Effect of conglutinin on phagocytic activity of bovine granulocytes.

In the present study we investigated the effect of bovine conglutinin on the phagocytic activity of leukocytes. We measured both the chemotactic activity of conglutinin and its effect on the internalization of zymosan particles and E. coli by granulocytes. We also assessed the binding of conglutinin to various microorganisms isolated from clinical cases in cattle. We showed that conglutinin binds strongly to the surface of yeast cells and to mannan-rich zymosan particles, while weak binding was observed in the case of the bacterial strains tested, including those whose O antigen is composed of mannan. Conglutinin (1-10 microg/ml) neither acts as a chemotactic factor for peripheral blood leukocytes nor affects ingestion of E. coli by granulocytes. However, as flow cytometry based assay showed, conglutinin (0.1-1 microg/ml) increased ingestion of zymosan expressed as mean fluorescence intensity (MFI) of positive cells.

Properdin factor D. II. Activation to D by properdin.

The protein in the properdin pathway responsible for conversion of precursor factor D to D has been isolated and found to be identical with properdin. Sequential ion exchange and gel filtration chromatography demonstrated identity between properdin protein, measured by radial immunodiffusion, and the capacity to activate D to D, assessed by formation of the intermediate, EAC43B(D). Properdin, purified in this manner, was homogeneous on acid polyacrylamide disc gel electrophoretic analysis, with the band of protein corresponding to the position of eluates in the replicate gel capable of activating highly purified D. Demonstration of the homogeneity of purified D by alkaline disc gel electrophoresis, coupled with the linear stoichiometric hemolytic titrations of each factor, indicates that direct interaction between properdin and D generates D. Thus, activation of D by properdin represents a mechanism in the properdin pathway by which D becomes available for formation of the C3b-dependent C3 convertase.

Human plasma protein corona decreases the toxicity of pillar-layer metal organic framework.

This scenario was designed to investigate the protein corona pattern on the pillar-layer surface of a Cu-based metal-organic framework (MOF) in human plasma. The [Cu(L)(L/)].1.3DMA (MOF-1) {L = 4, 4/-bipyridine and L/ = 5-aminoisophthalic acid}, was synthesized through the sonochemical irradiation approach as well as characterized by various techniques like scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray powder diffraction and single-crystal X-ray diffraction. The space group was determined to be an orthorhombic space group (Pbam) by single-crystal X-ray diffraction. Single-crystal X-ray analyses on MOF-1 showed that Cu+2 ion was 6-coordinated. Besides, to study and clarify interactions between MOFs and biological milieu, human whole blood plasma was selected as a model. Fluorescence spectroscopy and SDS-PAGE techniques were employed to explore quantitative and qualitative in situ characterization of protein corona as well. Furthermore, cell viability in a cancerous cell lines was evaluated by MTT assay in the presence and absence of the corona. The results from SDS-PAGE illustrated that the most adsorbed quantity among plasma proteins belongs to fibrinogen (α, ß and γ chains), and this protein showed the maximum frequency on the MOF-1s surface, so the possible interactions of MOF-1s with fibrinogen also studied using fluorescence spectroscopy and corresponding data were plotted. According to the obtained data from MTT assay, these structures have concentration-dependent toxicity. In brief, based on the obtained data in the current study, the designed MOF can be introduced as a new desirable carrier for drug/gen delivery after further prerequisite assessments.

Analysis of hepatic transcriptome modulation exerted by γ-conglutin from lupins in a streptozotocin-induced diabetes model.

Lupinus albus γ-conglutin is proposed to positively affect glucose metabolism through inhibition of hepatic glucose production and insulin-mimetic activity; however, the action mechanism is not entirely known. Besides, most studies had focused on its effect on molecular targets directly related to glucose metabolism, and few studies have investigated how γ-conglutin may affect the liver gene expression or if it plays a role in other metabolic processes. Therefore, we investigated the influence of γ-conglutin on the liver transcriptome of streptozotocin-induced diabetic rats using DNA microarrays, ontological analyses, and quantitative PCR. Of the 22,000 genes evaluated, 803 and 173 were downregulated and upregulated, respectively. The ontological analyses of the differentially expressed genes revealed that among others, the mitochondria, microtubules, cytoskeleton, and oxidoreductase activity terms were enriched, implying a possible role of γ-conglutin on autophagy. To corroborate the microarray results, we selected and quantified, by PCR, the expression of two genes associated with autophagy (Atg7 and Snx18) and found their expression augmented two and threefold, respectively; indicating a higher autophagy activity in animals treated with γ-conglutin. Although complementary studies are required, our findings indicate for the first time that the hypoglycaemic effects of γ-conglutin may involve an autophagy induction mechanism, a pivotal process for the preservation of cell physiology and glucose homeostasis.

