Protein patterns in various malignant human brain tumors by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis with silver staining was used to study protein patterns in various malignant human brain tumors obtained at surgery. These samples included 20 high-grade astrocytomas (anaplastic astrocytomas and glioblastomas), one low-grade astrocytoma, six juvenile astrocytomas, four ependymomas, and five medulloblastomas. Histological correlates of the sampled tissue were carefully established prior to micropunch sampling. The molecular weight range of these gels was 14,000 to 100,000, and the isoelectric points ranged from 4.7 to 7.0. Proteins that have been identified include albumin, actin, tubulin, glial fibrillary acidic protein, vimentin, glutamic oxaloacetic transaminase, neuron-specific enolase, and the beta-subunit of the guanine nucleotide regulatory proteins. Each type of tumor was found to have a characteristic protein profile that set it apart from the other tumors studied. By providing a convenient tool for the display of a wide spectrum of tumor markers in a single study, two-dimensional gel electrophoresis protein profiles may be useful as diagnostic and prognostic adjuncts. Furthermore, several protein spots that were not noted in normal human cortex were identified in the various tumor gels. Antibodies can be raised against some of these tumor-associated proteins, and their further characterization could provide valuable insights into the biology of these tumors.

Effect of chronic treatment with clorgyline on the relative concentration of specific proteins in the hippocampus and parietal cortex of the rat.

The effect of the chronic administration of clorgyline, a type A inhibitor of monoamine oxidase, on the relative concentration of proteins from the brain of the rat was examined by analysis of two-dimensional electrophoretic gels. The results from this study showed that the administration of clorgyline for 3 weeks produced a significant elevation in the relative concentration of two proteins in the parietal cortex (mol. wt 23,000 and 30,000) and one protein in the hippocampus (mol. wt 25,000). In contrast, the relative concentration of three proteins (mol. wt 31,000, 42,000 and 45,000) was significantly reduced in the parietal cortex by chronic treatment with clorgyline. No protein in the hippocampus was found to be significantly reduced by treatment with clorgyline. Since a previous study has indicated that the relative concentration of three different proteins were significantly altered by the repeated administration of desipramine, the results from the present experiment indicate that different changes in proteins are produced by repeated treatment with the type A monoamine oxidase inhibitor, clorgyline, as compared to those produced by the tricyclic antidepressant, desipramine. These results support previous suggestions that different classes of antidepressant compounds may exert their effects through different mechanisms of action.

Localization of Ca(2+)-binding proteins of rat cortex on two-dimensional gels-I. Identification of calmodulin and the b subunit of calcineurin.

At least three Ca(2+)-binding proteins were detected in rat cortex by (45)Ca(2+) autoradiography of two-dimensional electrophoretograms. The identities of two of these Ca(2+)-binding proteins were determined to be calmodulin and the B subunit of calcineurin. The identification was based upon the following criteria: (1) co-localization on polyacrylamide gels with the appropriate purified proteins, (2) staining of nitrocellulose blots with specific antisera for calmodulin and calcineurin and (3) ability to bind Ca(2+). This information is useful in that it identifies two major brain proteins visible on silver-stained two-dimensional polyacrylamide gels. In addition, this data reveals the location of an unidentified Ca(2+)-binding protein of molecular weight ? 18,000 Da and pI 5.4 on these gels.

Localization of Ca(2+)-binding proteins of rat cortex on two-dimensional gels-II. analysis of Ca(2+) binding proteins in ammonium sulfate fractions of rat brain.

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and (45)Ca(2+)-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by (45)Ca(2+)-autoradiography in this study are unknown.

Effects of bilateral lesion of the locus coeruleus and of neonatal administration of 6-hydroxydopamine on the concentration of individual proteins in rat brain.

The role that norepinephrine plays in regulating the concentration of different proteins in the parietal cortex, hippocampus and cerebellum was assessed by investigating the effects of either a bilateral lesion of the locus coeruleus or neonatal administration of 6-hydroxydopamine. Two weeks after lesioning the locus coeruleus, the concentration of two different proteins was elevated in the hippocampus; a third protein was reduced in concentration in this brain area as a result of the lesion. Three proteins were affected in concentration in the cerebellum after the locus coeruleus lesion--two were elevated in concentration and one was reduced in concentration. No proteins were altered in concentration in the parietal cortex as a result of the lesion. Seventy days after neonatal treatment with 6-hydroxydopamine, a total of 6 proteins were found to be changed. Four of these (one in the hippocampus and 3 in the parietal cortex) were reduced in concentration while two proteins (both in the cerebellum) were elevated in concentration after neonatal treatment with the catecholamine neurotoxin. There was little overlap between those proteins affected in concentration by the bilateral lesion of the locus coeruleus and those changed by neonatal treatment with 6-hydroxydopamine. These results suggest that the concentration of a number of different proteins may, under normal physiological conditions, be regulated in vivo by norepinephrine in the brain.

