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Changes in some biological properties of Streptococcus salivarius isolates from infected rats.

Isolation of colonies with altered colonial morphology was frequently observed following infection of specific pathogen-free rats fed a caries-inducing high sucrose diet with two human strains of Streptococcus salivarius. These isolates produced unique rough colonies on mitis salivarius agar, in contrast to the smooth, mucoid colonial morphology of the parental strain. The rough variants were characterized by a marked increase in the synthesis of polysaccharide from sucrose, especially cell-associated water-insoluble glucan, whereas the smooth parental strains mainly produced cell-free fructan. Glucose-grown cells of rough variants but not smooth parental strains were aggregated upon addition of sucrose. Such variants were not detected during laboratory transfers of the human strains of S. salivarius nor by mutagenesis of the cultures.

Animals↗

Measurement of glomerular filtration rate using inulin prepared from Vernonia herbacea, a Brazilian native species.

Vernonia herbacea (Vell.) Rusby (Asteraceae) is a perennial herb native to the cerrado vegetation of tropical areas in Brazil, which accumulates inulin in the underground reserve organs. The aim of this paper was to determine whether the inulin extracted from V. herbacea could replace commercial inulin for the measurement of glomerular filtration rate (GFR). Underground organs of vegetative plants were collected from a preserved area of the Brazilian cerrado. The inulin fraction utilized was obtained by ethanol precipitation after discarding the high molecular mass fructans in the freeze-thawing precipitate. GFR was determined in male Wistar rats anesthetized with inactin (100 mg/kg), which received intravenously commercial inulin obtained from Dahlia sp (Sigma) or Vernonia herbacea inulin (30 mg/100 g) as a priming dose and 0.05 mg min-1 100 g-1 as a sustaining dose in isotonic saline at the rate of 0/055 ml/min. Clearance was determined during 3 periods, with urine collected from the bladder and blood from the carotid artery. There was no significant difference in the GFR measured by clearance in inulin from both sources even when the plasma concentration of inulin from V. herbacea was doubled. The mean arterial pressure did not vary after the application of both inulins, indicating that they do not produce systemic side effects. The filtered load and the excreted amount of inulin from V. herbacea were equal, showing that the substance is not influenced by tubular function. These results demonstrate that the inulin from V. herbacea can substitute for imported inulin for the determination of GFR and in experiments of kidney microperfusion as a marker of tubular water reabsorption.

Animals↗

The targeting of Bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N-terminus mature part.

We compared the ability of signal sequences from various Bacillus or yeast secreted proteins to direct Bacillus subtilis levansucrase into the secretion pathway of the yeast Saccharomyces cerevisiae. The efficiency of these sequences correlated with the overall hydrophobicity of their h-domain and was independent of their origin. Furthermore, the net charge of the proximal protein sequence downstream from the signal sequence contributed to the competence of the heterologous proteins to be secreted by yeast. Modification of this net charge allowed the protein to be translocated under the control of the yeast invertase signal sequence. Moreover, glycosylation of levansucrase did not modify significantly the fructosyl polymerase activity.

Acid Phosphatase↗

Role of epitope density in the induction of immunity and tolerance with thymus-independent antigens. IV. Selective tolerance and IgE response by DNP-levan conjugates.

Tolerance induction of IgE antibody-forming cells by dinitrophenylated levan (DNP-LE) conjugates was investigated in CBA mice. Antibody production was measured by passive coutaneous anaphylaxis on rat skin. Specific antibody neutralization was obtained with conjugates of different substitution degrees. Specific inhibition of IgE antibody production was obtained only with highly substituted conjugates. However, the epitope density required for inducing tolerance to antibody synthesis of the IgE class was lower than for other classes of immunoglobulins. The tolerance induced by DNP-LE was shown to affect only the anti-DNP B cells of IgE class without T cell participation. These results demonstrate that B cell precursors of all Ig classes are susceptible to tolerance induction and not to triggering by T-independent antigens.

Animals↗

Idiotype-anti-idiotype interactions and the control of the anti-beta (2 leads to 6) polyfructosan response in the mouse: specificity and idiotypy of anti-ABPC48 anti-idiotypic monoclonal antibodies.

