Wolff M. Kirsch

Bulk Isolation in Nonaqueous Media of Nuclei from Lyophilized Cells

Intact lyophilized nuclei are obtainable from a variety of tissues, either in situ or in culture, by freezing at -156�C, drying at -25�C, and mechanical disassociation in glycerol at 2�C. Centrifugal separation of nuclei is accomplished in an 85 : 15 by volume mixture of glycerol and 3-chloro-1,2 propanediol at 2�C. The method gives homogeneous nuclear preparations in high yield with preservation of labile and water-soluble constituents.

Immunocytochemical localization of S-100 protein in neurons and glia of hamster cerebellum.

Though the biochemical properties of S-1G0 protein have been extensively characterized13 16, the role of this unique molecule in the function of the nervous system remains essentially speculative. Histochemical attempts to ascertain the cytological distribution of this highly acidic protein have been frustrated by ease of solubilization in conventional tissue fixatives as well as limitations of previously applied histochemical methodology. Cytological localization of S-1(30 protein by the immunofluorescence method as visualized by darkfield microscopy suggest the molecule to be situated predominantly in glial cells as well as other neural structures 5, 8,23,24. The immunofluorescence technique for identification of tissue antigens has well known limitations which have been discussed elsewhere 15. This study describes the use of the peroxidase-labeled antibody method for the localization of S-1C0 protein in the tissue sections of hamster cerebellum. Advantages of this technique for localization of tissue antigens have been described in a previous publication is. Cerebellums of adult golden hamsters were removed immediately after decapitation and frozen with a dry ice-acetone mixture after embedding in O.C.T. (Ames Co.). Six-micron-thick sagittal sections cut on a cryostat were air dried and then subsequently fixed in cold acetone for 5 rain (temperature 4 °C). After washing for 15 min in phosphate buffered saline (PBS, without Ca 2+ and Mg 2+) sections were covered with antiserum to beef S-lC0 protein, which were obtained through the generosity of Dr. Blake Moore. Sheep gamma globulin against rabbit IgG was commercially obtained (Cappel Laboratories, Inc.) and conjugated with horseradish peroxidase by the method of Kawaoi and Nakane 9. In brief, the conjugation procedure is as follows: 5 mg of horseradish peroxidase (Sigma Type VI) is dissolved in 1.0 ml of freshly prepared 0.3 M sodium bicarbonate, 0.1 ml of 1 ~ fluorodinitrobenzene (Sigma) in absolute ethanol is mixed with the above solution, 1.0 ml of 8.04 M sodium periodate in distilled water added and incubated for 30 min at room temperature. One ml of 0.16 M ethyleneglycol is then added, mixed for 1 h and the solution dialyzed against

Glycolytic metabolites and co-factors in human cerebral cortex and white matter during complete ischemia

Abstract Human cerebral cortex and white matter obtained from patients with intracranial tumors were incubated ischemically for varying periods of time extending to 4 h in order to determine the order and rate of recruitment of energy reserves. An estimate of metabolic rate (∼P flux) was calculated on the basis of the disappearance rate of substrates with known energy potential ( P-creatine , ATP, glucose and glycogen). Extrapolation of this data to the in vivo status of anesthetized human brain gives predicted values for cerebral respiratory rate and glucose consumption which are in substantial agreement with that estimated by other techniques. No significant difference in the metabolic rate of human grey and white matter exposed to anesthetic agents could be detected. The order of recruitment of energy reserves in the ischemic cerebral tissue was P -creatine, ATP, glucose, with glycogen expended last at much slower rate. The rate of glycolysis increased by at least 10-fold in the ischemic tissue and appeared to be due primarily to facilitation at the phosphofructokinase and hexokinase steps of the Embden-Meyerhof pathway.

THE FINE STRUCTURAL LOCALIZATION OF S-100 PROTEIN IN RODENT CEREBELLUM

This report describes the first fine structural locations of S-100 protein in the hamster cerebellum. Innovations in tissue fixation as well as tracer preparation enable localization of S-100 protein to several critical cellular and subcellular sites. Certain neuronal elements, Purkinje cells, contain S-100 protein in both cytoplasm and nucleus. Specific membrane structures including plasma membranes and the nuclear membranes of Purkinje cells are strikingly positive. Localization of S-100 protein to the pericapillary astrocytic endfeet as well as regions of the synapse are of particular biological interest. The clear nuclear localization of the molecule suggests a role in the genetic expression of the developing mammalian brain.

