorption rate (p < 0.01). Absorption rate was significantly affected by food for both soluble aspirin (p = 0.028) and for solid paracetamol (p = 0.0003). Time to maximum concentration was not significantly affected by food for soluble aspirin (p = 0.17) but significantly lengthened for solid paracetamol (p = 0.0003). The extent of absorption was affected by food in terms of maximum concentration for both drugs (p = 0.0001), with a reduction of 49% in the fed state for solid paracetamol compared to 18% for soluble aspirin, the difference between the drugs being statistically significant (p = 0.0024). The overall bioavailability of soluble aspirin was unaffected by food and the bioavailability of salicylic acid was increased in the fed state, whereas that of solid paracetamol was lowered in the fed state. Greater inter-individual variation was seen in paracetamol concentrations compared with aspirin or salicylic acid levels. In conclusion, these results show that the absorption of soluble aspirin is largely unaffected by food, whereas, in the same volunteers, the absorption of solid paracetamol tablets is greatly affected. In some volunteers, maximum plasma concentrations of paracetamol following food did not reach levels previously reported to be required for effective analgesia, and this may have implications for pain relief in some individuals. The practice in some individuals of taking aspirin tablets after food to minimise potential gastric disturbance should not affect the level of analgesia.

Acetaminophen↗

PubMed1977

Phosphorylation of rabbit and human erythrocyte membranes by soluble adenosine 3':5'-monophosphate-dependent and -independent protein kinases.

Previous reports from this laboratory and others have established that both the rabbit and human erythrocyte membranes contain multiple protein kinase and phosphate acceptor activities. We now report that these membranes also contain phosphoryl acceptor sites for the soluble cyclic AMP-dependent and -independent protein kinases from rabbit erythrocytes. The rabbit erythrocyte membrane, which does not contain a cyclic AMP-dependent protein kinase, has at least four polypeptides (Bands 2.1, 2.3, 4.5, and 4.8) which are phosphorylated in the presence of the soluble cyclic AMP-dependent protein kinases I, IIa, and IIb isolated from rabbit erythrocyte lysates. The resulting phosphoprotein profile is very similar to that obtained for the cyclic AMP-mediated autophosphorylation of human erythrocyte membranes. The activities of the soluble cyclic AMP-dependent protein kinases toward the membranes have been studied at several pH values. Although the substrate specificity of the three kinases is similar, polypeptide 2.3 appears to be phosphorylated to a greater extent by kinase IIa than by I or IIb. This occurs at all pH values studied. Also apparent is that the pH profile for membrane phosphorylation is different from that of histone phosphorylation. The phosphorylation of membrane proteins can also be catalyzed by the soluble erythrocyte casein kinases. These enzymes are not regulated by cyclic nucleotides and can use either ATP or GTP as their phosphoryl donor. Polypeptides 2.1, 2.9, 4.1, 4.5, 4.8, and 5 of both human and rabbit erythrocyte membranes are phosphorylated in the presence of GTP and the casein kinases. This reaction is optimal at pH 7.5. Experiments were performed to determine whether the phosphorylation of the membranes by the soluble and membrane-bound kinases is additive or exclusive. Our results indicate that after maximal autophosphorylation of the erythrocyte membranes, phosphoryl acceptor sites are available to the soluble cyclic AMP-dependent and -independent protein kinases. Furthermore, after maximal phosphorylation of the membranes with one type of soluble kinase, further 32P incorporation can occur as a result of exposure to the other type of soluble kinase.

Animals↗

PubMed1986

Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens.

In this study, we used monoclonal antibodies to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cell free IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [3H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL 2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To our knowledge, this is the first demonstration of release or secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen.

Animals↗

PubMed1976

Demonstration of a precursor-product relationship between soluble and cross-linked elastin, and the biosynthesis of the desmosines in vitro.

