Integrating glutathione metabolism and mitochondrial dysfunction with implications for Parkinson's disease: a dynamic model.

UNLABELLED: Oxidative/nitrosative stress and mitochondrial dysfunction have been implicated in the degeneration of dopaminergic neurons in the substantia nigra during Parkinson's disease (PD). During early stages of PD, there is a significant depletion of the thiol antioxidant glutathione (GSH), which may lead to oxidative stress, mitochondrial dysfunction, and ultimately neuronal cell death. Mitochondrial complex I (CI) is believed to be the central player to the mitochondrial dysfunction occurring in PD. We have generated a dynamic, mechanistic model for mitochondrial dysfunction associated with PD progression that is activated by rotenone, GSH depletion, increased nitric oxide and peroxynitrite. The potential insults independently inhibit CI and other complexes of the electron transport chain, drop the proton motive force, and reduce ATP production, ultimately affecting the overall mitochondrial performance. We show that mitochondrial dysfunction significantly affects glutathione synthesis thereby increasing the oxidative damage and further exacerbating the toxicities of these mitochondrial agents resulting in neurodegeneration. Rat dopaminergic neuronal cell culture and in vitro experiments using mouse brain mitochondria were employed to validate important features of the model. MAJOR CONCLUSIONS: Using a combination of experimental and in silico modeling approaches, we have demonstrated the interdependence of mitochondrial function with GSH metabolism in relation to neurodegeneration in PD.

Dynamic modeling of alpha-synuclein aggregation for the sporadic and genetic forms of Parkinson's disease.

Excessive accumulation of alpha synuclein (a-syn) in the brain has been implicated in several degenerative neurological disorders, most notably Parkinson's disease. The aggregation of a-syn is the major component of intraneuronal inclusions, Lewy bodies, which are neuropathological features, observed in Parkinson's disease, Lewy body dementia, and other synucleopathies. Diverse cellular events can contribute to a-syn accumulation, aggregation, and to subsequent Lewy body formation. These factors include genetic mutations of synuclein, parkin, or the deubiquitinating enzyme, ubiquitin C-terminal hydrolase (UCH-L1), leading to reduced clearance of a-syn by the ubiquitin proteasomal pathway (UPP). Furthermore, intracellular insults include environmental factors and an age-related decrement in antioxidant defense systems that increase oxidative stress and can affect either the accumulation or clearance of a-syn. We have dynamically modeled a-syn processing in normal and in several disease states; focusing upon alterations in the aggregation and clearance of a-syn as influenced by the UPP and the oxidative stress pathways. Simulation of increased oxidative stress generates a free radical profile analogous to that reported in vivo following exposure to the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Varying model parameters of oxidative stress, UPP dysfunction, or both pathways, simulate kinetics of a-syn that corresponds with the neuropathology described for the sporadic and genetic forms of Parkinson's disease. This in silico model provides a mathematical framework that enables kinetic appraisal of pathway components to better identify and validate important pharmacological targets.

Comparison of vector space model methodologies to reconcile cross-species neuroanatomical concepts.

Generating informational thesauri that classify, cross-reference, and retrieve diverse and highly detailed neuroscientific information requires identifying related neuroanatomical terms and acronyms within and between species (Gorin et al., 2001) Manual construction of such informational thesauri is laborious, and we describe implementing and evaluating a neuroanatomical term and acronym reconciliation (NTAR) system to assist domain experts with this task. NTAR is composed of two modules. The neuroanatomical term extraction (NTE) module employs a hidden Markov model (HMM) in conjunction with lexical rules to extract neuroanatomical terms (NT) and acronyms (NA) from textual material. The output of the NTE is formatted into collections of term- or acronym-indexed documents composed of sentences and word phrases extracted from textual material. The second information retrieval (IR) module utilizes a vector space model (VSM) and includes a novel, automated relevance feedback algorithm. The IR module retrieves statistically related neuroanatomical terms and acronyms in response to queried neuroanatomical terms and acronyms. Neuroanatomical terms and acronyms retrieval obtained from term-based inquiries were compared with (1) term retrieval obtained by including automated relevance feedback and with (2) term retrieval using "document-to-document" comparisons (context-based VSM). The retrieval of synonymous and similar primate and macaque thalamic terms and acronyms in response to a query list of human thalamic terminology by these three IR approaches was compared against a previously published, manually constructed concordance table of homologous cross-species terms and acronyms. Term-based VSM with automated relevance feedback retrieved 70% and 80% of these primate and macaque terms and acronyms, respectively, listed in the concordance table. Automated feedback algorithm correctly identified 87% of the macaque terms and acronyms that were independently selected by a domain expert as being appropriate for manual relevance feedback. Context-based VSM correctly retrieved 97% and 98% of the primate and macaque terms and acronyms listed in the term homology table. These results indicate that the NTAR system could assist neuroscientists with thesauri creation for closely related, highly detailed neuroanatomical domains.

