Judit Csejtey

Isolation of metabolically active capillaries from rat brain

MANY substances circulating in the blood do not enter the central nervous system or achieve only limited steadystate concentrations in the brain. To account for these observations, a functional barrier is considered to separate the central nervous system from the vascular elements. Recent studies suggest that this blood-brain barrier may be related to the unique ultra structural anatomy of endothelial cells in small blood vessels of the brain. These cells are circumferentially sealed together by tight junctions, lack fenestrations and are surrounded by astroglial processes (REESE & KARNOVSKY, 1967). Despite this anatomical arrangement, some metabolites appear to be transported across the blood-brain carrier by stereospecific carrier-mediated transport systems (OLDENDORF, 1971). This suggests that transcapillary movement of specific molecules might occur through the cells of the capillary wall. Thus, metabolic study of capillaries isolated from brain may shed light on some of the factors that regulate the permeability of the blood-brain barrier. Recently, several laboratories have reported methods for preparing brain capillary segments (SIAKOTOS et al., 1969; J o o & KARNUCHINA, 1973; ORLOWSKI et al., 1974; BRENDEL et at., 1974). These studies have been limited by a requirement for large quantities of bovine brain or have not presented sufficient criteria of purification and metabolic activity. In this report, we describe a novel method for isolating brain capillaries from rat brain which are suitable for study in uitro. They appear to be free of neuronal and glial contamination, contain characteristic marker enzymes in high concentration, and are capable of carrying out glucose transport and oxidation. Brain capillaries were isolated in physiologic buffer without the addition of digestive enzymes in order to minimize damage to cellular metabolism and membrane transport. The cerebral cortices from 20 one-month-old rats were minced in 200ml of oxygen saturated Ringer’s solution containing 1.2 mM-MgCl,, 15 mwHepes buffer (pH 7.4), 5mM-glucose and 1% fraction V bovine serum albumin. The mince was passed serially through 670, 335 and 116pm nylon meshes and then centrifuged at loo0 x g for 10min. In order to remove cellular debris and myelin the pellet was suspended in 100 ml of the Same buffer now

PROTEOLYTIC ACTIVITY AND BASIC PROTEIN LOSS IN AND AROUND MULTIPLE SCLEROSIS PLAQUES: COMBINED BIOCHEMICAL AND EIISTOCHEMICAL OBSERVATIONS

Abstract— This combined histochemical and biochemical study has shown that acid proteinase activity (PH 3.5) is increased around histologically‐defined active plaques of multiple sclerosis (MS). Biochemical estimation showed that the enzyme is more active in most samples of ‘normal’ white matter in MS than in controls. A gradient of enzyme activity was observed: control white matter‐white matter distant from plaqueclose white matter‐edgsplaque. Both electrophoretic and histochemical techniques revealed a reduction or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base‐line of thin‐layer chromatoplates were shown to be predominantly phosphoinositides combined with encephalitogenic protein

Increased protease activity and changes in basic proteins and lipids in multiple sclerosis plaques

Abstract Various sections of multiple sclerosis (MS) brains, including the plaques and the edge of the plaques, have been studied. The lipids were determined by quantitative thin-layer chromatography and the values for cerebroside, cholesterol, and cholesterol ester were found to be in agreement with previous workers. There was little difference between MS and normal brains in their phospholipid content. The significant finding was the demonstration that with a partial or total destruction of the encephalitogenic protein around the plaques, a 4-fold increase in acid proteinase occurred. Previous reports ( Chao and Einstein 1968; Einstein et al. 1968a; 1970) have shown that the encephalitogenic protein is converted in vivo and in vitro to an active polypeptide of smaller molecular weight. These suggest, although they do not necessarily prove, that the acid proteinase which is a lysosomal enzyme(s) plays a role in MS by releasing the encephalitogen as such a polypeptide into the circulation and in this way initiating an immunological reaction.

Biochemical maturation of the central nervous system. II. Protein and proteolytic enzyme changes.

