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During the past decade, regenerative medicine has been the subject of intense interest due, in large part, to our growing knowledge of embryonic stem (ES) cell biology. ES cells give rise to cell lineages from the three primordial germ layers--endoderm, mesoderm, and ectoderm. This process needs to be channeled if these cells are to be differentiated efficiently and used subsequently for therapeutic purposes. Indeed, an important area of investigation involves directed differentiation to influence the lineage commitment of these pluripotent cells in vitro. Various strategies involving timely growth factor supplementation, cell co-cultures, and gene transfection are used to drive lineage specific emergence. The underlying goal is to control directly the center of gene expression and cellular programming--the nucleus. Gene expression is enabled, managed, and sustained by the collective actions and interactions of proteins found in the nucleus--the nuclear proteome--in response to extracellular signaling. Nuclear proteomics can inventory these nuclear proteins in differentiating cells and decipher their dynamics during cellular phenotypic commitment. This review details what is currently known about nuclear effectors of stem cell differentiation and describes emerging techniques in the discovery of nuclear proteomics that will illuminate new transcription factors and modulators of gene expression.

This study used heroin self-administration to investigate incubation of goal-directed heroin-seeking behavior following abstinence. Male Sprague-Dawley rats self-administered heroin on a fixed ratio 10 (FR10) schedule of reinforcement with licking of an empty spout serving as the operant behavior during 14 daily 3 h sessions. After this acquisition period, all rats received a 90 min extinction session following either 1 day or 14 days of home cage abstinence. When the extinction session occurred after only 1 day of home cage abstinence, rats with a history of heroin self-administration divided their responses equally between the previously "active" and "inactive" spouts. However, when the extinction session occurred following 14 days of home cage abstinence, the rats exhibited marked goal-directed heroin-seeking behavior by licking more on the previously "active" than "inactive" spout. These findings demonstrate that heroin-seeking behavior incubates over time, resulting in goal-directed heroin-seeking behavior in rats following 14 days but not 1 day of abstinence. Moreover, this facilitatory effect occurred in response to a different training schedule, lower total drug intake, and shorter periods of daily access than previously reported with heroin.

Enzymes are the metabolic catalysts that affect a multitude of physiological processes and responses. Tight control of enzyme activity is therefore essential in maintaining the steady state of all organisms. Keywords: feedback inhibition; phosphorylation; allosteric regulation; covalent modification; proenzymes

Only a few studies have used models of inflammatory bowel disease (IBD) with weanling animals. Previously, the effects of probiotics have not been assessed in such IBD models. The objectives of our study were 2-fold: to establish a suitable model of dextran sulfate sodium (DSS)-induced colitis in weanling rats and to determine the effects of the probiotic formulation VSL#3 on DSS-induced colitis in weanling animals.Rats were weaned on postnatal day 21 and administered 2%, 2.5%, or 3% (wt/vol) DSS in drinking water. In subsequent experiments, newly weaned animals were administered vehicle or VSL#3 (0.06, 0.6, or 6 mg) by orogastric gavage. These treatments were given to animals maintained on water (postnatal days 21-28) and then on DSS (postnatal days 28-35). Disease activity indices were determined on a routine basis. On day 35, rats were euthanized. The total colon length was determined. Other parameters of colitis were measured from the distal colon. These parameters included myeloperoxidase (MPO), interleukin (IL)-1beta, inhibitory kappaB-alpha (IkappaB-alpha), and histological assessment of crypt damage and inflammation.DSS 2% was optimal for inducing colitis in weanling rats without significant morbidity. VSL#3 treatments improved various parameters of 2% DSS-induced colitis in weanling rats. The 0.6- and 6-mg doses of VSL#3 were most effective for attenuating this colitis.The probiotic formulation VSL#3 improved DSS-induced colitis in weanling rats. This improvement of colitis involved changes in colonic IkappaB-alpha, IL-1beta, and MPO, which are suggestive of immune modulation by VSL#3.

Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day x 3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatments. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity-related genes is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures.