Seventeen hybridomas, secreting monoclonal anti-idiotypic antibodies (IDA) directed against the BALB/c ABPC48 idiotype, were isolated from one immunized BALB/c mouse. Several IDA also bind another Balb/c idiotype: UPC10. ABPC48 and UPC10 are both myeloma proteins with a beta (2 leads to 6)-polyfructosan (levan) specificity. The binding of every IDA to the ABPC48 idiotype can be completely inhibited by levan molecules, but at different concentrations. Mutual inhibition assays between the IDA made it possible to define six groups of IDA which bind at least three different idiotopes of ABPC48. Sixteen IDA have been studied by means of mouse anti-anti-idiotypic antibodies (Ab3) directed against two of them, IDA3 and IDA10. Anti-IDA3 Ab3 recognize idiotopes particular to IDA3 which are not found on other monoclonal anti-idiotypic antibodies (Ab2). Anti-IDA10 AB3 cross-reacts with several monoclonal Ab2, including Ab2 with different spectrotypes belonging or not to the same isotype and Ab2 with different specificities for the ABPC48 idiotype. Some IDA10 idiotopes are present in the polyclonal anti-ABPC48 antibody response of BALB/c, A/J and CBA mice showing that they are recurrent and that their expression is not linked to a particular Igh-C haplotype. In contrast, IDA3 idiotopes are not detected in the same anti-ABPC48 antisera.

Animals↗

Patterns of isotype expression by B cell clones responding to thymus-dependent and thymus-independent antigens in vitro.

It was found that the Type 2 thymus-independent (TI-2) antigens bacterial levan, trinitrophenyl-Ficoll, and pneumococcal carbohydrate vaccine (PnC) stimulate clonal expansion and antibody secretion in splenic fragments from either hemocyaninprimed or unprimed irradiated recipients bearing B cells from unprimed donors. The in vitro stimulation with TI-2 antigens leads to the expression of isotype switching and provides a more balanced variety of isotypes than is usually observed in vivo. Still, some characteristic patterns of isotypes expressed in vivo to either TI-2 or thymus-dependent (TD) antigens are preserved in vitro. Frequencies of phosphocholine (PC)-reactive B cells responding to either PnC or to PC-hemocyanin (PC-Hy) suggest an appreciable overlap in populations responding to these TI and TD forms of antigen. The existence of a population responsive to either form of PC determinant is supported by the observation that many clones arising in the presence of both forms of antigen express patterns of isotypes that appear as summations of those distinct patterns shown by clones responding to only one form or the other. These data suggest that PC-Hy- and PnC-responding cells may derive from a linear rather than a branched pathway of B cell development and that expression of isotype switching over the lifetime of a developing B cell clone may be regulated in a manner dependent on the form of the stimulating antigen.

Animals↗

Antibody response against poly (Glu60Ala30Tyr10) terpolymer and bacterial levan in kappa-deficient mice.

In murine species, the kappa (+)-bearing immunoglobulins dominate the antibody (Ab) repertoire with a kappa/lambda ratio of 95:5. The aim of the present study is to investigate the characteristics of the antibody response in kappa-deficient (K-/-) mice immunized with a T-dependent synthetic antigen, poly(Glu60Ala30Tyr10) (GAT) and a T-independent antigen, bacterial levan (BL). K-/- mice were obtained by targeted deletion of the J kappa C kappa gene segments. In response to GAT, K-/- mice respond by producing increasing amounts of anti-GAT Ig lambda 1 and Ig lambda 2 in the primary as well as secondary response, although anti-GAT specific monoclonal antibodies (mAb) raised in K-/- mice are mostly of IgM isotype. The GAT public idiotype, GATIdX, present on all GAT-specific Ab bearing kappa light chain, is not detected in the sera of K-/- mice or on any of the anti-GAT lambda 1 mAb. In response to BL, the amount of Ig lambda 1+ Ab in K-/- mice is comparable to the amount of Ig kappa + Ab in normal mice. However, lambda 2+ Ab are detected neither in wild-type nor in K-/- mice. Like kappa + Ab, the majority of lambda 1+ mAb are specific for beta 2-6 fructosan present in BL and rye levan and, to some extent, express the BL-specific idiotype, A48ld. Our results show that important compensatory mechanisms occur in kappa-deficient mice, restoring their ability to mount immune responses against a variety of T-dependent and T-independent antigens by the alternative usage of the clonally restricted lambda repertoire.

Animals↗

Effect of splenectomy on treatment of Lewis lung carcinoma by an immunomodulatory polysaccharide and a cytotoxic agent.

