Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

Background: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. Objective: the objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. Methods: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. Results: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. Conclusion: the microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations

CYTOPLASMIC AND PLASMA-MEMBRANE ULTRASTRUCTURE OF PARACOCCIDIOIDES-BRASILIENSIS YEAST-PHASE CELLS AS REVEALED BY FREEZE-ETCHING

Using a freeze-etch technique the cytoplasmic and plasma membrane ultrastructure of Paracoccidiodies brasiliensis yeast-phase cells was studied. The multinucleate yeast-phase cells which grow by simultaneous multiple budding, like those of Mucor sp. contain several nuclei, mitochondria, well-developed ER, small vacuoles and lipid droplets. Complex structures with no apparent connexion to the plasma membrane of P. brasiliensis usually lack inveginations, but invaginations which do occur are always rod-shaped which indicates P. brasiliensis to be of either ascomycetous or basidiomycetous origin.

QUANTIFICATION OF IMMUNOGLOBULIN-A IN CHORIOAMNIOTIC MEMBRANE OF PATIENTS WITH PREMATURE RUPTURE OF MEMBRANES

Objective: the proposal was to study the presence of immunoglobulin A (IgA) in the chorioamniotic membrane of healthy postpartum women with premature rupture of the chorioamniotic membrane (FROM). Method: A single radial immunodiffusion technique was used to quantify the IgA in the chorioamniotic membrane tissues. Results: the level of IgA was approximately 10 times higher in patients whose membranes had been ruptured for > 10 h (24.58 mg/dl). These results were compared with those of a previously published study where the mean of amount of IgA was 2.52 mg/dl in membranes of patients with rupture < 10 h. Our results show that IgA began to rise after 10-15 h following rupture. Conclusion: Although more studies need to be performed our data indicate that the increasing IgA in our patients after 10 h of latency probably represents the beginning of an ascending colonization of bacteria which could be the source of future infection.

The effects of copper ions on the synthesis of periplasmic and membrane proteins in Acidithiobaeillus ferrooxidans as analyzed by SDS-PAGE and 2D-PAGE

The effects of 200 mM copper ions on the synthesis of membrane and periplasmic proteins were investigated in iron-grown cells of Acidithiobacillus ferrooxidans (At. ferrooxidans). Total membrane protein profiles of cells grown in the absence of copper ions (unadapted cells) and in the presence of copper ions (copper-adapted cells) were compared by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Crude preparations of outer membrane and periplasmic proteins were analyzed by SDS-PAGE. The synthesis of proteins was diminished or increased in the presence of copper ions. Low molecular weight proteins (< 14 kDa) were significantly repressed by copper. These proteins are probably acidic proteins located in the outer membrane. An over-expression of a periplasmic protein of about 17 kDa was detected in the copper-adapted cells and was assumed to be rusticyanin, a 16.5-kDa periplasmic copper protein present in At. ferrooxidans cells and involved in the electron-transport chain of the iron oxidation pathway. To our knowledge, this is the first report of a possible involvement of the rusticyanin and outer membrane proteins in the mechanism of copper resistance in At. ferrooxidans. (C) 2003 Elsevier B.V. All rights reserved.

Effect of long-term treatment with insulin and/or acarbose on glomerular basement membrane thickening in alloxan-diabetic rats

Acarbose is a competitive inhibitor of the intestinal alpha-glycosidases, that can delay absorption of intestinal carbohydrates causing their malabsorption. In the present paper we studied the effects of insulin, acarbose and their association on glomerular basement membrane thickening in alloxan-diabetic rats. Twenty-five male and female Wistar rats, approximately 3 months old at the beginning of the experiment, were assigned randomly to each of five experimental groups: normal control rats, alloxan-diabetic control rats, alloxan-diabetic rats treated with acarbose, alloxan-diabetic rats treated with insulin, and alloxan-diabetic rats treated with insulin plus acarbose. Alloxan was administered in a single iv dose of 42 mg/kg body weight. Insulin was given subcutaneously at doses of 18 to 30 IU/kg corrected daily on the basis of glycosuria and ketonuria. Acarbose was given mixed with rat chow in a dose of 50 mg/100 g chow. Body weight, water and food intake and diuresis, as well as blood and urine glucose were determined after 1, 3, 6, 9, and 12 months of treatment. Glomerular basement membrane (GBM) thickening was determined by electron microscopy at the same times. Clear clinical and laboratory signs of severe diabetes, with blood glucose levels above 200 mg/dl and urine glucose above 3000 mg/dl, were observed in all alloxan-diabetic control rats, in all periods of follow-up, whereas administration of insulin or acarbose reduced the blood glucose levels of treated groups. The most satisfactory control of blood and urine glucose was observed in animals treated with both insulin and acarbose. However, diarrhea was observed in diabetic rats treated with acarbose associated or not with insulin. GBM thickening was correlated with age in all groups. Beginning at six months after diabetes induction, the GBM of untreated diabetic rats was significantly thicker (mean +/- SEM, 4.446 +/- 0.45 mm) than that of normal rats (2.977 +/- 0.63 mm). Both insulin and acarbose prevented GBM thickening and their combination induced thickening similar to the age-dependent thickening observed for normal rats of the same age. We conclude that acarbose when combined with insulin may be a good option in the control of diabetes and its renal complications.