The challenges and rewards of integrating diverse neuroscience information.

The design of database models and schemas for storing, cross-referencing, and retrieving neuroscience information faces issues that are similar but more complex than most of the other biomedical disciplines, such as genomics and proteonomics. Specifically, the visualization and manipulation of very large and diverse image data, such as digital brain atlases and functional magnetic resonance images, play a unique role in neuroscience while much of the associated information is textually recorded. Nongraphical information can include the annotation of large brain structures ranging from anatomical regions to intracellular structures, the description of cellular functional properties, and their various interrelationships, such as fiber connections. It is necessary that the heterogeneous and distributed types of data be cross-referenced to each other so that this diverse information can be efficiently retrieved, shared, and exchanged among the different neuroscientific disciplines. Continued advances in computers and Internet technologies appear to indicate that increasingly large data sets will be maintained on local or regional file servers and that informational interoperability will be achieved using a networked information system infrastructure. The authors and others have proposed and implemented models of semantically organized information systems that utilize centrally stored and highly structured archival information to index, cross-reference, and retrieve diverse, Web-based data sets.

The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase.

Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.

Terminology Query Language: a server interface for concept-oriented terminology systems.

Designers of medical computing applications increasingly require terminology support for their systems. Yet, terminology systems today lack standard methodologies for providing terminology support. This invariably means increased implementation time and expense for system developers who need to use terminologies in their applications. We introduce Terminology Query Language (TQL), a simple query language interface to server implementations of concept-oriented terminologies. TQL is a declarative, set-based query language built on a generic entity-relationship (E/R) schema. TQL defines a common query-based mechanism for accessing terminology information from one or more terminology servers over a network connection.

Role of the sarcoplasmic reticulum in regulating the activity-dependent expression of the glycogen phosphorylase gene in contractile skeletal muscle cells.

Nerve-evoked contractile activity in skeletal muscle regulates transcript and protein levels of many metabolic genes in a coordinate fashion, including the muscle isozyme of glycogen phosphorylase (MGP). Cellular signaling mechanisms mediating the activity-dependent modulation of MGP transcript levels were investigated in a spontaneously contractile rat skeletal muscle cell line (Rmo). Mechanisms regulating MGP mRNA levels in Rmo myotubes were compared with those previously shown to modulate the gene encoding the alpha subunit of the acetylcholine receptor (alphaAChR). Reducing the resting membrane potential from -78 to -30 mV, either electrochemically (KCl) or by increasing Na(+) permeability (veratridine): (1) prevented activation of transverse tubules, (2) impeded calcium release by the sarcoplasmic reticulum (SR), and (3) blocked Rmo contractility. MGP mRNA levels decreased to 30% of control levels and alphaAChR levels increased to 350% following 24 h of depolarization. Differing mechanisms appear to mediate this voltage-dependent regulation of MGP and alphaAChR. Inhibition of SR calcium efflux selectively decreased MGP mRNA levels by 30-50% when using dantrolene, thapsigargin, or a dose of ryanodine shown to inactivate Ca(2+)-induced SR Ca(2+) release (CICR). By contrast, blockade of voltage sensors in transverse tubules with nifedipine, a dihydroaminopyridine (DHAP) antagonist, selectively increased alphaAChR mRNA levels by twofold. These data indicate that the voltage-dependent regulation of AChR gene expression differs from that modulating the MGP gene. KCl-induced depolarization and dantrolene both inhibit pulsatile SR Ca(2+) efflux in Rmo myotubes, but by differing mechanisms. Depolarization and dantrolene comparably reduced MGP mRNA levels and decreased MGP transcript stability from a t(1/2) of 24 h to 14.5 and 16 h, respectively. Reduced transcript stability can account for the observed reduction in mRNA levels of MGP in noncontractile Rmo myotubes and could be a significant regulatory mechanism in skeletal muscle that coordinates the activity-dependent expression of MGP with other glycogenolytic genes.