Heterologous antilymphocyte globulin: studies in vitro.

Incubation of dog-lymphocyte cultures with globulin from antiserum to lymphocytes resulted in a greater percentage of transformed cells than incubation with phytohemagglutinin. Viability counts demonstrated that this globulin and phytohemagglutinin had approximately equal degrees of cytotoxicity.

Detection of IFN-alpha produced in the presence of plasma gamma-globulin fraction proteins.

Interferon-alpha was detected in IFN pool produced by human leukocytes in the presence of gamma-globulin fraction proteins, copper and zinc cations, and metal-modified gamma-globulins. The cytokine appeared in culture medium at early terms (24 h) of incubation, is characterized by acid resistance, and is neutralized by antibodies to IFN-alpha. The content of IFN-alpha in supernatants of induced leukocytes reached 60-90 pg/ml and correlated with antiviral activity of the samples. Zinc bound to human serum gamma-globulin attenuated and copper stimulated the realization of IFN-inducing characteristics of the protein at early terms of incubation.

Altered development of neuronal progenitor cells after stimulation with autistic blood sera.

Changes of brain structure and functions in people with autism may result from altered neuronal development, however, no adequate cellular or animal models are available to study neurogenesis in autism. Neuronal development can be modeled in culture of neuronal progenitor cells (NPCs) stimulated with serum to differentiate into neurons. Because sera from people with autism and age-matched controls contain different levels of numerous biologically active factors, we hypothesized that development of human NPCs induced to differentiate into neurons with sera from children with autism reflects the altered early neuronal development that leads to autism. The control and autistic sera were collected from siblings aged below 6 years that lived in the same environment. The effect of sera on differentiation of NPC neurospheres into neuronal colonies was tested in 72-h-long cultures by morphometry, immunocytochemistry and immunoblotting. We found that sera from children with autism significantly reduced NPCs' proliferation, but stimulated cell migration, development of small neurons with processes, length of processes and synaptogenesis. These results suggest that development of network of processes and synaptogenesis--the specific events in the brain during postnatal ontogenesis--are altered in autism. Further studies in this cell culture model may explain some of the cellular alterations described in autistic patients.

Anti-influenza A virus activities of mannan-binding lectins and bovine conglutinin.

Mannan-binding lectin (MBL) and bovine conglutinin (BKg) belong to the collectin family, which is involved in first-line host defense against various infectious agents. We have previously reported that human MBL inhibited type A influenza viral hemagglutination, infection and spreading to adjacent cells without complement activation. In this study, we investigated the direct antiviral activities of bovine MBL, rabbit MBL and BKg. All collectins used in this study inhibited viral infectivity and hemagglutination at concentrations of 0.02-0.3 microg/ml. They also demonstrated inhibitory activity against viral spreading. Like human MBL, bovine MBL and BKg showed antiviral activities at their physiological concentrations. These results suggest that mammalian MBLs and BKg may inhibit the spread of influenza A virus through the bloodstream.

Stimulation of cholesterol ester exchange by lipoprotein-free rabbit plasma.

A fraction in normal and hypercholesterolemic rabbit plasma of density greater than 1.25 stimulates the exchange of cholesterol esters between very low density and low density lipoproteins from hypercholesterolemic rabbit plasma. The exchange does not result from lecithin:cholesterol acyltransferase activity. The active factor appears to be a high molecular weight globulin with an isolelectric point of 5.2.

The effect of serum protein fractions on liposome-cell interactions in cultured cells and the perfused rat liver.

