Isolation of a histone-specific proteinase from brain cell nuclei of rats]

A method for isolating histone proteinase of rat brain chromatin is described, including ammonium sulphate fractionation gel filtration on sefacryl 5200, ion exchange chromatography on mono S and affinity chromatography with benzamidine sefarose. The enzyme molecular weight equals 25 kDa. It is purified 15621 times in comparison with initial nuclear extract.     

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.     

Isolation and some chemical properties of oligodendroglia from calf brain

Abstract— The method of Norton and Poduslo (1970) for isolating brain cells has been adapted for the isolation of oligodendroglia from the white matter of calf brain. The cells were obtained in greater than 90 per cent purity, and in a yield of 11 × 10⁶ cells/g of white matter. This number of cells represented a recovery of 11 per cent of the total cells in the tissue and therefore a considerably higher recovery of the original number of oligodendroglia. The average cell contained 5, 2 pg of DNA, 2–0 pg of RNA and 6, 7 pg of lipid. The lipid comprised cholesterol, galactolipid (both cerebroside and sulphatide) and phospholipid in the molar ratio of 1:0, 45:2, 3. Gangliosides were present in a concentration similar to that found in isolated rat neurons, The myelin-specific enzyme, 2′, 3′-cyclic nucleotide 3′-phosphohydrolase, was present at a level nearly equal to that in myelin, and eight-fold higher than the levels in rat neurons or astrocytes. The isolated oligodendroglia differed considerably from isolated astrocytes in size, morphology and chemical composition.     

In vivo measurement of pyridine nucleotide fluorescence from cat brain cortex.

A modification of the methods is described which makes it possible to measure pyridine nucleotide fluorescence from the brain cortex in vivo without interference from movement and hemodynamic artifacts. Movement artifacts were eliminated by the use of a window technique. Fluorescence changes due to changes in hemoglobin oxygenation have been eliminated by measuring fluorescence at an isobestic wavelength of the hemoglobin-oxyhemoglobin reaction. The interference due to changes in red blood cell concentration has been studied by simultaneous measurements of fluorescence and ultraviolet reflection. Hemodilution revealed a linear relationship between the fluorescence from the pyridine nucleotide and reflected ultraviolet light. The ratio between the light absorption changes was approximately unity under the particular optical geometry employed in this study. This method has been used to measure fluorescence changes produced by nitrogen anoxia. The technique is discussed in relation to previous methods and the effects of anoxia are compared to previous findings.     

Isolation and characterization of peroxisomes from the renal cortex of beef, sheep, and cat

The isolation and characterization of highly purified and structurally well-preserved peroxisomes from the renal cortex of different mammalian species (beef, sheep, and cat) is reported. Renal cortex tissue was homogenized and a peroxisome-enriched light mitochondrial fraction was prepared by differential centrifugation. This was subfractionated by density-dependent banding on a linear gradient of metrizamide (1.12-1.26 g/cm3) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at a mean density of 1.225 cm3. Ultrastructural morphometric examination revealed that peroxisomes made up 97 to 98% of the isolated fractions. By biochemical analysis the contamination with marker enzymes of mitochondria and lysosomes was extremely low. The specific activity of catalase was enriched, depending on the species, between 28- and 38-fold over the homogenate. Peroxisome preparations from all three species exhibited a high but varying level of activity for cyanide-insensitive lipid beta-oxidation. In beef and sheep preparations a small amount of esterase activity cosediments with peroxisomes. These peroxisomes show distinct structural membrane associations with smooth elements of ER. Urate oxidase, a marker enzyme for rat liver peroxisomes, is found only in peroxisomes prepared from beef kidney cortex, with sheep and cat preparations being negative. This correlated with the occurrence of polytubular inclusions in the beef kidney peroxisomes. The large size and the angular shape of isolated peroxisomes as well as the presence of paracrystalline matrical inclusions imply that the majority of peroxisomes are derived from the epithelial cells of the proximal tubule of the kidney cortex. The significant differences found in the characteristics of the renal peroxisomes in three different species investigated, demonstrate the remarkable adaptability and plasticity of this organelle.     

Whole cell recordings from brain of adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown. The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.     

Electrical characteristics of neurons in the cat motor cortex

Electrical characteristics of motor cortical neurons were studied in acute experiments on immobilized cats. Values of the input resistances varied from units to tens of megohms (mean 11.11±3.93 MΩ). The threshold current is a hyperbolic function of input resistance of the corresponding neurons and negative correlation was found between the axonal conduction velocity and input resistance. The time constant (τ₀) of the membrane was 7.1±3.46 msec. A time constant τ₁, of 1.65±0.36 msec, could also be distinguished in some neurons. Electrotonic lengths of dendrites of the cortical neurons were calculated by the use of Rall's model: mean 3.66±0.94 (in units of length constant).