Leucine transport from the ventricles and the cranial subarachnoid space in the cat

Abstract— The clearance of [¹⁴C]leucine from the ventricular and cranial subarachnoid space was studied in cats subjected to ventriculo-cisternal and ventriculo-craniosubarachnoidal perfusions. Clearance from both the ventricles and the subarachnoid space was mediated by transport mechanisms with nonsaturable and saturable components. Net clearance from the subarachnoid space was considerably greater than from the ventricles. Analysis of the transport kinetics revealed the affinity constants (K_t) to be comparable for both compartments but transport sites appeared to be more numerous in the subarachnoid space (greater V_max). Proportionally, the amount of [¹⁴C]leucine retained by brain declined as the concentration of leucine in the perfusate was increased. Since at high concentrations the CSF transport systems and presumably cellular uptake were inhibited (self-saturated) it was assumed that the lower brain levels of [¹⁴C]leucine reflected a proportionately greater loss of [¹⁴C]-leucine from the brain interstitial space to blood.     

Endotoxin removal from protein solutions

Endotoxins liberated by gram-negative bacteria are frequent contaminations of protein solutions derived from bioprocesses. Because of their high toxicity in vivo and in vitro, their removal is essential for a safe parenteral administration. A general method for the removal of endotoxins from protein solutions is not available. Methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. Various techniques described in the patent literature are not broadly applicable, as they are tailored to meet specific product requirements. Besides ion-exchangers and two-phase extraction, affinity techniques are applied with varying success. Also, taylor-made endotoxin-selective adsorber matrices for the prevention of endotoxin contamination and endotoxin removal are discussed for this purpose. After giving an overview of the properties of endotoxins and the significance of endotoxin contamination, this review intends to provide an overall picture of the various methods employed for their removal. Avenues are pointed out how to optimise a method with regard to the specific properties of endotoxins in aqueous solution.     

The removal of pyroglutamic acid from monoclonal antibodies without denaturation of the protein chains

Typically, the removal of pyroglutamate from the protein chains of immunoglobulins with the enzyme pyroglutamate aminopeptidase requires the use of chaotropic and reducing agents, quite often with limited success. This article describes a series of optimization experiments using elevated temperatures and detergents to denature and stabilize the heavy chains of immunoglobulins such that the pyroglutamate at the amino terminal was accessible to enzymatic removal using the thermostable protease isolated from Pyrococcus furiosus. The detergent polysorbate 20 (Tween 20) was used successfully to facilitate the removal of pyroglutamate residues. A one-step digestion was developed using elevated temperatures and polysorbate 20, rather than chaotropic and reducing agents, with sample cleanup and preparation for Edman sequencing performed using a commercial cartridge containing the PVDF membrane. All of the immunoglobulins digested with this method yielded heavy chain sequence, but the extent of deblocking was immunglobulin dependent (typically>50%).     

SDS removal from protein by polystyrene beads

The applications of sodium dodecyl sulfate (SDS) to molecular weight determination (1,2) and for the separation of protein subunits (3) have been of immense value in biochemical studies [see Waehneldt (4) for a general review]. The tight stoichiometric binding of SDS to polypeptide chains has proven to be a nuisance if one desires to recover the activity of the isolated polypeptides. Removal of the SDS has been affected by the use of anion exchangers in the presence (5,6) and absence of urea (6). However, the residual levels of SDS or urea are often quite unsatisfactory for further protein studies.We have attempted to adapt the procedure of Holloway for the removal of Triton X-100 by Bio-Beads to the removal of SDS. We chose bovine serum albumin as a test protein since it has well-established strong binding properties for linear-chain fatty acids (7).     

Phagocytosis of erythrocytes in the subarachnoid space at spinal nerve exits

Summary Interdigitating cells (IDC) in the thymus of the spotless starling, Sturnus unicolor, were examined by electron microscopy. They occur principally in the thymic medulla and corticomedullary border. They possess an irregular nucleus and a perinuclear area of cytoplasm, containing most of the membranous organelles, surrounded by a peripheral electron-lucent zone. Clusters of smooth Golgi vesicles and complicated labyrinthine membrane-membrane contacts are the most characteristic cytological features. Birbeck granules are absent. Lymphocytes, plasma cells and even myoid cells can be found embedded in the cytoplasm. Immature elements, intermediate between epithelial-reticular cells and interdigitating cells, are tentatively identified as prointerdigitating cells. The functional significance of IDCs, and their phylogenetic significance in the vertebrate immune system, is discussed.     

Scanning electron microscopy of the subarachnoid space in the dog: evidence for a non-hematogenous origin of subarachnoid macrophages.

Injection of viable BCG into the subarachnoid space of immunized and non-immunized dogs produced a 10-fold increase in the populations of pial free cells. In immunized animals injected three days previously with BCG, stereoscopic SEM revealed that many pial cells had rounded up and were protruding into the subarachnoid space. With continued rounding these cells took on amoeboid characteristics, with shapes that suggested a capacity for cell movement. Internally, these pial cells possessed an increased volume of perinuclear cytoplasm and organelles. Reactive pial cells could be distinguished from macrophages of presumed hematogenous origin on the basis of their surface morphology. These findings suggested that pial cells had the ability to alter their normal structural and behavioral characteristics and to become macrophage-like under these conditions of secondary challenge by BCG.     

