Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus

Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of γ-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in γ-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in γ-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed γ-glutamyl transpeptidase activity localized in the capillary walls.     

Comparative studies on membranes from bovine choroid plexus and rat kidney cortex.

Comparative subcellular fractionation studies on rat kidney and bovine choroid plexus using differential centrifugation and free flow electropheresis were undertaken because of the morphological and functional similarities of the epithelial cells of both tissues. The activities of three enzymes commonly used as markers for brush border membranes in kidney were measured in fractions of each tissue. γ-Glutamyl transpeptidase, alkaline phosphatase, and 5'-nucleotidase copurified in membrane fractions of renal cortex collected by differential centrifugation. Application of a similar fractionation procedure to choroid plexus gave relatively similar results, except for alkaline phosphatase, the yield of which was substantially reduced in a fraction enriched with two marker enzymes. Further fractionation of γ-glutamyl transpeptidase and alkaline phosphatase activities in these membrane fractions was achieved using free flow electropheresis. The two enzymes from kidney exhibited discrete peaks with a small separation, while the electropheretic pattern of γ-glutamyl transpeptidase from choroid plexus was biphasic. Alkaline phosphatase was observed to migrate with the more basic γ-glutamyl transpeptidase peak.     

The choroid plexus removes beta-amyloid from brain cerebrospinal fluid

beta-Amyloid (Abeta) concentration in the cerebrospinal fluid (CSF) of the brain may be regulated by the choroid plexus, which forms a barrier between blood and brain CSF. Abeta uptake from CSF was determined as its volume of distribution (V(D)) into isolated rat choroid plexus tissue. The V(D) of [125I]Abeta1-40 was corrected by subtraction of the V(D) of [14C]sucrose, a marker for extracellular space and diffusion. Abeta uptake into choroid plexus was time and temperature dependent. Uptake of [125I]Abeta was saturable. Abeta uptake was not affected by addition of transthyretin or apolipoprotein E3. In studies with primary culture monolayers of choroidal epithelial cells in Transwells, Abeta permeability across cells, corrected by [(14)C]sucrose, was greater from the CSF-facing membrane than from the blood-facing membrane. Similarly, cellular accumulation of [125I]Abeta was concentrative from both directions and was greater from the CSF-facing membrane, suggesting a bias for efflux. Overall, these results suggest the choroid plexus selectively cleanses Abeta from the CSF by an undetermined mechanism(s), potentially reducing Abeta from normal brains and the brains of Alzheimer's disease patients.     

Isolation of a highly enriched sarcolemma membrane fraction from canine heart.

A highly enriched sarcolemma preparation was isolated by differential centrifugation of a canine ventricular homogenate followed by centrifugation of a membrane fraction layered over 22% (w/v) sucrose. Ouabain binding, ouabain-sensitive potassium phosphatase activity and 5'-nucleotidase activity were enriched 19--27 fold over the homogenate whereas Ca2+-ATPase and succinate dehydrogenase activities were 0.75 and 0.36, respectively, of that for the homogenate. The isolation procedure was relatively rapid and yielded about 2.0 mg protein/100 g of ventricular muscle. The highest salt concentration used in the procedure was 0.6 M KCl and no detergents were employed. Initial characterization studies suggested that the sarcolemma-enriched fraction consists predominantly if not totally of freely permeable membrane vesicles and that the sarcolemma does not manifest a Ca2+-ATPase activity, at least within the limits of the assay procedures employed. This preparation was concluded to be about 1.5- to 4-fold more highly enriched with sarcolemmal markers than preparations obtained by previously published procedures. Accordingly, the preparation provides an improved basis for the probe of calcium movements that occur across the sarcolemma in association with the excitation-contraction-relaxation sequence of the mammalian myocardial cell.     