Effect of 5,7-dihydroxytryptamine on the concentration of individual proteins in different areas of the rat brain.

Proteins which are apparently regulated in concentration in two different areas of the rat brain by the indole neurotransmitter serotonin were identified using two-dimensional gel electrophoresis combined with computerized scanning densitometry. Reduction in central serotonin levels produced a decrease in the concentration of 3 different proteins (2 in the parietal cortex, 1 in the hippocampus). Two proteins, both in the hippocampus, were elevated in concentration following serotonin depletion. These results demonstrate that there exist in the brain a limited number of proteins whose concentration is influenced by serotonin.

Subfornical organ: effects of salt loading and water deprivation on in vitro radioamino acid incorporation into individual proteins.

The subfornical organ of the brain has a role in the regulation of fluid balance in higher animals. In this study the effects of salt loading and water deprivation on specific proteins in this organ were investigated. For 4 days, 3 groups of rats were given an appropriate fluid diet (control, 2% NaCl and water deprived), with all groups having free access to food. Animals were killed by decapitation, and the subfornical organ was quickly dissected out and incubated for 6 h in a medium containing [35S]methionine and [35S]cysteine. Proteins from these organs were then separated by two-dimensional electrophoresis, and the resulting autofluorographs were analyzed by scanning densitometry. The results show that the incorporation of labeled amino acids into 8 proteins was changed due to the experimental manipulations.

Effect of reduction of cholinergic input on the concentration of specific proteins in different cortical regions of the rat brain.

The effect of lesioning the nucleus of the tractus diagonalis on the concentration of specific proteins in the hippocampus and the occipital cortex was assessed. Rats received either a sham or an electrolytic lesion and were killed 9 or 35 days later. Tissue samples were removed by microdissection and proteins were separated by two-dimensional gel electrophoresis. Gels were stained with silver, and then analyzed by quantitative computerized scanning densitometry. Of the 143 proteins analyzed, only four were found to be altered in concentration in both brain areas as a result of the lesion. Protein 82 (molecular weight 39,000, pI 6.5) was reduced 71% in the hippocampus and 50% in the occipital cortex 9 days after the lesion, while protein 109 (molecular weight 32,000, pI 6.4) was elevated 140% in the hippocampus and 130% in the occipital cortex at the same time point. Protein 6 (molecular weight 58,000, pI 5.7) was unchanged 9 days after the lesion but was elevated in concentration in both the hippocampus and the occipital cortex 35 days after lesioning. Protein 74 (molecular weight 39,000, pI 5.8) was elevated in concentration both 9 and 35 days after lesioning in the occipital cortex, but only at day 35 in the hippocampus. These results demonstrate that the concentration of these four proteins may be regulated by the cholinergic input to the hippocampus and the occipital cortex. The possibility exists that one or more of these proteins may be related to either the muscarinic or nicotinic cholinergic receptor in rat brain.

An apparent genetic polymorphism for a protein present in the hypothalamus of Sprague-Dawley rats.

Using two-dimensional gel electrophoresis, an apparent genetic polymorphism was detected in the hypothalamus of a group of inbred Sprague-Dawley rats. The proteins involved in this polymorphism have a molecular weight of 57,000 daltons and isoelectric points ranging from 6.1 to 6.3. These proteins met four criteria that should be met before a positional shift on two-dimension gels can be attributed to a genetic polymorphism. This is the first report of the existence of a genetic polymorphism in the brains of a group of inbred Sprague-Dawley rats. The functional significance of this polymorphism is currently under investigation.

Isolation of specific proteins affected by estradiol in the arcuate-median eminence of prepuberal female rats.

Prepuberal female rats (25 days of age) were injected with estradiol benzoate (EB 10 micrograms/rat, s.c. in oil) or oil vehicle. Forty-eight hours after treatment, all animals were decapitated, their brains removed and sectioned. The arcuate nucleus of the hypothalamus and median eminence were microdissected and processed for isoelectric focusing followed by slab gel electrophoresis. The resulting two-dimensional electrophoretic gels were analyzed to quantitate the specific proteins resolved using a scanning microdensitometric method. Out of 235 proteins measured, 8 proteins were found to be significantly increased and 4 were decreased by EB treatment. The proteins which increased in concentration ranged in molecular weight from 15 to 43 kDa and isoelectric points (pI) of 4.9 to 7.0. The 4 proteins decreased by the EB treatment were 44, 67, 74 and 80 kDa in molecular weight and their pI's ranged from 6.5 to 7.1. It is suggested that these proteins might be involved in some of the neuroendocrine effects that are induced by estradiol in this region of the brain.

