Mapping and quantitation of proteins from discrete nuclei and other areas of the rat brain by two-dimensional gel electrophoresis

Heydorn, WE; Creed, G. Joseph; Goldman, David; Kanter, Diane M.; Merril, C R; Dm, Jacobowitz

doi:10.1523/jneurosci.03-12-02597.1983

Cited by 57 publications

(34 citation statements)

References 31 publications

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“…pH gradients of the first-dimension gels were not routinely measured; their approxi mate ranges were 3.5-6.7, and the acidic end of the gel is at the right. Identities of actin (A), pi 5.6, and (3-tubulin (T), pi 5.4-5.5, are based on a comparison with the data of Strocchi et al (1982) and Heydorn et al (1983). Also shown are auto radiographs (panels B-F) of 35S-labeled proteins synthesized in vitro from polysomes of pentobarbital-anesthetized control (8) or hyper thermic (E) rats or from operated manipulated control (C) or ischemic (F) rats (n = 4-5 rats/experimental or control group).…”

Section: Resultsmentioning

confidence: 99%

Synthesis of Heat Shock Proteins in Rat Brain Cortex after Transient Ischemia

Dienel¹,

Kiessling²,

Jacewicz³

et al. 1986

J Cereb Blood Flow Metab

125

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Summary: Cell-free protein synthesis and two-dimen sional gel autoradiography were used to characterize early postischemic protein synthesis in rat neocortex. Severe forebrain ischemia was induced for 30 min (four vessel occlusion model) and followed by 3 h of recircula tion. Polysomes were isolated from the cerebral cortex, translated in vitro in a reticulocyte system, and analyzed by two-dimensional gel electrophoresis. The translation products of postischemic polysomes included a major new protein family (70 kDa) with multiple isoelectric variants that was found to comigrate with the 68-to 70-Energy failure during transient ischemia blocks protein synthesis, but when recirculation occurs, protein synthesis is impaired for at least 12-24 h after restoration of energy stores (Kleihues et aI., 1975;Cooper et al., 1977;Dienel et al., 1980; Nowak et aI., 1985). In contrast to reduced produc Dr. Kiessling's present address is Abteilung Neuropathologie, Pathologisches Institut, Albertstr. 19,7800 Freiburg, F.R.G.Abbreviations used: LSD, lysergic acid diethylamide; mRNA, messenger ribonucleic acid. 505kDa "heat shock" protein synthesized from polysomes of hyperthermic rats. Two other stress proteins (93 and 110 kDa) also appeared to be synthesized in increased amounts after ischemia. A complement of proteins that was indistinguishable from that of controls was also syn thesized after ischemia, indicating that messenger ribonu cleic acid coding for most brain proteins is preserved after ischemia and is bound to polysomes. Key Words: Brain ischemia-Heat shock proteins-Hyperthermia Protein synthesis. other brain proteins . Hyperthermia and other injurious or noxious treatments that in crease ornithine decarboxylase activity in rat brain, liver, and kidney (Dienel and Cruz, 1984) are also known to stimulate the synthesis of heat shock pro teins in rat tissues (brain, liver, heart, adrenals, etc.), in tissue culture cells, and in many other or ganisms (Ashburner and Bonner, 1979;Currie and White, 1981; Cosgrove et aI., 1982; Schlesinger et aI., 1982). In addition, a 71-kDa heat shock protein is synthesized in rat brain, liver, and kidney after carotid ligation of one animal (Currie and White, 1981) and in rat brain slices subjected to the isch emia of decapitation and tissue-slicing injury (White, 1980;1981).The purpose of this study was to determine if ri bonucleic acids (RNAs) coding for heat shock pro teins are being translated shortly (3 h) after post ischemic reperfusion when the rate of protein syn thesis is reduced by >50% and when ornithine decarboxylase activity is just beginning to increase (Dienel et aI., 1985a). We show that disaggregated poly somes derived from 3-h postischemic rat brain contain the messenger RNA (mRNA) coding for the normal complement of brain proteins and, in