Abstract In an earlier study, the maturation of the central nervous system was followed by analyzing the lipids 5 . Since in the myelin the lipids are complexed with proteins, we have extended our studies to the proteins of the brain, cord, and myelin and have followed the development from embryonic to mature rabbit brains. It was found that the brain weights are quite uniform in the same age group, and that the greatest increase occurs between 14 and 60 days. For assaying protein changes, the technique of acrylamide electrophoresis was found eminently suitable. It was applied in two forms: one, after complete solubilization of the myelin for demonstration of the proteolipids and the encephalitogenic proteins; two, after acid extraction for the demonstration of all basic proteins, including the encephalitogenic protein. This basic, encephalitogenic protein appears later than the proteolipids, although both are constitutents of the myelin. The embryonal brain contains a fast-moving basic protein, most likely a histone, which is either not present or in minimal quantity after birth. Brain and cord myelin proteins were compared and it was found that the sequence of protein synthesis in the early stage differed in that the encephalitogenic protein appeared earlier in the cord than in the brain.

Brain, CSF, and tumor pharmacokinetics of ?-difluoromethylornithine in rats and dogs

SummaryWe have determined the pharmacokinetic parameters for diffusion of α-[5-14C]-difluoromethylornithine (DFMO) from blood to brain, blood to cerebrospinal fluid (CSF), 9L rat brain tumor to adjacent brain, and blood to the subcutaneously-implanted 9L tumor in rats, and within the CSF of beagle dogs. DFMO diffusion across the blood-brain and blood-CSF barriers is quite restricted in both rats and dogs, but diffusion across the defective capillary system of both subcutaneous and intracerebral 9L tumors in rats is not. Under steady-state plasma conditions in rats, uptake of DFMO by the intracerebral 9L tumor and diffusion from tumor 5–6 mm into adjacent brain is not restricted; tissue/plasma ratios were approximately 1. Therapeutic efficacy will therefore not be limited by transport of DFMO into tumor or to the extracellular environment of tumor.

Suppression of experimental allergic encephalomyelitis by chemically modified encephalitogen

Abstract The results of enzymatic and chemical degradation studies of the basic myelin protein suggested that tryptophan is essential for encephalitogenic activity in the guinea pig. The object of the present investigation was to modify the protein through its tryptophan residue in such a way that it becomes non-encephalitogenic, yet suppresses experimental allergic encephalomyelitis (EAE). Accordingly, the single tryptophan residue of the protein was modified with specific reagents such as 2-hydroxy-5-nitrobenzylbromide (HNB-BR), 2-nitrophenylsulfenyl chloride (NPSCl) and performic acid. Injection of the modified proteins did not result in neurological disease, although some guinea pigs did exhibit histological changes in the brain. Starting 3 days after injection with the active inoculum, the treatment with the modified proteins was carried out for four weeks, using a total of 900 μg og the modified proteins. The guinea pigs treated with HNB and performic acid oxidized protein did not show neurological signs of the disease, but from the NPSCl group, two out of 10 developed EAE, although at a later data compared with those guinea pigs which were injected with the unmodified encephalitogen. The results not only confirm previous findings that intact tryptophan in the protein is a prime requirement for the production of EAE in guinea pigs, but also provide evidence that tryptophan is not essential for suppression of the disease. The conclusion we may draw from these studies is that the encephalitogenic basic myelin protein has two active sites: one which contains tryptophan required for the paralytic effect, the other without tryptophan which provides protection.

Relationship of Octanol/Water Partition Coefficient and Molecular Weight to Cellular Permeability and Partitioning in S49 Lymphoma Cells

We have used modified standard methods and derived new formulae to quantitate cell permeability (P), cell/media partitioning (λ), and intracellular sequestration or binding rate constants (m) for cultured S49 murine lymphoma cells in suspension. Using 15 standard compounds and anticancer drugs, we found quantitative relationships among log P, log PO (octanol/pH 7.4 buffer partition coefficient), and molecular weight (MW) such that logP = −4.5 + 0.56log (PO(MW)−1/2). A good correlation among P, λ, and MW was also determined with λ = 0.67 + 5890 gm1/2 cm−1 sec (P (MW)1/2). These studies show that there is a strong partitioning (λ) dependence to molecular weight and permeability that can be predicted even for known carrier-transported and biotransformable compounds. Furthermore, results of this study show that the slope of the plot of permeability and lipophilicity is not necessarily unity as has been postulated from the results of other studies.