Direct evidence showing that a soluble form of elastin is the precursor of cross-linked elastin was obtained from pulse-chase experiments using chick embryo aortas and by demonstrating the conversion of soluble elastin into cross-linked elastin in a cell-free system. Acetic acid extracts of embryonic chick aorta pulse-labeled with [14C]lysine contain two radioactive proteins of molecular weights 74,000 and 138,000 which have been identified previously as soluble elastin and the pro-alpha chain of collagen, respectively. In pulse-chase experiments, the radioactivity incorporated in the soluble elastin during the pulse with [14C]lysine disappeared during a 24-hour chase with [12C]lysine and 89% of that which disappeared was accounted for in the desmosines of alkali-insoluble elastin. The disappearance of the radioactivity from the soluble fraction and its appearance in the desmosines of elastin were inhibited by beta-aminopropionitrile, a specific inhibitor of the cross-linking enzyme lysyl oxidase. In addition in vitro experiments, it was shown that the radioactivity in the desmosines of elastin can arise from that present in an acid-soluble precursor protein. This precursor protein is soluble elastin, as demonstrated by the formation of desmosines when a homogeneous preparation of soluble elastin was incubated with purified lysyl oxidase.

Amino Acids↗

PubMed1989

The role of soluble cross-linked fibrin in D dimer immunoreactivity of plasmic digests.

Monoclonal antibody DD/3B6 reacts with a site near the gamma gamma isopeptide cross link and has been used to detect cross-linked fibrin degradation products in plasma as a marker for clot lysis. However, interpretation of the results is potentially complicated, because soluble cross-linked fibrin polymers contain a gamma gamma cross-link and may also react with the antibody before or after plasmic degradation. We have prepared plasmic digests of fibrinogen, soluble cross-linked fibrin polymers, and cross-linked particulate fibrin clot, have characterized their composition by electrophoresis, and have compared their D dimer immunoreactivities. Undegraded soluble cross-linked fibrin polymer showed no reactivity below 20 micrograms/ml, indicating that the epitope recognized by DD/3B6 is poorly reactive before plasmic degradation. The D dimer immunoreactivity of plasmic digests of particulate or of soluble cross-linked fibrin increased with progressive degradation, with stage 3 digests having 20-fold to 40-fold higher immunoreactivity than stage 1 digests. A stage 3 digest of fully cross-linked particulate fibrin reacted fourfold and 10-fold more than stage 3 digests of soluble fibrin polymers and untreated fibrinogen, which contained 21% and 8.5% soluble fibrin, respectively. Thus, D dimer immunoreactivity in stage 3 digests correlated with the amount of soluble cross-linked fibrin polymer in the undegraded starting material. These findings demonstrate that degradation of soluble or particulate cross-linked fibrin increases D dimer immunoreactivity progressively, with maximal reaction attained with stage 3 digests.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-Linking Reagents↗

PubMed1987

Endocytosis of particulate and soluble IgG immune complexes: differential effects of cytoskeletal modulating agents.

We determined the ability of cytoskeletal modulating agents to affect endocytosis of particulate and soluble immune complexes. Either immunoglobulin G-opsonized 51Cr-labelled erythrocytes (E) or 125I-labelled soluble IgG anti-dinitrophenyl immune complexes (IC) were added to adherent murine peritoneal macrophages and the extent of internalization was measured. Either intracellular 51Cr radioactivity or trichloroacetic acid soluble 125I radioactivity was monitored as an indication of uptake by the cell. In addition to cytochalasin B and colchicine, the phospholipid methylation inhibitors, 3-deazaadenosine and 3-deaza(+/-)aristeromycin, were used. Whereas incubation of macrophages with colchicine alone resulted in 50% inhibition of uptake of E-IgG, there was no effect on the degradation of soluble IC. Both cytochalasin B and colchicine produced a 95% inhibition of E-IgG uptake by macrophages, but these drugs only minimally inhibited (17%) the degradation of soluble immune complexes. Both inhibitors of methylation produced a 50% decrease in phospholipid methylation in treated cells. However, only 3-deazaadenosine inhibited phagocytosis (50% of control for E-IgG and 75% of control for soluble IC). These data suggest that an intact cytoskeleton is necessary for the uptake of a particulate immune complex and much less important for the internalization and degradation of these model soluble immune complexes. In addition, inhibition of phospholipid methylation reactions alone do not impare the uptake and degradation of either a soluble or a particulate immune complex.

Adenosine↗