We have studied the interactions of liposomes with human skin fibroblasts and mouse P815Y mastocytoma cells in culture, and the perfused rat liver, with the following findings. 1. In all the systems studied serum was found to cause an increase in the uptake of a [14C]cholesterol label into cells from anionic and neutral liposomes and a decrease in the uptake of the label from cationic liposomes. 2. Evidence suggests that albumin enhances the exchange/transfer of [14C]-cholesterol between liposomes and cultured cells. 3. With [14C]cholesterol in the liposome bilayer and [3H]methotrexate entrapped in the aqueous spaces of the liposome, the alpha- and beta-globulin fractions of serum decreased the transfer of both labels from cationic liposomes into cultured cells and the perfused rat liver. The beta-globulin fraction caused increased leakage of methotrexate from fluid liposomes of all charges. 4. The alpha- and beta-globulin fractions of serum appear to enhance the uptake of anionic liposomes into the perfused rat liver.

Neuromuscular transmission in neonatal mice injected with serum globulin of myasthenia gravis patients.

Neuromuscular transmission was studied in neonatal mice following injection with serum globulin of patients with myasthenia gravis (MG). Compared to controls, these mice showed significant reduction in successive muscle action potentials evoked by repetitive nerve stimulation, amplitude of miniature endplate potentials, and postjunctional sensitivity to acetylcholine. There was no change in evoked isometric tension, quantal content of endplate potentials, or input resistance of the endplate membrane. These results confirm earlier reports of neuromuscular block in animals following injection of globulin of myasthenic patients, and demonstrate that decrease in amplitude of evoked potentials and of miniature endplate potentials is due to reduction in sensitivity to acetylcholine rather than in input resistance of the postsynaptic membrane. These findings are compatible with a postsynaptic defect in MG caused by a humorally mediated autoimmune mechanism.

The inhibiting effect of a plasma globulin on arterial thrombus formation, (as compared to dipyridamole).

The inhibiting effect on arterial white thrombus formation of a globulin prepared from beef plasma has been compared to dipyridamole in white Wistar rats. It was demonstrated that the globulin fraction had a greater effect in inhibiting thrombus formation as judged by the lag time [t(l)], the maximal thrombus value [m(T)], the maximal thickness of the thrombus [m(D)] and the maximal thrombus surface [m(O)].

Influence of coagulation on the activation of plasminogen by streptokinase and urokinase.

Human plasma was mixed with Ca++ or thrombin, and urokinase (UK) or streptokinase (SK) and a chromogenic substrate (S-2251: H-D-Val-Leu-Lys-pNA) specific to plasmin. The hydrolysis of S-2251 was higher when clot was formed by the addition of Ca++ or thrombin than in the absence of clot. The hydrolysis of S-2251 by euglobulin in the presence of UK was also higher when clot was formed, thus, inhibitors may not be related to the better activation of plasminogen, in the presence of fibrin clot. It may be suggested that plasminogen was better activated by activators (UK and SK) in the clot than in its absence.

Properties of washed human platelets.

We have shown previously that washed human platelets resuspended in Tyrode solution containing albumin and apyrase maintain their disc shape and their ability to aggregate upon the addition of low concentration of ADP, providing fibrinogen is added to the suspending medium. We have now examined their responses to other aggregating and release-inducing agents. Collagen, arachidonate, thrombin, immune serum globulin, the ionophore A23, 187 and phytohaemagglutinin from Phaseolus vulgaris caused aggregation and release of granule contents. The response to adrenaline was variable. Serotonin caused the platelets to change shape but no aggregation or release occurred. Addition of a small amount of plasma was necessary for ristocetin-induced aggregation. Polylysine caused immediate platelet-to-platelet adherence with little or no release of granule contents. Responses to collagen or thrombin were greater in a modified medium containing magnesium but no calcium; in this medium, aggregation caused by ADP or polylysine was followed by the release of granule contents whereas these agents caused aggregation without release in a medium with both calcium and magnesium. When protein was omitted from the suspending medium, platelet aggregation in response to ADP was variable. In this medium, collagen and thrombin caused more extensive release than in the albumin-containing medium. Aggregation by polylysine was accompanied by release and extensive lysis in the protein-free medium. Thus, the composition of the final resuspending medium has a major effect on the responses of washed human platelets to aggregating agents.