Deletion of Epstein-Barr virus latent membrane protein 1 gene in United States and Brazilian Hodgkin's disease and reactive lymphoid tissue: High frequency of a 30-bp deletion

A 30-basepair (bp) deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene has been reported in nasopharyngeal carcinoma and EBV-associated malignant lymphomas. Prior studies have found the deletion in about 10% to 28% of cases of Hodgkin's disease (HD), particularly in cases with aggressive histology. We studied the prevalence of 30-bp LMP1 gene deletion in EBV-positive HD in the United States (US) (12 cases) and Brazil (26 cases) with comparison to reactive lymphoid tissues (21 cases) and HD without EBV-positive Reed-Sternberg cells (15 cases). We studied the status of the LMP1 gene by Southern blot hybridization of polymerase chain reaction (PCR) products obtained after amplification with primers spanning the site of the deletion. We also performed EBV typing, EBER1 in situ hybridization, and LMP1 protein immunohistochemistry. EBV was detected in 12/26 (46%) cases of HD from the US and 26/27 (96%) cases of Brazilian HD. The 30-bp LMP1 gene deletion was observed in 4/12 (33%) cases of EBV-positive HD from US, and 12/26 (46%) cases of Brazilian EBV-positive HD, including 3 cases of type B EBV, as compared with 12/21 (57%) reactive lymphoid tissues and 9/15 (60%) cases of EBV-negative HD. US and Brazilian HD showed a higher prevalence of the 30-bp LMP1 gene deletion, compared with studies of others. The unexpected finding of high incidence of 30-bp deletion in LMP1 gene in reactive lymphoid tissue and HD without EBV-positive Reed-Sternberg cells suggests that this deletion may not be relevant to HD pathogenesis in most cases. Copyright (C) 1997 by W.B. Saunders Company.

Immunosuppression in paracoccidioidomycosis: T cell hyporesponsiveness to two Paracoccidioides brasiliensis glycoproteins that elicit strong humoral immune response

To assess human cellular immune response to paracoccidioidomycosis (PCM), lymphocyte proliferative responses to purified antigens from Paracoccidioides brasiliensis were determined in healthy persons previously infected by the fungus (positive donors), in healthy noninfected persons (controls), and in PCM patients. Affinity-purified gp70 and gp43, the two major antigens in humoral immune responses, were used, Both induced lymphocyte proliferation (gp43 species-specific) in positive donors but not in controls; healthy persons previously infected by Histoplasma capsulatum reacted to gp70 and not to gp43, A similar cross-reactivity in antibody response to gp70 was previously reported; however, antibody response to gp43 has been considered specific, Lymphocytes from PCM patients, who, unlike positive donors, have high levels of anti-gp43 and anti-gp70 antibodies, proliferated poorly with gp70 and gp43 but better with other stimuli, This dichotomy between humoral and cellular antigen-specific responses suggests a Th2 immune response in PCM, which may be related to failure to control the infection.

Study of structural surface modified tin oxide membrane prepared by sol-gel route sintered at 400 degrees C

This work describes the chemical modification by Tiron(R) molecules of the surface of SnO2 nanoparticles used to prepare nanoporous membranes. Samples prepared with Tiron(R) content between 1 and 20 wt% and fired at 400 C were characterised by X-Ray Powder Diffraction (XRPD), Extended X-ray Absorption Fine Structure (EXAFS), N-2 adsorption isotherms analysis and permeation experiments. XRPD and EXAFS results show a continuous reduction of crystallite size by increasing the Tiron(R) contents until 7.5 wt%. The control exercised by Tiron(R) modifying agent in crystallite growth allows the fine tuning of the average pore size that can be screened from 0.4 to 4 nm as the amount of grafted molecules decreases from 10 to 0 wt%. In consequence, the membrane cut-off can be screened from 1500 to 3500 g.mol(-1).

Structural biology of membrane-acting peptides: Conformational plasticity of anticoccidial peptide PW2 probed by solution NMR

The bottleneck for the complete understanding of the structure-function relationship of flexible membrane-acting peptides is its dynamics. At the same time, not only the structure but also the dynamics are the key points for their mechanism of action. Our model is PW2, a TRP-rich, cationic peptide selected from phage display libraries that shows anticoccidial activity against Eimeria acervulina. In this manuscript we used a combination of several NMR techniques to tackle these difficulties. The structural features of the membrane-acting peptide PW2 was studied in several membrane mimetic environments: we compared the structural features of PW2 in SDS and DPC micelles, that were reported earlier, with the structure properties in different lipid vesicles and the peptide free in water. We were able to unify the structural information obtained in each of these systems. The structural constraints of the peptide free in water were fundamental for the understanding of plasticity necessary for the membrane interaction. Our data suggested that the WWR sequence is the region responsible for anchoring the peptide to the interfaces, and that this same region displays some degree of conformational order in solution. For PW2, we found that affinity is related to the aromatic region, by anchoring the peptide to the membrane, and specificity is related to the N- and C-termini, which are able to accommodate in the membrane due to its plasticity. (C) 2007 Elsevier B.V. All rights reserved.