Chronic lactic acidosis in an adult: A new syndrome associated with an altered redox state of certain NAD/NADH coupled reactions

Abstract Chronically elevated blood lactic acid, pyruvic acid and increased L:P ratios have been found in a twenty-eight year old woman with episodic acidosis. The patient has no other associated disease. Alcohol ingestion increases the hyperlacticacidemia and exacerbates the patients symptoms of weakness and easy fatiguability. Moderate exercise increases blood lactic acid levels from 3,1 to 10.2 μM per ml and lowers arterial blood pH from 7.4 to 7.26. Hyperuricemia is present due to depressed uric acid clearance. Certain NAD/NADH coupled metabolic reactions are clearly shifted towards the reduced state (lactate/pyruvate, α-glycerophosphate dihydroxyacetone phosphate and galactose-glucose interconversion). Skeletal muscle and liver demonstrate normal total NAD/NADH content and partition of these pyridine nucleotides. Four members of the patients maternal family have an abnormal lactate response to the combination of alcohol ingestion and exercise, suggesting that this defect may be an inherited disorder.

γ-Carboxyglutamic acid in eukaryotic and prokaryotic ribosomes☆

Abstract γ-carboxyglutamic acid (GLA) has been assayed in ribosomes of wheat germ and E. coli, and in E. coli ribosomal sub-units, by amino acid analysis of total protein. Results, as nMoles GLA/mg protein, were 18.1 (wheat germ), 58.4 (E. coli), 39.6 and 81.5 (E. coli 30S and 50S sub-units, respectively.) Results for wheat germ and previously reported mammalian ribosomes were highly similar. Hence the level of GLA in eukaryotic ribosomes appears to be approximately constant, but low compared to bacterial (E. coli) ribosomes.

The occurrence of γ-carboxyglutamic acid in mammalian ribosomes☆

Abstract γ-carboxyglutamic acid (GLA) has been identified and estimated in mammalian ribosomes (mouse, rat, man, cultured cells) by amino acid analysis of total extracted protein. Assay results varied from 5.7–19.1 nMoles GLA/mg protein (6.0–9.9 residues GLA/1000 residues GLU), depending on purity and origin of ribosomes. GLA was not eliminated by purification. GLA in mouse hepatoma ribosomes was increased 3-fold upon preparation of puromycin-dissociated sub-units and sedimentation on gradients containing 1.0 M KCl. This tightly bound GLA (17.7 nMoles/mg sub-unit protein) could be present in one or more ribosomal proteins incorporated in the nucleoli, or in GLA-containing proteins acquired in the cytoplasm.

Loss of hepatoma ribosomal RNA during warfarin therapy

40–50% decreases in cytoplasmic ribosomal RNA were observed in mouse hepatoma implants, but not livers, after 4–5 daily warfarin injections. Similar treatment greatly depressed rates of invivo14C-orotate incorporation into hepatoma ribosomal RNA in the cytoplasm. Labelling of mature 18S and 28S RNA in the nucleus appeared to be unaffected. Possible mechanisms for this warfarin effect are briefly discussed.

Thoughts on the Biology and Therapy of Malignant Gliomas

Further specifying SZENT-GYORGI’s definition of cancer (1), a malignant glioma can be considered as a tumor mass composed of glial cells manifesting disordered proliferation. We remain ignorant as to what composes and comprises order in either a normal or malignant glial cell. Furthermore, great uncertainty surrounds the molecular mechanisms controlling the growth and replication of malignant glial cells, and whether cell division in normal glia is identical to mitotic events in better studied systems. If true, as WATSON has stated, that ability to effectively treat a disease depends upon our ability to understand the perturbation of molecules underlying the disorder, it is no surprise that the past record of accomplishment with malignant gliomas leaves a great deal to be desired (2). Despite this fundamental lack in basic scientific information, there are certain undeniable signals that real progress is about to be recorded in the management of malignant gliomas using an empirical approach. In a cogent analysis of predictive factors for effective chemotherapy of human cancer, SKIPPER (3) and ZUBROD (4) have shown that two objective parameters serve to herald therapeutic success. These signals, either in humans or experimental animals, consist of an increased incidence of remissions as well as a longer duration of remission. It is most likely that we can expect no dramatic or sudden breakthroughs, but must accept the inevitability of gradual progress in planned, persistent drug testing, armed with a better understanding of both cell kinetics and therapeutic agent scheduling as exemplified by the recent work of TATOR and WASSENAAR (5). Excellent experimental models for cerebral gliomas are now available, and these systems have provided evidence for tumor control, but not cure, with various drug protocols.

An assay for organic phosphorus fractions in microgram quantities of tissue

Abstract A fluorometric technique for the determination of acid-soluble phosphorus, lipid phosphorus, and nucleic acid phosphorus in 0.5 to 1.0 μg quantities of tissue is described. The assay is based on the stoichiometric generation of NADPH from P i by the use of purified crystalline enzymes, a feature which confers the advantage of extension to even greater sensitivity by application of the “NADPH cycling” system. Assays of mouse brain and mouse brain tumor were performed at both the microgram and milligram level and compared. The fluorometric assay offers about fivefold increase in sensitivity with a sacrifice of analytical accuracy of approximately 6% (4% vs. 10%) when compared to an established spectrophotometric procedure.