Effects of a morphine-rabbit anti-morphine antibody mixture on guinea-pig isolated ileum.

Incubates of morphine with serum globulins obtained from sera of rabbits immunized with a morphine-bovine serum albumin conjugate produced immediate Schultz-Dale contractions when added to superfused, electrically stimulated guinea-pig ileal strips. Incubates of morphine with Krebs-Henseleit solution produced relaxation and depression of tone, and inhibition of electrically induced contractions. It is concluded that the spasm of guinea-pig ileum produced by incubates containing morphine-binding serum globulins and morphine resulted from transient passive sensitization and an acute anaphylactic type of response.

Proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin.

The interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. Mouse alpha-macroglobulin formed complexes with thrombin, clotting factor Xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pancreatic elastase. These complexes lost the proteolytic activities against high-molecular-weight substrates, but protected the active sites of the enzymes from inactivation by their proteinaceous inhibitors. Mouse murinoglobulin showed essentially the same properties except (i) that it did not form a complex with the clotting factor Xa, and (ii) that it did not protect plasma kallikrein, neutrophil elastase or submaxillary proteinase from inactivation by their proteinaceous inhibitors, although it formed complexes with these proteinases. No interaction was detected between Clostridium histolyticum collagenase and murinoglobulin or alpha-macroglobulin. These results indicate (i) that murinoglobulin has a proteinase-binding spectrum similar to that of alpha-macroglobulin, but is weaker in the ability to protect the bound proteinases from inactivation by the proteinaceous inhibitors than alpha-macroglobulin and (ii) that mouse alpha-macroglobulin has essentially the same inhibitory spectrum as the human homologue.

Hamster alpha-macroglobulin and murinoglobulin: comparison of chemical and biological properties with homologs from other mammals.

alpha-Macroglobulin and murinoglobulin were purified to homogeneity from Syrian hamster plasma and their properties were compared with those of their respective homologs from other mammals. The trypsin-inhibiting capacity of hamster murinoglobulin was much weaker than those of rat and mouse murinoglobulins. Hamster alpha-macroglobulin was cleaved by trypsin at a number of sites whereas the human homolog was split essentially only in a "bait" region into two fragments of similar size. Hamster alpha-macroglobulin treated with methylamine differed from that treated with trypsin in the electrophoretic mobility, intensity of fluorescence induced by binding of bis(8-anilino-1-naphthalenesulfonate), and plasma clearance pattern, whereas virtually no difference was observed between the human homologs treated in the same manner. The reaction of hamster alpha-macroglobulin with methylamine, as measured by the generation of thiol groups and the decrease in trypsin-protein amidase activity, was much slower than that of the human homolog. Trypsin in a complex with hamster alpha-macroglobulin retained its fibrinolytic activity, but this was not the case for human or rabbit alpha-2-macroglobulin. These results suggest that, compared with the human homolog, hamster alpha-macroglobulin is more loosely packed in the native state, undergoes conformational change more slowly on treatment with methylamine, and less efficiently hinders the access of proteinaceous substrates to trapped proteinase. The serum concentration of hamster alpha-macroglobulin was 6.9 mg/ml, or about 3-fold higher than that of the human type, and showed little change during the acute-phase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of blood product medium supplements on the activity of cefotaxime and other cephalosporins against Enterococcus faecalis.

