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A study was made of K+, Na+, Ca++ and Mg++ binding and ion-exchange properties of S-layer proteins from Bacillus subtilis. Our results indicate that the proteins of S-layer have two binding sites with pK of 3.17 and 2.41. To study the capacity of S-layer subunits to bind K+, Na+, Ca++ and Mg++, potentiometric titrations were performed according to Albert on proteins in solution in the presence and the absence of cations. It appears that the proteins possess dissociable groups with different affinity for various cations.

In this study, we describe the role of porins extracted from the outer membrane of S. typhimurium on the interaction with the host organism. The tests were performed on CD-1 mice immunized both with whole S. typhimurium cells and with purified porins. The antibody titres obtained in mice treated with purified porins and in those treated with whole Salmonella are 1/320 and 1/80 respectively. Cell-mediated response was found to be stimulated in both mice groups: our results show that T lymphocytes determine an increase in H3-thymidine incorporation and MIF production. An increase in lymphocytes with membrane immunoglobulins was observed in the lymphocyte population from the spleens of the treated mice. Purified porins added to macrophages cultures exerted a toxic effect at concentrations between 10 and 20 micrograms/ml. Subinhibiting concentrations (5 micrograms/ml) led to modifications in the surface tension of macrophages layers adhering to the slides. Moreover, subinhibiting porins concentrations were found to modify macrophage functionality as shown by evaluation of the phagocytic index and of intracellular killing, both of which were found to have diminished with respect to controls. When purified porins were added in vitro to populations enriched with T and B lymphocytes, at concentrations between 1 and 5 micrograms/ml they were found to be mitogenic with respect to B lymphocytes. The results we obtained showed that lymphocytes with IgM increase in porins tests. Eventually, purified porins added to Hela and Hep-2 cells were shown to exert a toxic effect which became detectable in the final concentration of 5 micrograms/ml and 1 micrograms/ml for Hela and Hep-2 cells respectively.

Some our previous research works on bacterial adhesion to vaginal cells in the different phases of the menstruum showed that adhesion changes depending on changing environmental conditions. We therefore considered interesting to extend our investigations to anaerobic flora, in the light of recent observations intended to attribute an important role to anaerobic flora in the pathogenesis of vaginitis. The results obtained so far indicate that the maximum adhesion capability is found in the middle of the menstruum. The very low adhesion of bacteria belonging to the Leptothrix genus remains substantially unaltered throughout the menstruum. Low adhesion is also found in sporogenic bacteria, whereas the coccoid ones have a stronger adhesion, particularly about the middle of the menstruum. With lower pH values adhesion of the anaerobic flora is enhanced, whereas in the final phase of the menstruum, with higher pH values, adhesion is reduced. Competition tests evidence a stronger adhesion of coccoid as compared to bacillar types.

Salmonella typhimurium and Escherichia coli K12 mutants of R chemotype, with varying contents of major proteins, were studied with respect to serum-mediated killing. The mutants demonstrated a different susceptibility to serum lytic action. These results were related to phospholipid and fatty acid content, as well as different physico-chemical surface properties, such as outer membrane fluidity. Tests were carried out on all parameters considered in the literature to demonstrate the resistance to complement. Our results showed that in sensitive strains such as Salmonella strains SH6261, SH6378, SH5551, SH6017 and E. coli PC0479 tests taken alone were not sufficient to explain the resistance to complement. Therefore, complement susceptibility is probably determined by many factors influencing the microheterogeneity of the membrane system.

Sono state studiate le costanti apparenti di associazione di vari cationi per il cell-wall isolato di cellule diStaphylococcus aureus. E' risultato che gli ioni monovalenti presentano una bassa costante di associazione mentre gli ioni bivalenti e trivalenti presentano valori nettamente superiori delle costanti di associazione. Si conclude che mentre i cationi monovalenti sono legati da deboli legami elettrostatici, i cationi di valenza superiore formano con i gruppi dissociabili legami ionici stabili.

The conditions of adenovirus aggregations are analyzed. Adenovirus type 2 was propagated on Hep2 cells; virion aggregation analysis was performed by sedimentation velocity on sucrose gradients. The results show that aggregation depends on: 1) Ionic strength: aggregate formation was verified between 0.005 M and 0.05 M NaCl; 2) pH: at pH 7.1 virus particles are dispersed, 90 per cent of particles are aggregated at pH 4--5; at pH lower than 4 aggregation is not reversible; 3) Particle concentration: the dilution of a stock containing 5 X 10(11) particles/ml decrease rate of aggregate formation until all aggregation is inhibited; 4) Temperature: the degree of aggregation in the 25 to 37 degrees C range is constant but decreases when the temperature falls below 20 degrees, at +4 C there is no aggregation. The aggregation reaction presents a positive deltaH of 17.9 Kcal/mol and a deltaG of --1.32 Kcal/mol.

