Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.

Modulation of costimulatory molecules CD80/CD86 on B cells and macrophages by stress proteins GroEL, GroES and DnaK.

The aim of this study was to evaluate the effect of the Heat Shock Proteins GroES, GroEL and DnaK on the expression of the costimulatory molecules CD80/CD86 in B cells and macrophages. The interactions among these molecules are able to highly influence the immune response through the regulation of cytokine liberation which, on their own, are able to regulate the immunological response by a feedback mechanism. Our results showed that, on B cells, GroES and GroEL stimulated the expression of CD86 but did not induce the increase of the CD80 expression. CD86 peak expression showed a peak after 24-48 h of culture and decreased 60h after the stimulation. GroES and GroEL also stimulated the expression of CD80 and CD86 on macrophages. The same HSPs did not modify the expression of CD80 and CD86 on cells having characteristics of activated macrophages, the A-THP-1 cell line. DnaK did not induce any increase in the expression of CD80 and CD86 on lymphocytes or macrophages.

Histopathological features and modulation of type IV collagen expression induced by Pseudomonas aeruginosa lipopolysaccharide (LPS) and porins on mouse skin.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is responsible for serious infections in the immunocompromised host. Many skin lesions induced by P. aeruginosa have been described. Few investigations have been performed on the local action of P. aeruginosa components. OBJECTIVES: To shed light on the "in vivo" activity of lipopolysaccharide (LPS) and porins extracted from P. aeruginosa, by verifying their effects after inoculation in mouse skin through the observation of histological changes and immunohistochemical expression of collagen IV. RESULTS: Both substances were able to induce a similar inflammatory process and a characteristic reversible change in collagen IV distribution. Interestingly, a fibroblast increase was observed at 24 h in the skin treated with porins, while it appeared later in the skin treated with LPS. Besides these changes, porins particularly increased collagen edema, together with disgregation of hypodermal structures. Moreover "in vitro", porins were able to stimulate fibloblasts 3T3 to convert 72 kDa type IV collagenase into the activated 62 kDa form and to release the 92 kDa collagenase. CONCLUSION: LPS and porins, released by gram-negative bacteria during cell growth and lysis, interact with the host at target cells, such as keratinocytes, fibroblasts and immunocompetent cells, thus contributing significantly to the pathogenesis of P aeruginosa skin infections.

Ultrastructural and biochemical studies on Candida albicans after prolonged incubation in sea water.

Candida albicans yeast cells suspended in sterilized sea water and cultivated in Brain Heart Infusion broth were compared. Viability, chemical composition, surface hydrophobicity and ultrastructural characteristics showed variations after incubation in sea water. The yeast cells developed some ultrastructural changes after about a month in sea water. The surface hydrophobicity of the yeast cells was gradually reduced, starting from day 16, and continued to decline throughout the 32 days in sea water. A decrease in total carbohydrate, lipid and protein contents was also observed and corresponded with ultrastructural modifications.

Toxic effect on human spermatozoa by Chlamydia trachomatis purified lipopolysaccharide.

We have investigated the effect that lipopolysaccharide extracted from Chlamydia trachomatis has on human spermatozoa. A lipopolysaccharide of 0.1 microgram ml-1 caused a spermatozoa mortality rate of 65 +/- 4% evaluated by eosin exclusion test. The toxic activity occurred rapidly even after brief incubation times, reaching the maximum (100% mortality) within 60 min.

Defense mechanisms of IFN-gamma and LPS-primed murine microglia against Acanthamoeba castellanii infection.

In the central nervous system (CNS), cytokine-primed microglia play a central role in host's defense against Acanthamoeba castellanii infection. In this study, the effect of recombinant interferon (rIFN)-gamma and Salmonella enterica serovar enteritidis lipopolysaccharide (LPS), both inflammatory stimuli, on A. castellanii infection in murine microglia was examined. Priming of microglia with rIFN-gamma and LPS synergistically triggered, in a dose-dependent manner, amebastatic activity in these cells. More than 52%, 88% or 95% of this function was then abrogated by anti-IL-1beta (but not anti-IL-1alpha), IL-6 or TNF-alpha neutralizing antibodies, suggesting that these endogenously produced cytokines may participate in the antimicrobial capacity. Consistent with these findings, the priming of microglia with rIFN-gamma and LPS elicited the release of proinflammatory interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. Since L-canavanine affected amebastatic activity only during the priming process but not during the infection process, NO-dependent pathway appears to be not the sole antiparasitic mechanism involved in this function. These data suggest that rIFN-gamma and LPS, likely through a proinflammatory network, up-regulate the release of IL-beta, IL-6 and TNF-alpha, which could trigger antimicrobial activity against A. castellanii infection in the brain.

Transforming growth factor beta-1 and interferon-alpha in the AIDS dementia complex (ADC): possible relationship with cerebral viral load?