The activities of cefotaxime and other aminothiazoyl oxime cephalosporins against Enterococcus faecalis were enhanced by addition of 5% sheep blood to Mueller-Hinton agar. This effect was not seen with aztreonam (aminothiazoyl oxime monobactam), cefotiam (aminothiazoyl, nonoxime), or other cephalosporins, and it was specific to the syn-configuration of the oxime moiety. Enhancement of cefotaxime activity was demonstrable against approximately 50% of 86 clinical isolates and could only be shown at low bacterial inocula. Human serum, serum alpha 1-, beta- and gamma-globulin fractions and albumin often antagonized or did not affect significantly the antimicrobial activity of cefotaxime, while the alpha 2-globulin fraction usually enhanced drug activity. The in vivo activity of cefotaxime against E. faecalis was examined in a rat peritoneal abscess model. The test organism was resistant to cefotaxime by standard methods (MIC greater than 128 micrograms/ml) but was inhibited by 1.0 microgram/ml when rat serum was presented in the medium. Cefotaxime reduced titers of bacteria within abscesses after 5 days of therapy (5.77 +/- 0.68 log10 CFU/g) in comparison with those in control animals (7.38 +/- 0.28 log10 CFU/g, p less than 0.05). Moxalactam, the in vitro activity of which was not augmented by serum, proved ineffective in the animal model. While these observations do not have direct therapeutic relevance, they offer a possible explanation for the relatively infrequent occurrence of enterococcal superinfection in patients treated with cefotaxime.

Inhibition of hemorrhagic and edematogenic activities of snake venoms by a broad-spectrum protease inhibitor, murinoglobulin; the effect on venoms from five different genera in Viperidae family.

In order to obtain basic data on the effect of broad-spectrum protease inhibitor against local symptoms of Viperidae snake envenomation, inhibitory capacity of rat murinoglobulin on local hemorrhagic and edematogenic activities of venoms from Crotalus atrox, Bothrops jararaca, Lachesis muta muta, Trimeresurus flavoviridis and Echis carinatus sochureki were examined. Murinoglobulin, pre-incubated with the crude venoms at 37 degrees C for 15 min, inhibited hemorrhagic activity of all five venoms to various extents. The activity of C. atrox was almost completely inhibited at the murinoglobulin/venom ratio (w/w) of 20. The activity of B. jararaca, Lachesis muta muta and T. flavoviridis venoms was considerably inhibited at the ratio of 20 (77.2, 80.0 and 86.2% inhibition, respectively), however some of the activity still remained even at the ratio of 40 (84.2, 79.8 and 86.2% inhibition, respectively). Among the five venoms, E. c. sochureki venom is quite resistant to murinoglobulin treatment and statistically significant inhibition was only found at the ratio of 40 (64.1% inhibition). Fibrinolytic and gelatinase activities were more susceptible to murinoglobulin inhibition. The treatment at the ratios of 10 and 20 almost completely inhibited respectively the fibrinolytic and the gelatinase activities of all the venoms. Murinoglobulin treatment also significantly inhibited the edematogenic activity of L. muta muta, T. flavoviridis and Echis carinatus sochureki. The treatment of murinoglobulin at the ratio of 40 considerably suppressed the swelling up to 60 min after subcutaneous injection of L. muta muta and E. c. sochureki venoms, and up to 30 min after T. flavoviridis venom injection. Murinoglobulin is a potent inhibitor against local effects of multiple snake venoms in Viperidae family.

Cellular delivery and retention of photofrin: III. Role of plasma proteins in photosensitizer clearance from cells.

Human plasma proteins, albumin, globulins and low density (LDL), high density (HDL) and very low density (VLDL) lipoproteins were tested for their effects on retention of Photofrin and three other photosensitizers in cultured cells. This was assessed by incubating the cells, subsequent to the exposure to Photofrin, in the photosensitizer-free medium containing various concentrations of different plasma proteins. Photofrin clearance levels differed with individual plasma proteins and also were dependent on concentration of these proteins in the incubation medium. All of the proteins except VLDL promoted clearance of Photofrin taken up by the cells in the presence of 5% human serum. Subsequent to some Photofrin exposure conditions (in the presence of 5% fetal bovine serum, or in protein-free medium), albumin, in contrast to LDL, HDL and globulins, exhibited decreased capacity for promoting the photosensitizer clearance from the cells. The VLDL showed very little or no effect in promoting cellular clearance of Photofrin, tetraphenyl porphine tetrasulfonate (TPPS4), and di- and tetrasulfonated chloroaluminum phthalocyanine (AlPcS2 and AlPcS4, respectively). The LDL seem to be particularly effective in promoting clearance of Photofrin and AlPcS2 from the cells, whereas albumin and globulins were shown to be more effective than LDL and HDL in promoting the cellular clearance of TPPS4.