We have analysed the expression of the HMGA2 gene in a panel of normal and neoplastic thyroid tissues by immunohistochemistry and quantitative RT-PCR. HMGA2 protein was detectable in four out of 21 follicular carcinomas, 30 out of 45 papillary carcinomas, and 11 out of 12 undifferentiated carcinomas. As far as follicular thyroid adenomas are concerned, only three cases of the 31 analysed showed HMGA2 protein expression, whereas it was absent in seven normal thyroid tissues and in 12 hyperplastic nodules. Quantitative RT-PCR showed that almost all the papillary thyroid carcinomas and 13 out of 16 follicular thyroid carcinomas express much higher HMGA2 specific mRNA levels in comparison to normal thyroids and adenomas. Therefore, our data support the quantitative RT-PCR analysis of HMGA2 expression, rather than immunohistochemistry, as a powerful tool for the diagnosis of thyroid neoplasias. (c) 2008 Elsevier Ltd. All rights reserved.

Our results indicate that benzodiazepine (Bz) treatment time, greater than 2–3 months, induce a decrease of both specific and nonspecific responses. Mice treated for different times with diazepam or chlordemethyldiazepam showed decreased survival to experimental Salmonella typhimurium infections after three months of treatment. Adherence, expressed as the polymorphonuclear cells (PMN) capacity to attach to nylon wool, was impaired after 7 days of treatment. Longer treatments further increase this impairment. PMN from mice treated with Bz for 90 days also demonstrate on impaired chemotaxis and phagocytosis for Saccharomyces cerevisiae . Monocytes from mice treated for 7 days secreted more IL-1 α then controls; the antibody titer in mice given to prolonged treatment progressively diminished compared to controls. Con A or LPS stimulated lymphocytes showed an increase of H 3 -thymidine incorporation from mice treated for a short time and conversely a decreased incorporation when taken from mice that underwent longer treatments. Benzodiazepines were therefore found to affect PMN chemotaxis and phagocitosis, general immunity and survival of mice to infections.

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.

SUMMARY: The sensitizing effect and the local and general toxicity related to membrane components of the archaeobacterium Sulfolobus solfataricus was studied. Cell envelope fragments were biologically active but this activity was lost upon separation of the lipid and protein components. The envelope fragments exerted lethal effects on mice sensitized with D-galactosamine that were prevented by pretreatment with anti-TNF-α serum. This lethal activity occurred in both LPS-responder (BALB/cByJ) and LPS-nonresponder (C3H/HeJ) mouse strains. In addition, Sulfolobus envelope fragments tested in rabbits caused a local Shwartzman reaction, and showed pyrogenic activity. In vitro, the envelope fragments that act on spleen lymphocytes of the LPS-responder (BALB/cByJ) and LPS-nonresponder (C3H/HeJ) mice caused an uptake of [3H]thymidine similar to that caused by concanavalin A. A similar toxic activity to that exerted by eubacteria is therefore exerted by this non-pathogenic archaeobacterium despite the difference in surface chemistry.

Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. The results are as follows. a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested. b) The phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids. c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs. d) The cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at C16 cause a decrease in the formation of phagolisosomes in the macrophages tested.

We studied the exchange of phospholipids between Escherichia coli K-12 cells and the suspension medium containing inactivated guinea-pig serum. In this medium, the release of 3H-labelled phospholipids was proportional both to the quantity of serum and to the temperature of incubation. No phospholipids were released when no guinea-pig serum was added to the medium, or the incubation temperature was 4°C. The release of phospholipids into the medium was accompanied by an uptake of serum phospholipids by the cells, as demonstrated by incorporation of labelled phospholipids from the suspension medium. We conclude that an exchange occurs between the cellular phospholipids and those of the medium. Control tests with 3 H-thymidine showed that cellular lysis was not involved.

Rabbits immunized for 6 months with different amounts of sterile human serum showed weight loss and a decrease in leukocytes. The lymphocyte population reacting to ConA contained autoreactive cells capable of causing 51Cr release from labeled cells from a culture consisting of splenic and peripheral lymphocytic cells from the same animal.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the outer membrane bilayer of two mutant strains of Salmonella thyphimurium, i.e., SH 5014 and SH 6261, at different temperatures was analyzed in terms of continuous Lorentzian lifetime distributions. The results were compared with those obtained for the free fluorophore in an isotropic nonviscous solvent. The incorporation of DPH in the outer membrane fragments resulted in a broadening of the lifetime distribution which was attributed to the microenvironmental heterogeneity of the membrane bilayer for the extrinsic fluorophore. The differences observed between the two types of membrane bilayers were interpreted in terms of a different molecular organization and, to a lesser extent, in terms of a different fluidity. The comparison between the DPH lifetime distributions obtained using two different excitation wavelengths, i.e., 280 and 350 nm, suggested that the structural organization of the membrane domains, which are richest in proteins, is almost identical in the two examined mutant strains. This observation indicates that the different susceptibility of the two mutant strains toward phagocytosis and complement-mediated lytic action may depend on the molecular organization and dynamics of the lipid regions far from those containing proteins.