Mechanisms involved in the pathogenesis of the AIDS-dementia complex are still unclear. The dichotomy between a small number of HIV-infected cells in the brain and their marked dysfunction could be related to a cellular amplification and/or activation of cerebral viral load by several cytokines. This link between cytokines and viral load could play a role in the generation of the clinical dementia syndrome. We have studied cerebral levels of transforming growth factor-beta1 and interferon-alpha, both in the mild and severe AIDS-dementia complex and also compared these cytokines with HIV RNA load in patients with different degrees of dementia. Our data indicate that production of different cytokines characterized the expression of clinical dementia. In the mild AIDS-dementia complex, there was a significant inverse correlation between interferon-alpha and transforming growth factor-beta1 (r = - 0.743; p < 0.001), and HIV-RNA was present in inverse proportion to transforming growth factor beta1 (r = - 0.751; p < 0.001). In patients with severe AIDS-dementia, transforming growth factor-beta1 was undetectable, while interferon-alpha level were higher than in mild AIDS dementia and correlated positively to cerebral HIV-RNA. No significant difference was evident between these cytokines in the serum of ADC patients and in the control samples. Our study suggests that a relationship is possible between productive HIV infection in the cerebral nervous system and a heterogenous and different expression of the immune response via a complex interaction of cytokines with a differential modulation of the dementia phenotype.

Beneficial effects of myristic, stearic or oleic acid as part of liposomes on experimental infection and antitumor effect in a murine model.

Liposomes consisting of dicetyl-phosphate, cholesterol, lecithin and stearic or myristic or oleic acid, exert a protective effect for mice against experimental infection by Salmonella typhimurium, and delay both the onset and mortality B16 melanoma in these animals. Liposomes labelled with 3H-myristic acid were used as probes in the spleen and liver. We found that the treatment schedule rather than route of administration of liposomes, is important. The results show that in order to induce protection, preventive treatment must start at least three days before. Longer treatments do not increase the degree of protection, and treatments started at the same time as, or following experimental infection or tumor transplantation, have no effect.

Interactions between cytokines and growth hormone for resistance to experimental.

Cytokine-activated human vein endothelial cells (HUVEC) may play an important role in resistance to Toxoplasma gondii infection. In this study, it was investigated the role of rTNF-alpha and GH in the induction of antitoxoplasmal activities in HUVEC. Co-treatment of HUVEC with rTNF-alpha plus GH induced both toxoplasmastatic activity and the intracellular killing of T. gondii (p <0.01 each vs untreated cells). Thus, these functions were inhibited by both neutralizing antibodies to IL-6 and GM-CSF (but not to IL-3) suggesting that these cytokines participate in the inhibitory process. Consistent with this hypothesis, the treatment of HUVEC with rIL-6 or rGM-CSF in the presence of rTNF-alpha, limited T. gondii multiplication in a dose-dependent manner (p <0.01 each vs untreated cells). In order to elucidate the inhibitory mechanism of HUVEC, it was assessed by L-arginine analogs (e.g., NG-monomethyl-arginine) whether NO2 molecules originating from HUVEC were directly or indirectly involved in the rTNF-alpha/GH-dependent induction of toxoplasmastatic activity. A good correlation was found between toxoplasmastatic activity and NO2 release during the activation phase, before infection of the HUVEC with T. gondii, but no correlation was found between the parasitostatic activity and NO2 release during the infection phase. These data indicate that NO2- itself does not directly affect toxoplasmastatic activity. Besides, the reduction of intracellular killing by monoclonal antibodies to ICAM-1 suggest that this adhesin plays a role in controlling T. gondii entry into cells.

Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity.

N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+)-ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cervical bacterial flora in 40 women using intrauterine devices (IUD)]. / Studio della flora batterica cervicale in 40 donne portatrici di dispositivo intrauterino (IUD).

PIP: 40 women in apparent good health between the ages of 22 and 35 were divided into 2 groups. The 1st had copper IUDs inserted; the 2nd group received inert IUDs, all at the end of their menstrual period. Cervical smears of the trans-cul type to control the cervical flora were taken immediately before IUD insertion, and then after 7, 30, and 60 days. They were prohibited from using any vaginal medicine with germicidal effects. The samples were then examined in cultures and conditions allowing the growth of either aerobic or anerobic germs. The conclusion was that there is a change in purity of the bacterial flora in all women 7 days after IUD insertion until the 30th day, whereas at the 3rd control (day 60), a return to normalcy was observed. Signs of rejection of a foreign body were not observed in any woman, nor did any accidental pregnancies occur. (author's modified)^ieng

Changes in the intrinsic fluorescence of the human erythrocyte monosaccharide transporter upon ligand binding.