Stretch-activated ion channels contribute to membrane depolarization after eccentric contractions.

We tested the hypothesis that eccentric contractions activate mechanosensitive or stretch-activated ion channels (SAC) in skeletal muscles, producing increased cation conductance. Resting membrane potentials and contractile function were measured in rat tibialis anterior muscles after single or multiple exposures to a series of eccentric contractions. Each exposure produced a significant and prolonged (>24 h) membrane depolarization in exercised muscle fibers. The magnitude and duration of the depolarization were related to the number of contractions. Membrane depolarization was due primarily to an increase in Na(+) influx, because the estimated Na(+)-to-K(+) permeability ratio was increased in exercised muscles and resting membrane potentials could be partially repolarized by substituting an impermeant cation for extracellular Na(+) concentration. Neither the Na(+)/H(+) antiport inhibitor amiloride nor the fast Na(+) channel blocker TTX had a significant effect on the depolarization. In contrast, addition of either of two nonselective SAC inhibitors, streptomycin or Gd(3+), produced significant membrane repolarization. The results suggest that muscle fibers experience prolonged depolarization after eccentric contractions due, principally, to the activation of Na(+)-selective SAC.

Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes.

Malignant gliomas exhibit alkaline intracellular pH (pH(i)) and acidic extracellular pH (pH(e)) compared with nontransformed astrocytes, despite increased metabolic H(+) production. The acidic pH(e) limits the availability of HCO(-)(3), thereby reducing both passive and dynamic HCO(-)(3)-dependent buffering. This implies that gliomas are dependent upon dynamic HCO(-)(3)-independent H(+) buffering pathways such as the type 1 Na(+)/H(+) exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pH(i) (pH(i) = 7.31-7.48) than normal astrocytes (pH(i) = 6.98), and increased rates of recovery from acidification, under nominally CO(2)/HCO(-)(3)-free conditions. Inhibition of NHE1 in the absence of CO(2)/HCO(-)(3) resulted in pronounced acidification of gliomas, whereas normal astrocytes were unaffected. When suspended in CO(2)/HCO(-)(3) medium astrocyte pH(i) increased, yet glioma pH(i) unexpectedly acidified, suggesting the presence of an HCO(-)(3)-dependent acid loading pathway. Nucleotide sequencing of NHE1 cDNA from the gliomas demonstrated that genetic alterations were not responsible for this altered NHE1 function. The data suggest that NHE1 activity is significantly elevated in gliomas and may provide a useful target for the development of tumor-selective therapies.

Cloning and expression of the Na+/H+ exchanger from Amphiuma RBCs: resemblance to mammalian NHE1.

The cDNA encoding the Na+/H+ exchanger (NHE) from Amphiuma erythrocytes was cloned, sequenced, and found to be highly homologous to the human NHE1 isoform (hNHE1), with 79% identity and 89% similarity at the amino acid level. Sequence comparisons with other NHEs indicate that the Amphiuma tridactylum NHE isoform 1 (atNHE1) is likely to be a phylogenetic progenitor of mammalian NHE1. The atNHE1 protein, when stably transfected into the NHE-deficient AP-1 cell line (37), demonstrates robust Na+-dependent proton transport that is sensitive to amiloride but not to the potent NHE1 inhibitor HOE-694. Interestingly, chimeric NHE proteins constructed by exchanging the amino and carboxy termini between atNHE1 and hNHE1 exhibited drug sensitivities similar to atNHE1. Based on kinetic, sequence, and functional similarities between atNHE1 and mammalian NHE1, we propose that the Amphiuma exchanger should prove to be a valuable model for studying the control of pH and volume regulation of mammalian NHE1. However, low sensitivity of atNHE1 to the NHE inhibitor HOE-694 in both native Amphiuma red blood cells (RBCs) and in transfected mammalian cells distinguishes this transporter from its mammalian homologue.