The inflammatory activity of porins purified from Salmonella typhimurium has been investigated. Porins (0.3 to 30 micrograms) injected into the rat paw induced a dose-related edema that was not due to lipopolysaccharide contamination and did not appear to be dependent on the activation of the complement system. The edema induced by 30 micrograms of porins was comparable to that caused by 1 mg of carrageenin and was inhibited by indomethacin (5 mg/kg) and dexamethasone (0.1 mg/kg). Porins (1 to 10 micrograms/ml) induced a concentration-related release of histamine from rat peritoneal cells. These results are discussed in the light of the possible pathogenic role of porins in infections.

Major outer membrane proteins of Eikenella corrodens, an organism frequently isolated from patients with periodontal disease, were tested for some biological activities. Mouse peritoneal macrophages, exposed at low concentrations of the above-mentioned proteins (between 0.05 and 5 micrograms/ml), showed evident and marked morphological modifications consisting of increases in the size and vacuolation of the cells. Higher concentrations showed a toxic effect. Low concentrations resulted in a selective release of lysosomal enzymes without any significant release of lactatedehydrogenase, and cytoplasmic marker; while concentrations of 25-50 micrograms/ml, which were toxic in trypan-blue exclusion test, increased LDH release. Eikenella corrodens major proteins increased the platelet aggregation of ADP and thrombin. The residual complement activity of serum samples incubated with various amounts of proteins at 37 degrees C for 30 minutes appeared strongly reduced with respect to controls, thus showing a consumption of the complement components. These results suggested that Eikenella corrodens major proteins may play a role in the development of periodontal lesions.

The effect of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes (PMNs) has been studied. Various cytological, biochemical, metabolic, and physical correlates of both biological activities have been found to be markedly reduced by the presence in the medium of micromolar concentrations of protein SV-IV. Moreover, the Scatchard analysis of the labeled SV-IV binding to PMN cell surface has demonstrated that such binding is specific. The binding sites contain only saturable components, completely displaceable by unlabeled SV-IV. The number of the specific sites has been calculated to be 87,000/cell, with a Kd of 1.72 x 10-7 M. The molecular mechanism of the inhibitory effect is discussed along with the possible biological and clinical implications of the experimental findings.

The adherence of polymorphonuclear neutrophils was examined in 16 children affected by enteritis, pneumonia, hepatitis and infectious mononucleosis. The results were compared with those obtained in 30 healthy adult volunteers and in 15 healthy children of the same age. Adhesiveness was significantly higher in adults than in healthy children, and significantly higher in healthy children than in children with viral infection. In 7 patients tested one month after regression of the disorder, PMN adhesiveness had returned to normal.

The cardiovascular effects of porins were evaluated using porins isolated from Salmonella typhimurium SH5014. In dogs porins depress arterial systemic pressure, vasomotor reactivity of norepinephrine and peripheral vagal stimulation. They are capable of modifying the sinocarotidal baroreceptor reactivity. In mice porins increase the cardiotoxic effects of isoprenaline, thyroxine, emetine and of p-nitrophenol. In rats porins increase the arrhythmogenic and lethal effects of BaCl2 and also give rise to renal lesions, probably at the tubular level.

A study was conducted on the granulocytic phagocytosis of bacterial aggregates obtained under ideal environmental conditions. For the strains studied, aggregation was favored by low salt concentrations, low pH and temperatures between 30 degrees C and 40 degrees C. Our results show that the phagocytic capacity of granulocytes depends on the type and size of these aggregates. Those formed by a smaller number of cells are more easily phagocytized than the larger ones.

The adherence of bacterial cells to valvular prostheses has been studied. Bacteria were selected on the. basis of their surface features (fimbriae, hydrophobicity and specific receptors). It was found that only strains having fimbriae and high cell surface hydrophobicity adhered to bioprostheses, while they did not adhere to metallic prostheses to any significant extent. Adherence to bioprostheses depended on the exposure time and it was affected by the saline concentration of the suspension medium. Furthermore, different bacterial binding capacity was observed for bioprostheses from different companies.