Cholinergic projection sites of the nucleus of tractus diagonalis.

The cholinergic projection sites of the basal portion of the nucleus of the tractus diagonalis (td) were studied following bilateral stereotaxic lesions. Choline acetyltransferase (ChAT) activity was measured in various cortices, hippocampus and amygdala. Significant decreases in ChAT activity were observed in the anterior and posterior cingulate cortex, the frontal cortex, occipital cortex, hippocampus and dentate gyrus. It is concluded that (1) the hippocampus is innervated by the basal portion of the td and not from the medial septal nucleus, (2) the td contains cholinergic cell bodies which project widely to the cortex and hippocampus. It is suggested that these cholinergic projections have important influences on emotional behavior via connections with the cortex and limbic system.

Lesions of the paraventricular nucleus area of the hypothalamus disrupt the suprachiasmatic leads to spinal cord circuit in the melatonin rhythm generating system.

The circadian rhythm in melatonin production in mammals is regulated by a suprachiasmatic (SCN) leads to spinal cord leads to pineal circuit. In the present investigation the possible participation of the paraventricular nucleus of the hypothalamus (PVN) in the SCN leads to spinal cord segment of this circuit was investigated in the rat. Bilateral lesions of the PVN area were produced and one to two weeks later melatonin production was evaluated by measuring the activities of the two pineal enzymes required for the formation of melatonin from serotonin, indoleamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), and urinary 6-hydroxymelatonin, the major melatonin metabolite. In some cases pineal melatonin was also measured. Control animals received sham-PVN lesions. Histological examination of the lesions indicated that the PVN were bilaterally destroyed 100% in 12 animals. The nighttime pineal melatonin and urinary 6-hydroxymelatonin values in this group were reduced about 90%, nighttime pineal NAT activity was reduced about 98%, and HIOMT activity about 75%. The urinary 6-hydroxymelatonin values of PVN-lesioned animals and animals with denervated pineal glands were similar. In animals with hypothalamic lesions involving less than 30% of the PVN, nighttime values of NAT, HIOMT, and urinary 6-hydroxymelatonin were normal; in animals with 30 to 95% PVN damage these parameters were altered to a small degree. These studies, together with histochemical observations, indicate the SCN neurons responsible for pineal circadian rhythms project to the PVN area of the hypothalamus.

Effect of desmethylimipramine and reserpine on the concentration of specific proteins in the parietal cortex and the hippocampus of rats as analyzed by two-dimensional gel electrophoresis.

The effect of desmethylimipramine (DMI) and reserpine on the concentration of specific proteins in the parietal cortex and the hippocampus of rats was assessed using two-dimensional gel electrophoresis combined with computer-assisted scanning densitometry. Chronic administration of DMI for 3 weeks was found to produce a significant reduction in the concentration of two proteins in both brain regions examined. Both of these proteins have a molecular weight of approximately 57,000 daltons and isoelectric point of 6.2 to 6.3. A third, smaller protein (MW 28,000 daltons, isoelectric point 5.9) was increased in concentration in rats treated repeatedly with DMI. Acute drug treatment was, in all three cases, found to be without effect. In contrast, chronic treatment of rats with reserpine produced effects on these three proteins in the hippocampus which were quantitatively opposite to those obtained after chronic DMI administration. Again, acute drug treatment was without effect. These results demonstrate that chronic, but not acute, administration of agents affecting noradrenergic reactivity can also have an effect on the concentration of specific proteins within the central nervous system and are of interest in view of the known effects of these drugs on neurotransmitter and enzyme systems in the central nervous system.

Sex differences in specific proteins in the preoptic medial nucleus of the rat hypothalamus.

Using two-dimensional gel electrophoresis and a highly sensitive silver stain, specific proteins in adult male and female rat brain were examined. Based on previous studies, the preoptic medial nucleus (POM) of the hypothalamus served as the area of interest, with the parietal cortex acting as control. A significant difference between the sexes was found in the concentration of two proteins in the POM, a difference which was not found in the parietal cortex.

Identification of neuron-specific enolase and nonneuronal enolase in human and rat brain on two-dimensional polyacrylamide gels.