The effect of ligands on the tryptophan fluorescence of the purified monosaccharide transporter from human erythrocytes has been investigated. Cytochalasin B, D-glucose, and ethylideneglucose quench the fluorescence of the protein at longer wavelengths by 17%, 13%, and 8%, respectively. Propyl glucoside, another ligand, has no effect on the protein fluorescence. Values of the dissociation constants for cytochalasin B, D-glucose, and ethylideneglucose were determined from the concentration dependence of fluorescence change; these agree with the values obtained from the effects of these compounds upon the binding of [3H]cytochalasin B measured by equilibrium dialysis. There is no correlation between the effect of each ligand on the fluorescence of the transporter and the conformational state expected for its complex on the basis of other evidence. The fact that the quenching is greatest at longer wavelengths suggests that an exposed tryptophan residue(s), possible located at the ligand binding sites, is the perturbed one.

Equilibria and kinetics of ligand binding to the human erythrocyte glucose transporter. Evidence for an alternating conformation model for transport.

Cytochalasin B (CB), n-propyl beta-D-glucopyranoside (PG), and 4,6-O-ethylidene-D-glucose (EG) are known to bind asymmetrically to the human erythrocyte glucose transporter. The first two compounds bind to the inner (cytoplasmic) surface of the transporter, while the latter binds to the outer surface. Equilibrium measurements of the inhibition of CB binding to the glucose transporter reported herein indicate that the ternary complexes of CB transporter with EG, PG, or D-glucose are not formed. Moreover, measurements of CB binding in the presence of both EG and PG or in the presence of high concentrations of D-glucose show that a ternary complex of transporter and sugars bound simultaneously on both sides of the membrane probably does not occur. Finally, the kinetics of dissociation of radiolabeled CB from the transporter in the presence of CB, glucose, PG, and EG have been determined. With the exception of the case of EG, the kinetics fit a simple scheme of rate-limiting unimolecular dissociation, and in no instance do they suggest the existence of a ternary complex of sugar, CB, and transporter. These data are consistent with a model for transport in which the substrate binding site exists alternately at the cytoplasmic and external faces of the membrane, as the result of protein conformational change.

Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.

Porins and lipopolysaccharide induce apoptosis in human spermatozoa.

Treatment of human spermatozoa with porins or lipopolysaccharide (LPS) increases spontaneous apoptosis in these cells. Porins and LPS were extracted from Salmonella enterica serovar Typhimurium and Pasteurella multocida and were mixed with human spermatozoa for detection of levels of apoptosis.

The elevation of cellular phosphatidic acid levels caused by polyomavirus transformation can be disassociated from the activation of phospholipase D.

Middle T (mT), the oncogene of murine polyomavirus, causes transformation of rat fibroblasts by activating a number of signal transducing pathways usually used by polypeptide growth factors and their receptors. Here, we report data regarding the activation of signal transducing pathways involving phospholipase D (PL-D). The hydrolysis of phospholipids by PL-D produces phosphatidic acid (PA), a compound with multiple biological effects. The PA content of cells expressing wild-type mT, introduced via a number of different methods, is approximately 50% higher than their untransformed counterparts. This increase in cellular PA content is associated with an approximately 65% increase in PL-D activity in cells expressing wild-type mT. We have also examined the effects of a number of site-directed mutants of mT, on both cellular PA levels and on PL-D activity. Mutants that do not produce mT (Py808A) or that produce a truncated, nonmembrane bound mT (Py1387T) have PA levels similar to that of control cells. Cells expressing the 322YF mutant of mT (which abolishes interaction of mT with phospholipase C gamma1) show increases in both PA levels and PL-D activity that are similar to those seen with wild-type mT. Expression of mutants that abolish the interaction of mT with either shc or with phosphatidylinositol 3-kinase (250YS and 315YF, respectively) cause an increase in PL-D activity comparable to that seen with wild-type mT. However, the PA content of cells expressing these mutants is not elevated. These results suggest that mT causes activation of cellular PL-D, but this activation alone is not sufficient to cause an increase in cellular PA content. Therefore, wild-type mT must affect another, as yet unknown, step in PA metabolism.

The action of LPS porins and peptidoglycan fragments on human spermatozoa.

This study examines the action of the cell wall components of enterobacteria on the vitality of human spermatozoa. Lipopolysaccharides extracted from Escherichia coli K12 killed about 80% of the spermatozoa at a concentration of 50 micrograms/ml. Porins extracted from E. coli, Proteus mirabilis and Salmonella typhimurium killed between 80% and 100% of the spermatozoa at a concentration of 50 micrograms/ml. Muramic acid and N-acetylmuramic acid caused about 60% mortality at a concentration of 50 micrograms/ml. The possibility that the products of cellular lysis in the course of gram-negative infections cause temporary sterility is discussed.

[Chlorhexidine gel for control of subgingival bacterial plaque: experimental microbiological evaluation]. / Il gel di clorexidina nel controllo della placca batterica subgengivale: valutazione sperimentale microbiologica.

The Authors studies effects of chlorexidine gel 1% on subgingival microbial flora in a group of periodontal patients. Microbial findings appear to demonstrate some activity of chlorexidine gel 1%.