One of the most exciting and contentious issues in biomedical science today is the potential use of stem cells as a therapeutic intervention for a wide range of diseases with pathologies that involve cellular destruction. Stem cell therapy is currently practiced, or under active research, to repair the effects of neuromuscular degeneration, autoimmune response, ischemic attack, malignant carcinoma, congenital defects, metabolic disorders, and alcohol-related pathologies. When stimulated in a proliferative environment, stem cells may also provide the raw material for large-scale ex vivo tissue regeneration. There are several sources of human stem cells for laboratory culture, but the primary source of pluripotent human embryonic stem cells is the inner cell mass of in vitro fertilized embryos. Despite the potential rewards to medicine, experimentation using cells from viable human fetuses has triggered global political and ethical controversy that has slowed down the pace of research. An alternative source of human embryonic stem cells that circumvents some of these issues would significantly enhance research in this area. Parthenogenetically derived stem cells may represent such a resource. Parthenogenesis involves activation of the oocyte without sperm, and produces a nonviable blastocyst. That is, the activated egg develops to the blastocyst (and so can be used to generate stem cells), but it will not produce a viable pregnancy in mammals. The unique genesis of the embryo, and its arrested development at blastocyst, may afford sufficient moral and political latitude to enable its acceptance as an experimental entity. Research is ongoing to assess the genetic and phenotypic similarity of parthenogenetic stem cells (PSCs) to other stem cell types from mammalian models and to human stem cells derived from fertilized embryos. Keywords: Chimera; Genomic Imprinting; Parthenogenesis; Parthenote; Teratoma; Uniparental Disomy

A challenge of pediatric research is the limited ability to obtain tissue samples from small patients. To confront this problem, blood biomarkers can be used as surrogate markers of disease processes and aid in patient monitoring and disease detection. Furthermore, proteomic analysis of plasma samples is one approach for large-scale discovery of disease biomarkers. This study examined the use of plasma for disease process biomarkers in pediatric patients undergoing cardiopulmonary bypass (CPB) surgery. Proteomic studies of plasma are limited by the presence of a few high abundance proteins that mask the presence of lower abundance proteins of interest. Plasma immunoaffinity depletion (removing 6 of the highest abundance proteins of little pathological importance) increases sensitivity of detection for proteins such as those related to inflammation, remodeling, and damage. Using two-dimensional in-gel fluorescence electrophoresis, changes in the expression levels of proteins that occur as a result of CPB can be identified. In the present study, plasma depletion removed 83% of the plasma protein mass, allowing approximately 1400 spots to be observed by two-dimensional in-gel fluorescence electrophoresis. Of the detected spots, 79 (5.7%) were altered by CPB. These data illuminate the strength of plasma proteomics in identification of candidate biomarkers of CPB-associated disease processes.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.

While human embryonic stem cells (hESCs) hold tremendous therapeutic potential, they also create societal and ethical dilemmas. Adult and placental stem cells represent two alternatives to the hESC, but may have technical limitations. An additional alternative is the stem cell derived from parthenogenesis. Parthenogenesis is a reproductive mechanism that is common in lower organisms and produces a live birth from an oocyte activated in the absence of sperm. However, parthenogenetic embryos will develop to the blastocyst stage and so can serve as a source of embryonic stem cells. Parthenogenetic ESCs (pESCs) have been shown to have the properties of self-renewal and the capacity to generate cell derivatives from the three germ layers, confirmed by contributions to chimeric animals and/or teratoma formation when injected into SCID mice. Therefore, this mechanism for generating stem cells has the ethical advantage of not involving the destruction of viable embryos. Moreover, the cells do not involve the union of male and female and so genetic material will be derived exclusively from the female oocyte donor (with the attendant potential immunological advantages). This chapter describes the biology underlying parthenogenesis, as well as provides detailed technical considerations for the production of pESCs.

