Internalization of Staphylococcus aureus by bovine endothelial cells is associated with the activity state of NF-kappaB and modulated by the pro-inflammatory cytokines TNF-alpha and IL-1beta.

Bacterial internalization is an important process in the pathogenesis of infectious diseases in which nuclear factor kappaB (NF-kappaB) plays a prominent role. We present pharmacological evidence indicating that in bovine endothelial cells (BEC) the internalization of Staphylococcus aureus, a pathogenic bacterium that causes mastitis in bovine cattle, was associated with the activation of NF-kappaB. The internalization of S. aureus increased when BEC were stimulated with alpha-tumour necrosis factor (TNF-alpha) or beta-interleukin 1 (IL-1beta) which are known activators of NF-kappaB. SN50 (an inhibitor peptide of NF-kappaB nuclear translocation) and BAY 11-7083 (a chemical that inhibits the IkappaBalpha phosphorylation) caused significant reduction in S. aureus intracellular number, indicating that its internalization was associated with the NF-kappaB activity. Furthermore, specific inhibition of c-Jun N-terminal kinase with SP600125 (SP) or p-38 with SB203580 (SB) did not cause any change in the S. aureus intracellular number compared with the untreated control. Finally, TNF-alpha treatment of BEC after the addition of both SP and SB, induced a significant increase in S. aureus internalization above the control value. These data indicate that NF-kappaB activity is associated with S. aureus internalization and suggest that this transcription factor may play a role in the pathophysiology of bovine mastitis caused by this bacterium.

Correlation between formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites determined by a comet assay, increased MDA, and decreased glutathione during long exposure to thinner inhalation.

Thinner inhalation causes toxic effects in a variety of organs, principally in the central nervous system. Some studies have shown oxidative stress effects of thinner inhalation, such as: activation of free radical processes, decrease of antioxidants, and oxidation products of proteins and lipids but not of DNA. The aim of this study is to investigate the effect of thinner inhalation on DNA. We used the comet assay in conjunction with the enzyme formamidopyrimidine glycoslyase (Fpg). Our results show a significant increase in Fpg-sensitive sites in DNA of lymphocytes from rats exposed to thinner fumes compared to lymphocytes from control rats (p < 0.05). Moreover, DNA damage detected with Fpg shows a high correlation with increased malondialdehyde (MDA) and decreased glutathione (GSH), two widely used biomarkers of oxidative stress. The most abundant base oxidation product found in DNA is 8-oxoguanine; it is the main substrate of Fpg and the most commonly used biomarker for oxidative DNA damage. This suggests that oxidative DNA damage is at least partly responsible for the DNA damage detected by Fpg. We propose the comet assay in combination with Fpg as a sensitive biomarker to monitor exposure to thinner inhalation. Limitations of this method are discussed.

Equilibrium between monomeric and dimeric mitochondrial F1-inhibitor protein complexes.

Mg-ATP particles from bovine heart mitochondria have more than 95% of their F1 in complex with the inhibitor protein (IF1). The F1-IF1 complex was solubilized and purified. The question addressed was if this naturally occurring complex existed as monomers or dimers. Size exclusion chromatography and electron microscopy showed that most of the purified F1-IF1 complex was a dimer of two F1-IF1. As determined by the former method, the relative concentrations of dimeric and monomeric F1-IF1 depended on the concentration of protein that was applied to the column. Apparently, there is an equilibrium between the two forms of F1-IF1.

Structure and chemical composition of the cell walls from the haploid yeast and mycelial forms of Ustilago maydis.

Isolated or in vivo cell walls from the yeast and mycelial forms of haploid Ustilago maydis were not stained by the normal osmium procedure for electron microscopy. KMnO4 stained mycelial walls, revealing a layered structure with a loose electron-dense layer at the cell surface, but stained only the outer surface layer of yeast walls. Walls were purified from extracts obtained by ballistic and ultrasonic disruption. Chemical analysis showed that composition of yeast and mycelial walls was similar. Yeast walls contained higher amounts of neutral sugars and protein, whereas mycelial walls contained more chitin and phosphate. No chitosan or uronic acids were detected. Higher proportions of xylose and mannose were present in yeast walls, whereas the amounts of glucose and galactose were higher in mycelial walls. Fucose, arabinose, and ribose were detected in yeast walls only. Electrophoretic patterns of proteins extracted with SDS, beta 1, 3-glucanase, or chitinase were similar in walls of both morphologies, although some differential bands were identified. Most antigenic proteins appeared in the covalently bound fraction of the wall. Some were common to both morphologies, but others were stage specific.

[The inhalation of industrial solvents. The effect of the chronic inhalation of thinner on the rat liver and on an isolated mitochondrial fraction]. / Inhalación de disolventes industriales. Efecto de la inhalación crónica de tíner sobre el hígado de rata y la fracción mitocondrial aislada.

Industrial solvent inhalation has become one of the most important health problems worldwide. Workers frequently are exposed to the vapours of these type of compounds but perhaps the most important problem related with the industrial solvents is the voluntary inhalation, that, under certain conditions and according to the WHO becomes a pharmaco-dependency or a drug-addiction. In the last few decades this drug addiction has grown to impressive figures, this is an indication that the problem has to be studied, in order to gain insight on the possible mechanism, to which these compounds affect the organism and the physiology of individuals with this health problem. The pioneer studies of Costero and Barroso among others, pointed out on the extent of the effects of this drug addiction. Our work is focussed on the effects that long term thinner inhalation induces on the liver and on the subcellular mitochondrial fraction of rats.