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Section: Resultsmentioning

confidence: 99%

Synthesis of Heat Shock Proteins in Rat Brain Cortex after Transient Ischemia

Dienel¹,

Kiessling²,

Jacewicz³

et al. 1986

J Cereb Blood Flow Metab

125

View full text Add to dashboard Cite

show abstract

“…Following the procedure o f O'Farrell [26] with minor modifications for brain tissue [ 14], homogenized tissue proteins from the three brain nuclei were separated by two-dimensional gel electrophoresis. Briefly, tissue samples, approximately 70 pg of protein, were mixed with urea, loaded onto tube gels and separated first by isoelectric point.…”

Section: Two-dimensional Gel Electrophoresismentioning

confidence: 99%

“…The slab gels were fixed and then stained with silver. These procedures are described in detail elsewhere [14,17],…”

Section: Two-dimensional Gel Electrophoresismentioning

confidence: 99%

Quantification of Proteins in Discrete Brain Regions of Androgen-Insensitive Testicular Feminized <i>Tfm</i> Mice

Olsen

Heydorn²,

Rodriguez-Sierra

et al. 1989

Neuroendocrinology

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The effect of the testicular feminization mutation (Tfm) on the concentration of specific proteins in the medial preoptic area (MPO), ventromedial hypothalamus (VMH) and parietal cortex (CX) was examined. Adult Tfm and Swiss-Webster male mice were decapitated, the brains were removed and sectioned. Proteins from the three microdissected areas were separated by two-dimensional gel electrophoresis. Gels were stained with silver and then analyzed by quantitative computerized scanning densitometry. Of the 195 proteins quantified, the Tfm mutation significantly influenced the concentration of 16 proteins measured from gels of MPO tissue, 21 from VMH gels and 11 from CX. Of these, three proteins were affected in all brain regions; and three additional proteins were shown to vary in both MPO and VMH. One protein higher in the MPO and VMH of Tfm mice was identified as the glial fibrillary acidic protein. It is suggested that the proteins influenced by the Tfm mutation are regulated by steroids, most likely androgens. Thus, these proteins may prove to be important in hormone-regulated physiological functions.

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“…The 2-DE procedure of O'Farrell [21) was followed with minor modifications [12,13). In the isoelectric focusing dimension.…”

Section: Polyacrylamide 2-dementioning

confidence: 99%

“…After film development, quantitative analyses of individual protein spots were performed as described previously [ 12,29). Each protein spot was assigned a permanent index number which was used for cataloging data about that protein.…”

Section: Scanning Densitometrymentioning

confidence: 99%

Incorporation of Amino Acids into Proteins of the Hypothalamus of Prepuberal Female Rats after Estradiol Treatment

Rodriguez-Sierra

et al. 1987

Neuroendocrinology

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The arcuate nucleus-median eminence complex (AM) undergoes major structural and functional changes during normal puberty or if exposed to a pulse of estradiol in the prepuberal period. Those changes are expressed by increased synaptogenesis and by a drastic alteration in the feedback control of anterior pituitary gland hormone release. In this study we investigated the effects of estradiol benzoate (EB) on specific proteins in this hypothalamic area. Prepuberal, 25-day-old female rats were administered 10 µg of EB s.c. in oil or sesame oil vehicle. The animals were decapitated either 17 or 42 h after treatment. The brains were removed, blocked and serially sections at 300 µm using a Vibratome. The AM was dissected out and incubated for 6 h in a medium containing ³⁵S-methionine and ³⁵S-cysteine. Proteins from the AM were separated by two-dimensional gel electrophoresis, and the gels were exposed to X-ray film. The resulting autofluorographs were analyzed by scanning densitometry. The results show that the incorporation of labeled amino acids was increased in 10 proteins and decreased in 2 proteins in rats killed 17 h after EB. At 42 h after EB, 6 proteins showed an increased incorporation of amino acids and two proteins showed a decrease. Our results suggest that one or several of these proteins might be involved in the neuroendocrine and structural changes observed in the AM during puberty.

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Mapping and quantitation of proteins from discrete nuclei and other areas of the rat brain by two-dimensional gel electrophoresis

Cited by 57 publications

References 31 publications

Synthesis of Heat Shock Proteins in Rat Brain Cortex after Transient Ischemia

Synthesis of Heat Shock Proteins in Rat Brain Cortex after Transient Ischemia

Quantification of Proteins in Discrete Brain Regions of Androgen-Insensitive Testicular Feminized <i>Tfm</i> Mice

Incorporation of Amino Acids into Proteins of the Hypothalamus of Prepuberal Female Rats after Estradiol Treatment

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