Repeated amphetamine treatment produces a long-lasting augmentation of locomotor behavior in rats, a phenomenon known as behavioral sensitization. This process is thought to be a correlate of the addictive process in humans, and it is believed that there are drug-induced neuroadaptations that underlie these behavioral changes. One mechanism by which amphetamine can alter brain function is by affecting direct intercellular communication between neurons via gap junctions. The purpose of the present study was to examine the effect of an amphetamine treatment regimen known to produce changes in dye coupling between neurons, a functional correlate of gap junction function, on the expression of the neuronal gap junction-forming protein, connexin36. Here we report that withdrawal from an extended amphetamine regimen produces region-specific and time-dependent changes in connexin36 expression in rat nucleus accumbens and prefrontal cortex, brain regions known play roles in sensitization and addiction. This is, to our knowledge, the first demonstration of pharmacological manipulation of connexin36 in vivo.

This chapter reviews aspects of technologies and experimental design in transcriptomics. To better understand alcohol abuse and alcoholism, two key genetic elements prove to be important, these concepts suggest that: (1) genetic predispositions can place an individual at a risk for abuse and addiction. (2) There are genomic responses to chronic alcohol administration that contribute to clinical issues of tolerance, dependence (physical and psychological), and withdrawal. Aspects of both of these issues can involve altered levels of the messenger ribonucleic acid. That is, two individuals might express different levels of the same gene, thus placing one at a unique risk for developing alcoholism. Alternatively, chronic ethanol exposure will itself engender changes in the normal patterns of gene expression and create a new physiological state. The efficient, high screening of differences in the gene expression is the purview of the DNA array and constitutes the central technological feature of the new field of transcriptomics. This technology, while powerful, requires significant attention to detail, and considerable technical expertise.

Hybridization array technology makes it possible to compare global gene-expression patterns in any experimental context for which good-quality RNA can be generated. To date, DNA arrays have been used as a tool to compare functional genomic changes (differences in wholesale gene expression) in studies that cover an impressive variety of research disciplines including cancer, yeast genomics, and, more recently, neuroscience and behavior. The basic premise of the array experiment is that one interrogates a panel of probes (gene-specific cDNA fragments or gene-specific oligonucleotides that have been immobilized on a solid support) with RNAs (targets) from control and treated experimental samples that have been either radioactively or fluorescently labeled. Signal derived from either competitive (both samples on a single chip) or differential (one sample/one chip) hybridization is used to calculate relative gene expression. There are three widely used platforms available to perform array experiments (Affymetrix GeneChips, oligonucleotide arrays, and membrane-based cDNA arrays) and each platform offers advantages and limitations. The experimental description in this chapter explains, in detail, how to perform a hybridization array using the macroarray platform.

The electrical signal associated with nerve cells, mainly as a result of changes in the membrane potential during functional activity, can be recorded extracellularly to study central mechanisms underlying sensory processing. The secondary neurons in the spinal trigeminal complex receive inputs from peripheral neurons that innervate the orofacial region and forward information to the higher levels of the nervous system. Analyzing activity patterns of trigeminal neurons related to pain perception has proven to be an efficient method in studying orofacial pain mechanisms. Here we describe some basic techniques and tips for extracellular single neuron recording from the subnucleus caudalis of the trigeminal spinal nucleus in rats with orofacial injury. Two different rat models with temporomandibular joint inflammation and inferior alveolar nerve transection are described.

Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy. We herein describein vitroparthenogenetic development of monkey (Macaca fascicularis) eggs to the blastocyst stage, and their use to create a pluripotent line of stem cells. These monkey stem cells (Cyno-1 cells) are positive for telomerase activity and are immunoreactive for alkaline phosphatase, octamer-binding transcription factor 4 (Oct-4), stage-specific embryonic antigen 4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor rejection antigen 1-81 (TRA 1-81) (traditional markers of human embryonic stem cells). They have a normal chromosome karyotype (40 + 2) and can be maintainedin vitroin an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiatedin vitrointo dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells were injected into severe combined immunodeficient mice, teratomas with derivatives from all three embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (immunoreactive for nestin). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-dependent sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.

