Concentrations of Amino Acids in Extracellular Fluid After Opening of the Blood-Brain Barrier by Intracarotid Infusion of Protamine Sulfate

Abstract: This article evaluates the influence of an opening of the blood-brain barrier (BBB) on compounds in brain extracellular fluid. The concentrations of amino acids and some other primary amines were determined in dialysates sampled from the right parietal cortex of rats before and after an intracarotid infusion of protamine sulfate. Extravasated plasma proteins were visualized by Evans blue/albumin and immunohistochemistry. CSF albumin— an indicator of blood-CSF barrier opening—was quantified with immunoelectrophoresis. The brains were macroscopically edematous after 10 mg but not after 5 mg of protamine sulfate. The higher dose led to a 50% death rate. The concentrations of amino acids did not change 10 min after the BBB opening. No significant alterations in the amino acid concentrations were observed after the lower dose. The concentrations of glutamate, aspartate, GABA, glycine, taurine, and phosphoethanolamine increased significantly within 50–80 min after the infusion of 10 mg of protamine sulfate. CSF albumin levels were significantly increased 1 h after infusion. We conclude that a dysfunction of the BBB, of a degree known to induce brain edema (10 mg of protamine sulfate), significantly increases the extracellular concentration of excitatory amino acids, GABA, taurine, and phosphoethanolamine in the extracellular space.     

Host selection as a downstream strategy: polyelectrolyte precipitation of beta-glucuronidase from plant extracts.

Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors.     

Early Life Stress and Physical and Psychosocial Functioning in Late Adulthood

Background

Severe stress experienced in early life may have long-term effects on adult physiological and psychological health and well-being. We studied physical and psychosocial functioning in late adulthood in subjects separated temporarily from their parents in childhood during World War II.

Methods

The 1803 participants belong to the Helsinki Birth Cohort Study, born 1934–44. Of them, 267 (14.8%) had been evacuated abroad in childhood during WWII and the remaining subjects served as controls. Physical and psychosocial functioning was assessed with the Short Form 36 scale (SF-36) between 2001 and 2004. A test for trends was based on linear regression. All analyses were adjusted for age at clinical examination, social class in childhood and adulthood, smoking, alcohol intake, physical activity, body mass index, cardiovascular disease and diabetes.

Results

Physical functioning in late adulthood was lower among the separated men compared to non-separated men (b = −0.40, 95% confidence interval [95% CI]: −0.71 to −0.08). Those men separated in school age (>7 years) and who were separated for a duration over 2 years had the highest risk for lower physical functioning (b = −0.89, 95% CI: −1.58 to −0.20) and (b = −0.65, 95% CI: −1.25 to −0.05), respectively). Men separated for a duration over 2 years also had lower psychosocial functioning (b = −0.70, 95% CI: −1.35 to −0.06). These differences in physical and psychosocial functioning were not observed among women.

Conclusion

Early life stress may increase the risk for impaired physical functioning in late adulthood among men. Timing and duration of the separation influenced the physical and psychosocial functioning in late adulthood.     

Components of metabolic syndrome predicting diabetes: no role of inflammation or dyslipidemia

Objective: The diagnostic criteria and the clinical usefulness of the metabolic syndrome (MetSy) are currently questioned. The objective was to describe the structure of MetSy and to evaluate its components for prediction of diabetes type 2 (T2DM). Research Methods and Procedures: This was a case‐referent study nested within a population‐based health survey. Among 33,336 participants, we identified 177 initially non‐diabetic individuals who developed T2DM after 0.1 to 10.5 years (mean, 5.4 years), and, for each diabetes case, two referents matched for sex, age, and year of health survey. Baseline variables included oral glucose tolerance test, BMI, blood pressure, blood lipids, adipokines, inflammatory markers, insulin resistance, and β‐cell function. Exploratory and confirmative factor analyses were applied to hypothesize the structure of the MetSy. The prediction of T2DM by the different factors was evaluated by multivariate logistic regression analysis. Results: A hypothetical five‐factor model of intercorrelated composite factors was generated. The inflammation, dyslipidemia, and blood pressure factors were predicitive only in univariate analysis. In multivariable analyses, two factors independently and significantly predicted T2DM: an obesity/insulin resistance factor and a glycemia factor. The composite factors did not improve the prediction of T2DM compared with single variables. Among the original variables, fasting glucose, proinsulin, BMI, and blood pressure values were predictive of T2DM. Discussion: Our data support the concept of a MetSy, and we propose five separate clusters of components. The inflammation and dyslipidemia factors were not independently associated with diabetes risk. In contrast, obesity and accompanying insulin resistance and β‐cell decompensation seem to be two core perturbations promoting and predicting progression to T2DM.     