The effect of the spleen on the efficiency of chemotherapy (cyclophosphamide) and immunotherapy (the polysaccharide levan) of C57BL mice bearing the Lewis lung carcinoma was studied. The development of Lewis lung carcinoma caused a gradual splenomegaly in the C57BL mice. Splenectomy did not, however, affect tumor growth in the nontreated host. Levan induced a pronounced splenomegaly. Splenectomy reduced markedly the antitumoral effect of the polysaccharide. These results indicate that spleen elements participate in the inhibitory activity of levan. By contrast, splenectomy had no effect on the efficiency of treatment of cyclophosphamide.

Adjuvants, Immunologic↗

The inhibitory effects of high molecular levan on transport across the vascular wall stimulated by histamine.

Administration of levan to mice and rats inhibited the passage of intravenously injected trypan blue into the area of skin injected with histamine. In the mouse, the optimal dose of levan was about 5 mg/25 g body weight; higher doses, on the other hand, enhanced diffusion. In the rat, the inhibitory effect was directly related to the dose of levan, the optimal dose being five times higher than for mice. Treatment of rats with levan caused a marked reduction in the uptake and transport of colloidal carbon. In normal and histamine- or turpentine-injected cremaster muscles, treatment with levan prevented carbon from being adsorbed and endocytosed by the endothelial cells. Levan itself was frequently attached to the lumenal surface of endothelial cells, or less frequently was enclosed within pinocytic vesicles. Occasionally, transport across the interendothelial junction was also seen, but the impression gained was that the frequency and extent of patent junctions were reduced in animals receiving phlogistic stimuli and treated with levan. Carbon particles were generally aggregated in the central zone of vessels and were seldom seen near the endothelial cells, within endothelial cells or within any open junctions, presumably because of the coating effect of levan. With partly depolymerised levan, carbon particles were found in endothelial cells and intercellular spaces, but at lower concentrations than in non-levanised control animals. It is suggested that levan acts by altering the rheologic patterns in the microcirculation, by modifying the surface of endothelial cells and by influencing their behaviour, and, finally, by changing the constitution of the ground substance and basement lamina.

Animals↗

Histological study of homografts showing delayed rejection following levan administration.

The delaying effect of continued administration of levan on mouse skin homograft rejection was found to be due mainly to inhibition of thrombotic occlusion of the nutrient blood vessels. Levan administration did not appear to affect epidermal regeneration, but it seemed to inhibit degranulation of mast cells, resorption of plasma extravasated during the operation and growth of granulation tissue. The delayed rejection caused by levan treatment was apparently caused by infiltration of the graft by activated mononuclear cells from the blood stream. The phenomenon is believed to represent a late stage of the graft rejection process, its apperance depending on the survival of the graft beyond the stage of vascular occlusion.

Animals↗

The kinetics of mononuclear phagocyte infiltration in neoplasms: reactions to non-specific stimuli in a rat fibrosarcoma.

The kinetics of macrophage infiltration into a transplantable rat fibrosarcoma were investigated. Monocytes obtained from normal rats and labelled with 51Cr were injected into normal or fibrosarcoma-bearing rats which had previously been implanted with cotton pellets impregnated with BCG, B. pertussis carrageenan or levan. The subcutaneous tissues of normal and fibrosarcoma-bearing rats as well as the neoplasm itself were the sites for pellet implantation. The various additives induced an enhancement of macrophage infiltration into cotton pellets implanted for 1 week into subcutaneous tissues of normal rats. No significant effect was found in pellets implanted for 3 or 14 days. Macrophage infiltration into pellets in the neoplasm was consistently reduced when compared with controls. This occurred regardless of the length of time of implantation, the type of substance added to the pellet or (in the case of BCG or B. pertussis) whether the rats were pre-sensitised to the pellet additive or not. Pellets removed from the subcutaneous tissues of fibrosarcoma-bearing rats were either similar to or more often higher in radioactivity than those implanted into the neoplasm itself. The results indicate that the capacity of macrophages to respond to inflammatory stimuli is reduced in fibrosarcoma-bearing rats, but especially in the stroma of the neoplasm.

Animals↗

A novel biological function of macrophages associated with antigen discrimination properties.