The postgenomic era of large-scale gene expression studies is inundating drug abuse researchers and many other scientists with findings related to gene expression. This information is distributed across many different journals, and requires laborious literature searches. Here, we present an interactive database that combines existing information related to cocaine-mediated changes in gene expression in an easy-to-use format. The database is limited to statistically significant changes in mRNA or protein expression after cocaine administration. The Flash-based program is integrated into a Web page, and organizes changes in gene expression based on neuroanatomical region, general function, and gene name. Accompanying each gene is a description of the gene, links to the original publications, and a link to the appropriate OMIM (Online Mendelian Inheritance in Man) entry. The nature of this review allows for timely modifications and rapid inclusion of new publications, and should help researchers build second-generation hypotheses on the role of gene expression changes in the physiology and behavior of cocaine abuse. Furthermore, this method of organizing large volumes of scientific information can easily be adapted to assist researchers in fields outside of drug abuse.

Manganese (Mn) is an essential metal that, at excessive levels in the brain, produces extrapyramidal symptoms similar to those in patients with Parkinson's disease (PD). In the present study, Mn toxicity was characterized in a human neuroblastoma (SK-N-SH) cell line and in a mouse catecholaminergic (CATH.a) cell line. Mn was demonstrated to be more toxic in the catecholamine-producing CATH.a cells (EC50 = 60 microM) than in non-catecholaminergic SK-N-SH cells (EC50 = 200 microM). To test the hypothesis that the sensitivity of CATH.a cells to Mn is associated with their dopamine (DA) content, DA concentrations were suppressed in these cells by pretreatment with alpha-methyl-para-tyrosine (AMPT). Treatment for 24 h with 100 microM AMPT decreased intracellular DA, but offered no significant protection from Mn exposure (EC50 = 60 microM). Additional studies were carried out to assess if Mn toxicity was dependent on glutathione (GSH) levels. CATH.a cells were significantly protected by the addition of 5mM GSH (Mn EC50 = 200 microM) and 10mM N-acetyl cysteine (NAC) (Mn EC50 = 300 microM), therefore, indirectly identifying intracellular ROS formation as a mechanism for Mn neurotoxicity. Finally, apoptotic markers of Mn-induced cell death were investigated. DNA fragmentation, caspase-3 activation, and apoptosis-related gene expression were studied in CATH.a cells. No internucleosomal fragmentation or caspase activation was evident, even in the presence of "supraphysiological" Mn concentrations. cDNA hydridization array analysis with two differing Mn concentrations and time points, identified no noteworthy mRNA inductions of genes associated with programmed cell death. In conclusion, DA content was not responsible for the enhanced sensitivity of CATH.a cells to Mn toxicity, but oxidative stress was implicated as a probable mechanism of cytotoxicity.

Dopamine (DA) metabolism and oxidation produce both reactive oxygen species (ROS) and reactive quinones. These chemical species are implicated in dopamine neurotoxicity and neurodegeneration. In the present studies, human neuroblastoma (SK-N-SH) cells were exposed to toxic concentrations of dopamine (333 microM) in order to investigate molecular pathways involved in dopamine toxicity. cDNA hybridization array (microarray) technology demonstrated that GADD45 and GADD153 (growth arrest and DNA-damage inducible) gene expression was increased in dopamine-treated cells (333 microM for 18 h). Subsequent Northern and Western blot analysis confirmed these changes in GADD45 and GADD153 gene expression. The antioxidant, ascorbic acid, significantly reduced the increase in GADD45 gene expression but did not significantly reduce GADD153 gene expression. Currently, the precise function of the GADD gene products is not known. It is known, however, that these genes are upregulated in response to stress to allow cells time to repair macromolecular damage. In the present case, GADD gene expression (manifested as increased mRNA and protein levels) preceded dopamine-induced cytotoxicity. It appears that dopamine, through the formation of reactive oxygen species and quinones, may damage cellular macromolecules to the point of inducing GADD gene expression. Other genes that displayed changes, but that have not been subjected to post-hoc confirmation, include clusterin (increased), ubiquitin (increased), CD27 ligand (increased), CD27BP (increased), and rac-PK-beta (decreased).