The location of the enzymes neuron-specific enolase and nonneuronal enolase on two-dimensional gels generated from tissue samples obtained from fresh human and rat cortex has been identified. This identification is based upon the following criteria: comigration on polyacrylamide gels with the appropriate purified protein and staining on nitrocellulose protein blots of human and rat cortex using antibodies specific for each protein. The results show that our preparation of neuron-specific enolase from rat and human brain is highly pure, as only one spot is obtained on two-dimensional gels. Further, the antiserum to neuron-specific enolase is highly specific, as it reacts only with neuron-specific enolase on nitrocellulose blots derived from two-dimensional gels of cortical tissue. The location of these proteins is of interest because it positively identifies two major brain proteins on two-dimensional polyacrylamide gels of fresh cortical tissue. This information will be useful in a variety of future studies aimed at both identifying specific proteins on two-dimensional gels and observing the effects of experimental manipulations on brain and other neuronal proteins.

Incorporation of amino acids into proteins of the hypothalamus of prepuberal female rats after estradiol treatment.

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 micrograms 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 micron 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.

Identification and use of constitutive proteins for the normalization of high resolution electrophoretograms.

Quantitative inter-gel comparisons of proteins separated by high resolution two-dimensional protein electrophoresis present a number of problems. These problems may arise from: variations in pipetting and other mechanical manipulations of samples, protein loss during transfer from the first to the second gel dimension, variations in staining, and/or variations in film development during autoradiography, in the case of radioactively labeled proteins. This study presents a discussion of these issues and a normalization algorithm to deal with variations, which relies on a class of proteins present in most biological samples which by their nature may be considered internal standards. This class consists of proteins which are controlled by constitutive genes. Constitutive genes are genes that are expressed constantly. We have developed an algorithm which is currently available as a subroutine, 'FINDCONS', in the computerized densitometry and protein comparison analysis program, developed by Olson & Miller (1988). This algorithm identifies potentially 'constitutive' proteins. A normalization method employing these potentially 'constitutive' proteins was compared to several others by examining 2D-electrophoretograms of proteins from developing gypsy moths (Lymantria dispar L.) insect tissue. Following normalization, inter-gel comparisons of spots, which were 'identified' as 'constitutive', were observed to vary less in density than when no normalization method was used, or when normalization based on total integrated spot density was used. In addition to its use as a normalization tool, this algorithm and the subroutine FINDCONS may be useful as an aid in biological studies to identify 'constitutive' proteins.

The protein disease database of human body fluids: I. Rationale for the development of this database.

We are developing a relational database to facilitate quantitative and qualitative comparisons of proteins in human body fluids in normal and disease states. For decades researchers and clinicians have been studying proteins in body fluids such as serum, plasma, cerebrospinal fluid and urine. Currently, most clinicians evaluate only a few specific proteins in a body fluid such as plasma when they suspect that a patient has a disease. Now, however, high resolution two-dimensional protein electrophoresis allows the simultaneous evaluation of 1,500 to 3,000 proteins in complex solutions, such as the body fluids. This and other high resolution methods have encouraged us to collect the clinical data for the body fluid proteins into an easily accessed database. For this reason, it has been constructed on the Internet World Wide Web (WWW) under the title Protein Disease Database (PDD). In addition, this database will provide a linkage between the disease-associated protein alterations and images of the appropriate proteins on high-resolution electrophoretic gels of the body fluids. This effort requires the normalization of data to account for variations in methods of measurement. Initial efforts in the establishment of the PDD have been concentrated on alterations in the acute-phase proteins in individuals with acute and chronic diseases. Even at this early stage in the development of our database, it has proven to be useful as we have found that there appear to be several common acute-phase protein alterations in the plasma and cerebrospinal fluid from patients with Alzheimer's disease, schizophrenia and major depression. Our goal is to provide access to the PDD so that systematic correlations and relationships between disease states can be examined and extended.

The Protein Disease Database of human body fluids: II. Computer methods and data issues.

The Protein Disease Database (PDD) is a relational database of proteins and diseases. With this database it is possible to screen for quantitative protein abnormalities associated with disease states. These quantitative relationships use data drawn from the peer-reviewed biomedical literature. Assays may also include those observed in high-resolution electrophoretic gels that offer the potential to quantitate many proteins in a single test as well as data gathered by enzymatic or immunologic assays. We are using the Internet World Wide Web (WWW) and the Web browser paradigm as an access method for wide distribution and querying of the Protein Disease Database. The WWW hypertext transfer protocol and its Common Gateway Interface make it possible to build powerful graphical user interfaces that can support easy-to-use data retrieval using query specification forms or images. The details of these interactions are totally transparent to the users of these forms. Using a client-server SQL relational database, user query access, initial data entry and database maintenance are all performed over the Internet with a Web browser. We discuss the underlying design issues, mapping mechanisms and assumptions that we used in constructing the system, data entry, access to the database server, security, and synthesis of derived two-dimensional gel image maps and hypertext documents resulting from SQL database searches.