The ability of fresh or cultured subpopulation of adherent peritoneal exudate cells to perform discrete biological functions commonly associated with macrophages, namely antibody dependent cytotoxicity (ADCC) and phagocytosis, has been compared with the ability of the same cells to present TNP coupled to carbohydrate or protein carriers for a subsequent antibody response, or semi-allogeneic cells for T cell cytotoxic responses, in tissue culture. Data are presented to show that ADCC reactivity rapidly disappears on cultures of peritoneal cells, though phagocytic activity is more persistent throughout the 14 days of culture. When ADCC activity reappeared it was no longer restricted to those fractions active initially. Antigen handling of different TNP-protein conjugates was pronounced throughout the culture period and was not easily explained in terms of the ADCC/phagocytic activity of the macrophage subpopulation concerned. In contrast, however, antigen handling of carbohydrate antigens for presentation to T or B lymphocytes by the different macrophage populations varied during the culture period in fashions which were in some degree correlated with the phagocytic activity of the various cells but were also a function of the antigen under consideration. These data are interpreted in terms of a novel role for the macrophage in discrimination of carbohydrate antigens for presentation to lymphocytes.

Animals↗

Mechanism of the inhibitory effect of levan on experimental tumors.

The mechanism of the antitumoral effect of levan was studied in the C57BL mice-Lewis lung carcinoma system. Modulation of host immune response and a direct inhibitory effect on tumor cells were found. Local treatment was more effective when begun early. It reduced tumor incidence without affecting the size of developing tumors. Systemic treatment was more effective when started late, inhibiting equally the tumor size in all mice. Macrophages are involved in local, lymphocytes possibly in systemic, inhibition. A direct effect on tumor cells was suggested by a decrease in oncogenicity, following preincubation with levan. Levan augments the antitumoral effect of cyclophosphamide in vivo and in vitro.

Animals↗

Histological changes in spleen and lymph nodes of mice administered cyclophosphamide and levan.

Morphological changes in spleen and lymph nodes of C57B1 mice induced by the cytotoxic agent cyclophosphamide and the polysaccharide levan, separately and in combination were studied. In the spleen, an early decrease (phase 1) and a late increase (phase 2) in weight were found to result from all drug administrations. The hypocellularity of phase 1 was due to a depletion in the white pulp affecting mainly the B-region. Splenic weight decrease was parallel to B-cell depletion and most marked in animals exposed to cyclophosphamide with levan. The splenomegaly observed during phase 2 with all treatments was due to extramedullary hematopoiesis in the red pulp. In the lymph nodes, administration of cyclophosphamide and levan produced opposite effects on the B-cell region: cyclophosphamide eliminated the germinal centers for 8 days, but levan seemed to enhance B-cell activity. In animals given both cyclophosphamide and levan, inhibition of B-cell activity began earlier than with cyclophosphamide alone. Levan does not antagonize the suppressive effect of cyclophosphamide on the B-cell component at the early phase when the drugs are given together.

Animals↗

Electrophoretic studies of extracellular glucosyltransferases and fructosyltransferases from seventeen strains of Streptococcus mutans.

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60.

Electrophoresis, Polyacrylamide Gel↗

Renal clearances of 14C-inulin and polyfructosan in the rat. Effect of increased ureteral pressure.

Polyfructosan has been used as a substitute for inulin in GFR determinations. However, the validity of this substitution in conditions where renal tubular permeability to other substances, such as mannitol, sucrose and other substances, such as mannitol, sucrose and iothalamate, is increased has not been tested. Experiments were performed on 8 rats to compare the clearances of polyfructosan (CPF) and 14C-inulin (CIN) during hydropenia, 3% BW saline expansion, elevation of one ureteral catheter by 30 cm, and following return of increased ureteral pressure to the control level. No significant difference between CPF and CIN could be detected except in the kidney subjected to increased ureteral pressure. However, the magnitude of this difference--which may relate to the different molecular weights of the two compounds--was so small that we conclude that the use of CPF, as an index of GFR, is no less reliable than CIN under the conditions tested.

Animals↗

The glomerular filterability of polyfructosan-S in immature infants.

Gel chromatography was performed on 21 plasma, 5 urine and 3 spinal fluid samples from pre-term infants who had received intravenous polyfructosan-S (PF-S), in order to investigate whether all sizes of PF-S molecule are filtered at the glomerulus and penetrate deep extra-cellular fluid compartments. Plasma obtained after 24 h continuous infusion of PF-S had identical chromatograms to the aqueous solution, suggesting no glomerular hold-up of the largest molecules. PF-S had a broader chromatogram than inulin, suggesting that it has larger molecules. Urine and plasma samples obtained early after a single injection had an excess of high molecular weight polyfructoside, suggesting that small molecules penetrate deep extracellular fluid compartments more easily. Spinal fluid samples had an excess of low molecular weight polyfructoside, suggesting slow penetration of the largest molecules.

Chromatography, Gel↗