Repeated cocaine administration produces changes in gene expression that are thought to contribute to the behavioral alterations observed with cocaine abuse. This study focuses on gene expression changes in the frontal cortex, a component of reinforcement, sensory, associative, and executive circuitries. Changes in frontal cortex gene expression after repeated cocaine self-administration may lead to changes in the behaviors associated with this brain region. Rats self-administered cocaine for 10 days in a continuous access, discrete trial paradigm (averaging 100 mg/kg/day) and were examined for changes in relative frontal cortex mRNA abundance by cDNA hybridization arrays. Among the changes observed following array analysis, increased nerve-growth-factor-induced B (NGFI-B), adenylyl cyclase type VIII (AC VIII), and reduced cysteine-rich protein 2 (CRP2) mRNA were confirmed by quantitative RT-PCR. These changes share commonalities and exhibit differences with previous reports of gene expression changes in the frontal cortex after noncontingent cocaine administration.

The pathogenic mechanisms of the mutant huntingtin protein that cause Huntington's disease (HD) are unknown. Previous studies have reported significant decreases in the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brains of the R6/2 transgenic mouse model of HD. In an attempt to elucidate the cause of these neurochemical perturbations in HD, the protein levels and enzymatic activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, were determined. Enzyme activity was measured in brainstem homogenates from 4-, 8-, and 12-week-old R6/2 mice and compared with aged-matched wild-type control mice. We observed a 62% decrease in brainstem TPH activity (p = 0.009) in 4-week-old R6/2 mice, well before the onset of behavioral symptoms. In addition, significant decreases in TPH activity were also observed at 8 and 12 weeks of age (61%, p = 0.02 and 86%, p = 0.005, respectively). In the 12-week-old-mice, no change in immunoreactive TPH was observed. In vitro binding showed that TPH does not bind to exon 1 of huntingtin in a polyglutamine-dependent manner. Specifically, glutathione-S-transferase huntingtin exon 1 proteins with 20, 32 or 53 polyglutamines did not interact with radiolabeled tryptophan hydroxylase. Therefore, the inhibition of TPH activity does not appear to result from a direct huntingtin/TPH interaction. Receptor binding analyses for the 5-HT1A receptor in 12-week-old R6/2 mice revealed significant reductions in 8-OH-[3H]DPAT binding in several hippocampal and cortical regions. These results demonstrate that the serotonergic system in the R6/2 mice is severely disrupted in both presymptomatic and symptomatic mice. The presymptomatic inhibition of TPH activity in the R6/2 mice may help explain the functional consequences of HD and provide insights into new targets for pharmacotherapy.

We evaluated biochemical endpoints related to oxidative stress in brains of neonatal rats exposed to manganese (Mn). Oral Mn chloride (MnCl2) (0, 25, or 50 mg Mn chloride kg(-1) body weight per day) was given daily to neonatal rats throughout lactation (i.e. from postnatal day (PND) 1 to 21). As previously reported by [J. Appl. Toxicol. 20 (2000) 179], this treatment paradigm results in increased cerebral cortex (CTX) Mn concentrations in PND 21 rats from both Mn treatment groups. High dose Mn exposure also results in increased cerebellar Mn concentrations. This preliminary study determined whether this exposure paradigm also affects cerebrocortical or cerebellar metallothionein (MT) mRNA levels, glutamine synthetase (GS) activity, GS protein levels, as well as total glutathione (GSH) levels. High dose Mn exposure significantly increased (P < 0.05) total cerebrocortical GSH without accompanying changes in any of the other measured parameters. Therefore, it is unlikely that high dose Mn exposure is associated with oxidative stress in this experimental paradigm.

Alterations in gene expression caused by repeated cocaine administration have been implicated in the long-term behavioral aspects of cocaine abuse. The frontal cortex mediates reinforcement, sensory, associative, and executive functions and plays an important role in the mesocortical dopamine reinforcement system. Repeated cocaine administration causes changes in frontal cortex gene expression that may lead to changes in the behaviors subserved by this brain region. Rats treated non-contingently with a binge model of cocaine (45 mg/kg/day, i.p.) for 14 days were screened for changes in relative mRNA abundance in the frontal cortex by cDNA hybridization arrays. To confirm changes, immunoreactive protein was measured (via protein-specific immunoblots) in a second group of identically-treated animals. Protein levels of protein tyrosine kinase 2 (PYK2), activity-regulated cytoskeletal protein (ARC), as well as an antigen related to nerve growth factor I-B (NGFI-B-RA) were shown to be significantly induced after cocaine administration. Levels of NGFI-B mRNA were confirmed by real-time RT-PCR to be increased with cocaine administration. These observations are similar to previously reported cocaine-responsive changes in gene expression but novel to the frontal cortex. This study also validates the use of hybridization arrays for screening of neuronal gene expression changes and the utility of relative protein quantification as a post-hoc confirmation tool.