[Hardening and softening phenomena in beans: technological alternatives]. / Fenómeno de endurecimiento y ablandamiento del frijol: alternativas tecnológicas.

The effect of accelerated hardening and soaking solutions on cooking time and microstructure of common bean (Phaseolus vulgaris) was studied. Two varieties (Canario and Mayocoba) were grown in the same location. Three hardening procedures were used: 1) End A. Soaking in acetate buffer, pH = 4.0 at 37 degrees C for 5 hs, 2) End B. Storage at 37 degrees C, 100% RH for 28 days and, 3) End C storage at 13-33 degrees C, 76% RH for 120 days. The salt solutions used for soaking were: Soln 1 (1% NaCl+0.75% NaHCO3) and Soln 2 (0.75% NaHCO3). Cooking times were determined using a Mattson bean cooker. In both varieties, the three hardening procedures decreased (38-50%) cotyledons water holding capacity and increased significantly (2-4 times) cooking times. During soaking in salt solutions hardened beans reached maximum water absorption in four hours. Soaking in salt solutions decreased drastically (2.6-10.6 times) cooking times. Fresh, hardened and softened seeds were examined by light microscopy, observing ultrastructural differences among them. The methods used in this research might well represent the central components of an industrial technological procedure for the utilization of hardened beans.

Influence of hardening procedure and soaking solution on cooking quality of common beans.

Two common bean (Phaseolus vulgaris) varieties were seeded in the same location, harvested and cleaned. Three hardening procedures were used (soaking in acetate buffer, pH 4.1 at 37 degrees C for 5 h; storage at 37 degrees C, 100% RH for 28 days; and storage at 31-33 degrees C, 76% RH for 120 days) to have seeds in a hard-to-cook (HTC) state. The adverse effects of HTC condition, in terms of cooking time as assessed by a Mattson bean cooker, were practically eliminated by soaking seeds in salt solutions (1% NaCl + 0.75% NaHCO3; and 0.75% NaHCO3) instead of only water. Ultrastructural changes of cotyledon cells from fresh, HTC and softened seeds were observed. Results of this study may be used for the development of a technological procedure to utilize properly HTC beans generated by unefficient storage systems.

Loose binding of testicular mitochondrial ATPase to the inner membrane.

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N'-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F1 to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few "knobs" or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was 30/1 in liver mitochondria, whereas in the testis it was 3/1. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.

Location on chitin in the cyst wall of Entamoeba invadens with colloidal gold tracers.

Chitin was located in the cyst wall of Entamoeba invadens with colloidal gold-linked wheat germ agglutinin. Cysts stained differentially from trophozoites when encysting cultures were treated with the gold tracer; cysts acquired a wine-red coloration while, in general trophozoites remained unstained. Observation of cells with the electron microscope revealed that the tracer particles were bound specifically to the walls of the surface of the cyst when cells were exposed in suspension, and to the cyst wall cross-section, when cells were exposed to the tracer in thin section, indicating that chitin fibers were distributed on the surface as well as throughout the matrix of the cyst wall.

Identification of the structural component in the cyst wall of Entamoeba invadens.

Cyst walls of Entamoeba invadens were isolated and purified. Both whole cysts and purified walls appeared intensely fluorescent when stained with Calcofluor white M2R. Examination of positive replicas of purified cyst walls with the electron microscope revealed the presence of a microfibrillar structure. The main sugars detected in acid hydrolysates from the walls were hexosamines. X-ray diffraction analysis of purified cyst walls demonstrated that the crystalline polymer present was chitin.

[Staining of cysts of Entamoeba invadens, Entamoeba histolytica and Entamoeba coli with wheat germ agglutinin labelled with colloidal gold]. / Tinción de quistes de Entamoeba invadens, Entamoeba histolytica y Entamoeba coli con aglutinina de germen de trigo marcada con partículas de oro coloidal.

Cysts of Entamoeba invadens obtained under axenic conditions were stained with wheat germ agglutinin labelled with colloidal gold. Microscopic observation of encysting cultures revealed that upon staining, cysts acquired a red coloration, while trophozoites remained unstained. E. histolytica and E. coli cysts obtained from asymptomatic patients were also stained red by this technique. Electronmicroscopic examination of stained cells showed a layer of colloidal gold granules attached to the surface of cysts, while trophozoites remained free of gold granules. The results of the staining procedure developed, agreed with the known facts that wheat germ agglutinin is a lectin that binds specifically to chitin and its oligomers, and that chitin is present in the cyst wall of E. invadens. The results suggested that chitin is also present in the wall of cysts of E. histolytica and E. coli.

[Composition of the cell wall of Entamoeba invadens cysts]. / Composición de la pared celular de quistes de Entamoeba invadens.

Cyst walls isolated from Entamoeba invadens have a microfibrillar structure. The main sugars detected in acid hydrolysates from walls were hexosamines. X-ray diffraction analysis of the cyst walls demonstrated that the only crystalline polymer present was chitin.