Ependymal cyst of the subarachnoid space. Cytologic diagnosis and developmental considerations

Fluid aspirated from a subarachnoid cystic lesion that covered and compressed part of the left frontal lobe was examined cytologically and compared with histologic sections of the cyst wall. The fluid contained epithelial and histiocytelike cell populations. The epithelial cells were tall columnar, occurring singly or in clusters or sheets. Many cells were ciliated and their cytoplasm showed characteristic refractile granules. The differential diagnosis of this rare type of subarachnoid cyst and the mechanism of the development are discussed. Cytologic evaluation of the fluid of the subarachnoid cysts is potentially a more accurate method of classification of these lesions than is random biopsy of the cyst wall. It is of particular importance in cases with a history of growth, in which the progressive expansion results in attenuation of the diagnostic epithelial lining of the cyst.     

Enzymatic removal of oxidized protein aggregates from erythrocyte membranes

Erythrocytes oxidized or aged in the circulation undergo membrane protein aggregation and anti-band 3 autoantibody binding to the cell surface. When human erythrocytes were mildly oxidized in vitro with 0.1 mM Fe(III) at 37 degrees C for 3 h, the aggregation of nonionic detergent C(12)E(8)-insoluble membrane protein and the binding of anti-band 3 IgG to the cell surface were increased. Incubation of membranes isolated from the oxidized cells increased the amount of protein aggregates by 5-fold after 6 h, while incubation for a further 12 h sharply decreased the amount of aggregates. In the presence of diisopropyl fluorophosphate (DFP), however, the increased amount of aggregates was maintained in the subsequent incubation. Western blot analysis of the aggregates using rabbit anti-band 3 showed that band 3 protein aggregates increased in the initial stage of incubation and decreased upon subsequent incubation, whereas the increased band 3 protein aggregates did not subsequently decrease when membranes were incubated in the presence of DFP. Incubation of the oxidized cells at 37 degrees C for 18 h caused reduction of the membrane protein aggregates and the (125)I-anti-band 3 IgG binding to the cell surface, while incubation in the presence of DFP did not cause these reductions. The results suggest that the oxidation-induced cell membrane protein aggregates were probably removed by 80-kDa serine protease, namely, oxidized protein hydrolase (OPH), in the oxidized cell membranes [Fujino et al. (1998) Biochim. Biophys. Acta 1374, 47-54; (1998) J. Biochem. 124, 1077-1085; (2000) Biochim. Biophys. Acta 1478, 102-112], and as a result the increased anti-band 3 binding to the cell surface was reduced.     

6-OHDA-induced ectopia of external granule cells in the subarachnoid space covering the cerebellum

The present report describes the genesis, development and topographical distribution of ectopic cells of the external granular layer in the subarachnoid space covering the rat cerebellum. Following one intracisternal injection to newborn rats of 100 micrograms 6-hydroxydopamine (6-OHDA), the meningeal cells degenerate and are removed by phagocytosis within 24 h post injection (p.i.), leaving the cerebellar cortex without a pia-arachnoid cover. Defects appear in the basal lamina investing the cerebellar cortex 3 to 5 days p.i., and both external granule cells and 'sprouts' from Bergmann-glia endfeet grow into the subarachnoid space. The latter form large, flat glial lamellae and cover extensive areas of the denuded cerebellar surface, although they do not form a glial scar over the exposed neuropil of the cerebellar cortex. The numbers of ectopic external granule cells increase within the subarachnoid space both by proliferation and a continuous efflux of cells from the cerebellar cortex. They migrate, aggregate, and ultimately develop into granule, stellate and basket cells, the morphology of which is indistinguishable from their counterparts in situ; they make specific afferent and efferent connections, both among themselves and with the underlying cerebellar cortex and brainstem. The distribution of ectopic external granule cells and their derivatives is restricted to the anterior vermal fissures and the vermal-hemispheric junctions. The present results indicate that external granule cells and their derivatives are capable of both differentiating normally and surviving in the subarachnoid space if they become associated with glial cells and establish synaptic connections.     

The presence of arachnoiditis affects the characteristics of CSF flow in the spinal subarachnoid space: a modelling study

Syringomyelia is a neurological disorder characterised by high pressure fluid-filled cysts within the spinal cord. As syringomyelia is associated with abnormalities of the central nervous system that obstruct cerebrospinal fluid (CSF) flow, it is thought that changes in CSF dynamics play an important role in its pathogenesis. Using three-dimensional computational models of the spinal subarachnoid space (SAS), this study aims to determine SAS obstructions, such as arachnoiditis, change in CSF dynamics in the SAS. The geometry of the SAS was reconstructed from a series of MRI images. CSF is modelled as an incompressible Newtonian fluid with a dynamic viscosity of 1 mPa s. Three computational models simulated CSF flow in either the unobstructed SAS, or with the SAS obstructed by a porous region simulating dorsal or circumferential arachnoiditis. The permeability of this porous obstruction was varied for the model with dorsal arachnoiditis. The results show that arachnoiditis increases flow resistance in the SAS and this is accompanied by a modest increase in magnitude and/or shift in timing (with respect to the cardiac cycle) of the CSF pressure drop across the region of arachnoiditis. This study suggests that syrinx formation may be related to a change in temporal CSF pulse pressure dynamics.     

A rapid method for removal of detergents from protein solution

A simple and rapid technique is described for the removal of Triton X-100, deoxycholate, and cholate from protein solutions. The method involves a 2-min centrifugation of the sample on a Bio-Beads SM-2 bed prepared in a microcentrifuge tube and is suitable for multiple assays of 0.05- to 0.45-ml samples. Another advantage of this method is the high recovery of proteins without dilution of the sample.