Chronic cocaine use is known to elicit changes in the pattern of gene expression within the brain. The hippocampus plays a critical role in learning and memory and may also play a role in mediating behaviors associated with cocaine abuse. To profile the gene expression response of the hippocampus to chronic cocaine treatment, cDNA hybridization arrays were used to illuminate cocaine-regulated genes in rats treated non-contingently with a binge model of cocaine (45 mg/kg/day, i.p.) for 14 days. Validation of mRNA changes illuminated by hybridization array analysis was accomplished by measuring immunoreactive protein (via specific immunoblots). The induction of protein kinase Calpha, potassium channel 1.1, and metabotropic glutamate receptor 5 seen by hybridization arrays was confirmed at the level of protein. Immunoblot screening of previously described cocaine-responsive genes demonstrated increased levels of protein tyrosine kinase 2, beta-catenin, and protein kinase Cepsilon. While some of these changes exist in previously described cocaine-responsive models, others are novel to any model of cocaine use. The inductions of potassium channel 1.1, protein tyrosine kinase 2 and metabotropic glutamate receptor 5 are novel findings to hippocampal cocaine-responsive gene expression. These proteins have been shown to subserve learning and memory and/or long-term potentiation functions within the hippocampus. Additionally, these genes are known to interact with one another, forming a more complex pattern of gene expression changes. The findings suggest altered expression of genes with a number of different functions in the rat hippocampus after a 'binge' style of non-contingent cocaine administration. These changes in gene expression may play roles in neuronal plasticity and the behavioral phenomena associated with cocaine abuse.

Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7659-7663). Recent data showed that cysteine oxidation rather than tyrosine nitration is responsible for TH inactivation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., and Geddes, T. J. (1999) J. Neurosci. 19, 10289-10294). However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr(423) to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 +/- 0.9) x 10(3) m(-1) s(-1) at pH 7.4 and 25 degrees C for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Previously published results have established that chronic cocaine administration (30–45 mg/kg per day, 10–14 days) resulted in an upregulation of TH gene expression in dopaminergic pathways of rats. The present studies tested the effects of a tropane analog, PTT (2β-propanoyl-3β-(4-tolyl)-tropane), on TH expression. This drug has similar actions to cocaine, but possesses markedly different pharmacokinetics (20 times more potent at binding the dopamine transporter, markedly increased metabolic stability, and 10–20 times more potent in behavioral measures). Moreover, PTT demonstrates an increased selectivity for the dopamine (DA) and norepinephrine (NE) transporters compared with cocaine. In direct contrast to the previously reported effects of cocaine, 10 days of PTT administration (3.0 mg/kg per day, i.p.) produced a uniform downregulation of TH protein and activity gene expression. TH activity and immunoreactive protein where decreased by 54 and 69%, respectively in the nucleus accumbens. Within the ventral tegmental area, TH activity and protein were decreased by 33 and 19%, respectively. The underlying mechanisms for these fundamental differences are unclear, but likely reflect varying and selective affinities and lengths of occupancy at biogenic amine transporters.

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21-41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (Ndelta15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion Ndelta15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (Ndelta30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (Ndelta41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.

Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.

Cortical atrophy is well known in Alzheimer’s disease (AD), however, there are different interpretations of its location and extent. Neurodegeneration and atrophy are relatively consistent in medial temporal areas, including hippocampal, entorhinal, and perirhinal cortices, and they follow a uniform pattern. This is not the only region of atrophy, however, since it occurs in prefrontal, parietotemporal, occipital, and cingulate cortices. Furthermore, although gross focal atrophy is not always present, focal neurodegeneration may occur with focal changes in glucose metabolism. The concept of multifocal cortical degeneration leads to the view of multiple structural and functional subsystem disruption in AD.

