Innate immune recognition of flagellin limits systemic persistence of Brucella

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin‐deficient mutant of B. melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand‐off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.     

Oxidative Stress in Skin Fibroblasts Cultures of Patients with Huntington’s Disease

Oxidative stress and mitochondrial dysfunction should play a role in the neurodegeneration in Huntington’s disease (HD). The most consistent finding is decreased activity of the mitochondrial complexes II/III and IV of the respiratory chain in the striatum. We assessed enzymatic activities of respiratory chain enzymes and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with HD. We studied respiratory chain enzyme activities, activities of total, Cu/Zn- and Mn-superoxide-dismutase, glutathione-peroxidase (GPx) and catalase, and coenzyme Q₁₀ (CoQ₁₀) levels in skin fibroblasts cultures from 13 HD patients and 13 age- and sex-matched healthy controls. When compared with controls, HD patients showed significantly lower specific activities for catalase corrected by protein concentrations (P < 0.01). Oxidized, reduced and total CoQ₁₀ levels (both corrected by citrate synthase (CS) and protein concentrations), and activities of total, Cu/Zn- and Mn-superoxide-dismutase, and gluthatione-peroxidase, did not differ significantly between HD-patients and control groups. Values for enzyme activities in the HD group did not correlate with age at onset and of the disease and with the CAG triplet repeats. The primary finding of this study was the decreased activity of catalase in HD patients, suggesting a possible contribution of catalase, but not of other enzymes related with oxidative stress, to the pathogenesis of this disease.     

Effect of acute thioacetamide administration on rat brain phospholipid metabolism

Brain phospholipid composition and the [³²P]orthophosphate incorporation into brain phospholipids of control and rats treated for 3 days with thioacetamide were studied. Brain phospholipid content, phosphatidylcholine, phosphatidylethanolamine, lysolecithin and phosphatidic acid did not show any significant change by the effect of thioacetamide. In contrast, thioacetamide induced a significant decrease in the levels of phosphatidylserine, sphingomyelin, phosphatidylinositol and diphosphatidylglycerol. After 75 minutes of intraperitoneal label injection, specific radioactivity of all the above phospholipids with the exception of phosphatidylethanolamine and phosphatidylcholine significantly increased. After 13 hours of isotope administration the specific radioactivity of almost all studied phospholipid classes was elevated, except for phosphatidic acid, the specific radioactivity of which did not change and for diphosphatidylglycerol which showed a decrease in specific radioactivity. These results suggest that under thioacetamide treatment brain phospholipids undergo metabolic transformations that may contribute to the hepatic encephalopathy induced by thioacetamide.     

Synthesis and comparative molecular field analysis (CoMFA) of argentatin B derivatives as growth inhibitors of human cancer cell lines

Synthesis, characterization, anticancer activity, and comparative molecular field analysis (CoMFA) of 14 argentatin B (1) analogs are described. The effect of argentatin B derivatives on the growth of K562 (leukemia), PC-3 (prostate), U251 (CNS), and HCT-15 (colon) human cancer cell lines was determined using the sulforhodamine B test. The most active compound in this series, 2-formyl-(16beta,24R)-16,24-epoxy-25-hydroxycycloart-1-en-3-one (12), was about 35-50 times more potent than argentatin B (1). Structures were built using the X-ray crystallography of six derivatives for 3D modeling with Sybyl6.9. CoMFA of Log (1/IC50) in K562 cell line gave q2 = 0.507, r2 = 0.907, and three components. The standard deviation CoMFA contours indicate that increased activity is associated with a bulky group at C-2, a C1-C2 double bond, and low electronic density at C-25. Experimental Log P values for argentatin B and one derivative were 1-2 Log units more hydrophilic than the calculated CLog P values.     