Ex vivo analysis of leukocyte hydrogen peroxide production using a bi-plate model in mice

Implantation of artificial materials is followed by inflammation and wound healing, where phagocytic cells play an important role. The mechanisms whereby the implant surface may elicit and modulate leukocyte functions in vivo are not understood, partly due to the technical difficulties of examining the local inflammatory events in vicinity of the material-tissue interface with conventional biochemical and immunological techniques. In the present study a newly developed biplate implant was inserted subcutaneously in the mouse. Leukocytes from the local inflammatory exudate and leukocytes associated to the surface of the implants were retrieved after 1 and 6 days and separately assayed with respect to hydrogen peroxide (H₂O₂) production ex vivo. Implantation caused a local accumulation of predominantly mononuclear cells in the surrounding subcutaneous tissue. The H₂O₂ production was found to be low in both the subcutaneous exudate and the implant-associated leukocytes, irrespective of implant material and implantation times. However, ex vivo-stimulation with phorbol myristate acetate (PMA) caused an enhanced H₂O₂ production. These observations show that biplate implants do not maximally activate the oxidative metabolism of the recruited leukocytes. The exudate leukocytes were more responsive to PMA stimulation in comparison with implant-associated leukocytes, indicating that properties of the implant surface and possibly surface-associated proteins could modify the responsiveness of the phagocytic cells at the implant site. Our results suggest that the present biplate model may be suitable for further studies on local production of oxygen metabolites and function of leukocytes at implanted biomaterials. © 1996 Wiley-Liss, Inc.     

The poultry red mite,Dermanyssus gallinae,a potential vector of Erysipelothrix rhusiopathiae causing erysipelas in hens

Erysipelas is a bacterial disease caused by Erysipelothrix rhusiopathiae, which may infect swine as well as several other species of mammals and birds, including domestic fowl. In poultry, erysipelas may cause sudden high mortality due to septicemia. This communication describes the first isolation of E. rhusiopathiae from the haematophagous poultry red mite, Dermanyssus gallinae DeGeer (Acari: Dermanyssidae), that was collected on three farms where hen erysipelas was diagnosed. The bacteria were isolated from the integument as well as from the interior of the mites. Serotypes 1a and 1b of E. rhusiopathiae found in the mites corresponded with those isolated from the diseased birds. These findings imply that D. gallinae is a potential vector of E. rhusiopathiae. The current lack of effective measures to control D. gallinae causes recurring mite problems in poultry facilities once afflicted by this parasite. Consequently, mites containing E. rhusiopathiae may act as reservoir hosts of this bacterium, allowing it to persist in the poultry house between flock cycles as a source of infection for the replacement pullets. The zoonotic potentials of both E. rhusiopathiae and D. gallinae should also be considered.     

Orthogonal projections to latent structures as a strategy for microarray data normalization

Background  

During generation of microarray data, various forms of systematic biases are frequently introduced which limits accuracy and precision of the results. In order to properly estimate biological effects, these biases must be identified and discarded.     

Inhibition of Mitogen-Activated Kinase Signaling Sensitizes HeLa Cells to Fas Receptor-Mediated Apoptosis

The Fas receptor (FasR) is an important physiological mediator of apoptosis in various tissues and cells. However, there are also many FasR-expressing cell types that are normally resistant to apoptotic signaling through this receptor. The mitogen-activated protein kinase (MAPK) signaling cascade has, apart from being a growth-stimulating factor, lately received attention as an inhibitory factor in apoptosis. In this study, we examined whether MAPK signaling could be involved in protecting FasR-insensitive cells. To this end, we used different approaches to inhibit MAPK signaling in HeLa cells, including treatment with the MAPK kinase inhibitor PD 98059, serum withdrawal, and expression of dominant-interfering MAPK kinase mutant protein. All of these treatments were effective in sensitizing the cells to FasR-induced apoptosis, demonstrating that MAPK indeed is involved in the control of FasR responses. The MAPK-mediated control seemed to occur at or upstream of caspase 8, the initiator caspase in apoptotic FasR responses. Transfection with the constitutively active MAPK kinase abrogated FasR-induced apoptosis also in the presence of cycloheximide, indicating that the MAPK-generated suppression of FasR-mediated apoptotic signaling is protein synthesis independent. In cells insensitive to FasR-induced apoptosis, stimulation of the FasR with an agonistic antibody resulted in significant MAPK activation, which was inhibited by PD 98059. When different cell types were compared, the FasR-mediated MAPK activation seemed proportional to the degree of FasR insensitivity. These results suggest that the FasR insensitivity is likely to be a consequence of FasR-induced MAPK activation, which in turn interferes with caspase activation.