The neurotransmitter serotonin has been implicated in numerous physiological functions and pathophysiological disorders. The hydroxylation of the aromatic amino acid tryptophan is rate-limiting in the synthesis of serotonin. Tryptophan hydroxylase (TPH), as the rate-limiting enzyme, determines the concentrations of serotonin in vivo. Relative serotonin concentrations are clearly important in neural transmission, but serotonin has also been reported to function as a local antioxidant. Identification of the mechanisms regulating TPH activity has been hindered by its low levels in tissues and the instability of the enzyme. Several TPH expression systems have been developed to circumvent these problems. In addition, eukaryotic expressions systems are currently being developed and represent a new avenue of research for identifying TPH regulatory mechanisms. Recombinant DNA technology has enabled the synthesis of TPH deletions, chimeras, and point mutations that have served as tools for identifying structural and functional domains within TPH. Notably, the experiments have proven long-held hypotheses that TPH is organized into N-terminal regulatory and C-terminal catalytic domains, that serine-58 is a site for PKA-mediated phosphorylation, and that a C-terminal leucine zipper is involved in formation of the tetrameric holoenzyme. Several new findings have also emerged regarding regulation of TPH activity by posttranslational phosphorylation, kinetic inhibition, and covalent modification. Inhibition of TPH by L-DOPA may have implications for depression in Parkinson's disease (PD) patients. In addition, TPH inactivation by nitric oxide may be involved in amphetamine-induced toxicity. These regulatory concepts, in conjunction with new systems for studying TPH activity, are the focus of this article.

A technique is described for determining the apolipoprotein E genotype (apo E; alleles ϵ 2, ϵ 3, or ϵ 4) from tissues which have been fixed with 4–10% formaldehyde and archived. The procedure requires efficient extraction and exhaustive purification of DNA from the fixed tissue. Because the fixation process renders the DNA largely crosslinked and/or sheared (therefore unsuitable for traditional analysis), a nested polymerase chain reaction (PCR) is employed (using two apo E gene specific primer pairs) to specifically amplify the polymorphic region of the gene. The genotype was then determined using previously reported Hha I polymorphisms that occur as a direct result of the variant codons responsible for the three alleles. This protocol permitted the successful genotyping of 90% (34 out of 38) of the archived brain samples from Alzheimer's disease (AD) patients. These samples included such extremes as a sample that had been stored for 12 years in formalin. This procedure permits the retrospective analysis of samples that had been processed and stored well before the original characterization of apo E alleles as risk factors in AD. Finally, this approach is readily adapted to the analysis of any gene of interest, whether by restriction fragment length polymorphism or direct amplicon DNA sequencing. It is also a very robust assay for less stringent conditions such as DNA isolated from whole blood or frozen tissue.

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxylterminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37°C, TPH was much less stable (t 1/2=28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by at 1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.

Dopamine-induced DNA damage was studied in vitro in the presence of the enzyme tyrosinase. Dopamine auto-oxidizes to form dopamine quinone, a reactive molecule which spontaneously decomposes to form additional reactive species that can modify cellular macromolecules. The conversion of dopamine to reactive dopamine quinone is accelerated by the enzyme tyrosinase. The objective of this study was to evaluate whether dopamine autoxidation would lead to DNA-reactive intermediates and whether tyrosinase would increase the rate of this reaction. Incubation of DNA with [3H]dopamine resulted in the concentration-dependent covalent incorporation of the labeled catecholamine into precipitable nucleic acid (DNA adduct formation). The presence of tyrosinase increased the incorporation by as much as two orders of magnitude. Antioxidants markedly reduced this incorporation, suggesting that dopamine free-radicals were critical in DNA modification. DNA adducts formed by dopamine in the presence of tyrosinase were visualized using 32P-postlabeling and thin layer chromatography. The results suggest that DNA modification by dopamine is accelerated by tyrosinase which, in turn, could contribute to destruction of dopaminergic neurons in vivo.