A simplified caries risk test in stimulated saliva from elderly patients

Objectives: To evaluate and compare lactobacilli species (LB) and mutans streptococci (MS) caries risk identification by means of a Caries Risk Test Bacteria (CRT Bacteria test), with a conventional laboratory test (CLT), as well as their correlation with the dental caries experience [(decayed‐missing‐filled teeth (DMFT index)] in subjects 60 years of age and older. Design: Cross‐sectional study. Setting: Epidemiologic and Health Service Research Unit, Aging Area. XXI Century National Medical Center; Mexican Institute of Social Security (IMSS). Subjects: A total of 696 elderly subjects residing in southwestern Mexico City. Methods: The DMFT index was determined in 696 subjects and saliva collected by stimulation. This was processed with a CRT Bacteria test and a CLT (the gold standard) test. Subjects with high [≥10⁵ colony forming units (CFU)/ml] and low caries risk (<10⁵ CFU/ml) were assessed. Results: The CRT Bacteria test sensitivity (Sen) and specificity (Spe) were 0.97 and 0.86 for LB, respectively, with positive and negative predictor values of 0.92 and 0.95 respectively. For MS, Sen and Spe was 0.92 and 0.90, and 0.96 and 0.81 respectively. The receiver operating characteristic area under the curve (ROC area) in LB and MS was 0.94 and 0.89, respectively; thus, a correlation existed between the caries experience and the LB and MS caries risk, similar to that present with the CLT test. Conclusion: The CRT Bacteria test shows similar results to those of the CLT test in subjects 60 years of age and over.     

Reentrant waves in a ring of embryonic chick ventricular cells imaged with a Ca2+ sensitive dye

According to the classic model initially formulated by Mines, reentrant cardiac arrhythmias may be associated with waves circulating in a ring geometry. This study was designed to study the dynamics of reentry in a ring geometry of cardiac tissue culture. Reentrant calcium waves in rings of cultured embryonic chick cardiac myocytes were imaged using a macroscope to monitor the fluorescence of intracellular Calcium Green-1 dye. The rings displayed a variety of stable rhythms including pacemaker activity and spontaneous reentry. Waves originating from a localized pacemaker could lead to reentry as a consequence of unidirectional block. In addition, more complex patterns were observed due to the interactions between reentrant and pacemaker rhythms. These rhythms included instances in which pacemakers accelerated the reentrant rhythm, and instances in which the excitation was blocked in the vicinity of pacemakers. During reentrant activity an appropriately timed electrical stimulus could induce resetting of activity or cause complete annihilation of the propagating waves. This experimental preparation reveals many spontaneously occuring complex rhythms. These complex rhythms are hypothesized to reflect interactions between spontaneous pacemakers, wave propagation, refractory period, and overdrive suppression. This preparation may serve as a useful model system to further investigate complex dynamics arising during reentrant rhythms in cardiac tissue.     

In vitro effect of carbohydrates and enteric bacteria on adherence of Candida albicans

The adherence of Candida albicans to any cell is considered essential in the process that leads to colonization. Our objective in this study was to evaluate the effect of different carbohydrates and the presence of lactobacilli and Escherichia coli on the in vitro adherence of Candida albicans. The adherence to buccal epithelial cells was higher when growing at concentrations of galactose of 50, and 200 mM, as well as 50, 200, and 500 mM of sucrose, and 500 mM of mannose, compared with that obtained when growing in Sabouraud dextrose broth (p < 0.01). The presence of other microorganisms, such as Lactobacillus acidophilus and L. casei, caused a decrease in the in vitro adherence of C. albicans to buccal epithelial cells (p < 0.05), whereas E. coli did not modify this adherence at all.     

Study of supramolecular structures released from the cell wall of Candida albicans by ethylenediamine treatment

Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a β-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology. Received: 1 April 1996 / Accepted: 2 September 1996     

Influence of the incorporation of cholesterol on the doubling time and on the arrhenius and hill plots of two membrane-bound enzymes ofEscherichia coli K12

Escherichia coli was grown at 37°C with increasing cholesterol concentrations in the growth medium. Under these conditions, it was shown that neither the phospholipid species nor the fatty acid composition were affected by the incorporation of cholesterol. On the other hand, the doubling time (t_d) was increased; two membrane-bound enzymes, (Ca⁺⁺)-ATPase and D-lactate-dehydrogenase, were modified in the temperature-dependence activities, and the (Ca^±±)-ATPase changed its Hill coefficient for the inhibition by Na⁺. This last parameter was the most sensitive in detecting very low cholesterol incorporations to the cytoplasmic membranes.