D-aspartate oxidase in mammalian brain and choroid plexus

Abstract— Synaptosomes from guinea-pig cerebral cortex contain a fetuin: sialyl glyco-protein: glycosyl transferase; evidence is presented which indicates that both a sialyl transferase; evidence is presented which indicates that both a sialyl transferase and endogenous acceptors were located in the synaptosome ‘ghost’ fractions. Following solubilization of synaptosomes with Triton X-100 and the use of fetuin minus NANA as acceptor, 25 per cent of the transferase was recovered after centrifugation and column chromatography on Sephadex G-100 and G-200 with a 64·0-fold purification. The enzyme had a pH optimum of 6·3, required no divalent metal cation for activity, and exhibited high activity with either fetuin minus sialic acid, prothrombin minus sialic acid, Tamm-Horsfall glycoprotein minus sialic acid, or orosomucoid minus sialic acid as acceptor; neither BSM nor PSM minus NANA functioned as an effective acceptor. The fetuin:sialyl transferase using fetuin minus sialic acid and CMP-sialic acid as substrates a and b, respectively, gave the following kinetic constants when using the Cleland bisubstrate model: K_a= 35μM; K_b= 3 μM; K_ia, = 25 μM; K_ib= 25μM; and V₁= 92 pmoles. min^?1.mg^?1 of protein. The following divalent cations inhibited the reaction: Ba²⁺ > Hg²⁺ > Pb²⁺ > Cu²⁺.     

Isolation and characterization of two male-specific DNA fragments from the bovine gene

Two independent male-specific DNA fragments were isolated from the bovine genome by the technique of cloning by deletion enrichment. Each is represented in the order of one to five copies per haploid genome. Neither fragment was found to be polymorphic between two individuals of different breeds with 12 different restriction enzymes. A 'zoo blot' demonstrated that one fragment had a closely related sequence in the sheep genome, although no other hybridizing sequences were detected in a variety of animal DNAs tested with both probes. The DNA sequence of each fragment is presented; one fragment has a continuous open reading frame and a potential Z-DNA forming region.     

The choroid plexus and the paradox of interferons in the aging brain

The choroid plexus (CP) function is largely viewed as the source of cerebrospinal fluid (CSF) and as a barrier between the blood and the CSF. Other functions of the CP are becoming increasingly recognized as in the recent publication by Baruch et. al. who demonstrate increased expression of interferon type I mRNA signature (irf7, ifnß and ifit1) in CP of aged brains compared to younger brains, whereas interferon type II dependent genes (icam1, cxcl10, and ccl17) are reduced in the aging CP. The authors speculate an IFN-dependent mechanism that plays a role in the aging process and cognitive decline. This short communication summarizes the findings by the authors and highlights the seemingly paradoxical roles of IFN type I and type II in neuroinflammation.     

Isolation and characterization of calcineurin from bovine brain

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biondi bodies in the choroid plexus epithelium of the human brain

Summary Scanning electron microscopy (SEM) was used to examine choroid plexuses in the brain of two human adults aged 44 and 46, respectively, and 12 older subjects from 67 to 98 years of age. It was possible to obtain a three-dimensional view of the ring-like structures (Biondi bodies) located in the cytoplasm of choroid plexus epithelial cells in the older-age group. The filaments forming the rings were clearly visible. No such structures were found between epithelial cells. The intracellular location of the Biondi bodies and their state of preservation compared to other cytoplasmic elements suggest that they may have a destructive effect on epithelial cells of choroid plexuses. The same material was examined by transmission electron microscopy (TEM); the results obtained were in full agreement with the evidence obtained with SEM.     

Choline uptake across the ventricular membrane of neonate rat choroid plexus

The uptake of[³H]choline from thecerebrospinal fluid (CSF) side of the rat neonatal choroid plexus wascharacterized in primary cultures of the choroidal epithelium grown onsolid supports. Cell-to-medium concentration ratios were ~5 at 1 minand as high as 70 at 30 min. Apical choline uptake was facilitated; theK_m was ~50µM. Several organic cations (e.g., hemicholinium-3 and N¹-methylnicotinamide)inhibited uptake. The reduction or removal of externalNa⁺ or the addition of 5 mM LiClhad no effect on uptake. However, increasing externalK⁺ concentration from 3 to 30 mMdepolarized ventricular membrane potential (70 to 15 mV)and reduced uptake to 45% of that for the control. Treatment with 1 mMouabain or 2 mM BaCl₂ reduced uptake 45%, and intracellular acidification reduced uptake to ~90%of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled toNa⁺ influx but is sensitive toplasma membrane electrical potential.