Nerve-dependent factors regulating transcript levels of glycogen phosphorylase in skeletal muscle.

1. Muscle glycogen phosphorylase (MGP), the rate-limiting enzyme for glycogen metabolism in skeletal muscle, is neurally regulated. Steady-state transcript levels of the skeletal muscle isozyme of MGP decrease significantly following muscle denervation and after prolonged muscle inactivity with an intact motor nerve. These data suggest that muscle activity has an important influence on MGP gene expression. The evidence to this point, however, does not preclude the possibility that MGP is also regulated by motor neuron-derived trophic factors. This study attempts to distinguish between regulation provided by nerve-evoked muscle contractile activity and that provided by the delivery of neurotrophic factors. 2. Steady-state MGP transcript levels were determined in rat tibialis anterior (TA) muscles following controlled interventions aimed at separating the contributions of contractile activity from axonally transported trophic factors. The innervated TA was rendered inactive by daily epineural injections of tetrodotoxin (TTX) into the sciatic nerve. Sustained inhibition of axonal transport was accomplished by applying one of three different concentrations of the antimicrotubule agent, vinblastine (VIN), to the proximal sciatic nerve for 1 hr. The axonal transport of acetylcholinesterase (AChE) was assessed 7, 14, and 28 days after the single application of VIN. 3. MGP transcript levels normalized to total RNA were reduced by 67% in rat TA, 7 days after nerve section. Daily injection of 2 microg TTX into the sciatic nerve for 7 days eliminated muscle contractile activity and reduced MGP transcript levels by 60%. 4. A single, 1-hr application of 0.10% (w/v) VIN to the sciatic nerve reduced axonal transport but did not alter MGP transcript levels in the associated TA, 7 days after treatment. Application of 0.10% VIN to the sciatic nerve also did not affect IA sensory or motor nerve conduction velocities or TA contractile function. 5. Treatment of the sciatic nerve with 0.40% (w/v) VIN for 1 hr reduced axonal transport and decreased MGP transcript levels by 50% within 7 days, but also reduced sensory and motor nerve conduction velocities and depressed TA contractile function. 6. Myogenin, a member of a family of regulatory factors shown to influence the transcription of many muscle genes, including MGP, was used as a molecular marker for muscle inactivity. Myogenin transcript levels were increased following denervation and after treatment with TTX or 0.40% VIN but not after treatment with 0.10% VIN. 7. The results suggest that MGP transcript levels in TA are regulated predominantly by muscle activity, rather than by the delivery of neurotrophic factors. Intrinsic myogenic factors, however, also play a role in MGP expression, since denervation did not reduce MGP transcript levels below 30% of control TA. The dominant influence of activity in the regulation of MGP contrasts with the proposed regulation of oxidative enzyme expression, which appears to depend on both activity and trophic factor influences.

Role of E and CArG boxes in developmental regulation of muscle glycogen phosphorylase promoter during myogenesis.

Muscle glycogen phosphorylase (MGP) transcript and protein levels increase during skeletal muscle development in tandem with the products of other muscle genes responsible for glucose and glycogen metabolism. Previous studies demonstrated that a 269 bp region 5' to exon 1 of MGP is sufficient for developmental regulation in the C2C12 myogenic cell line (Froman et al., 1994). This genomic region (-209 to +60) contains four consensus E box motifs, a CArG-like sequence, and a GC-rich domain. Native MGP transcripts were not detected in pluripotent CH310T1/2 fibroblasts, but low levels of MGP mRNA were measured in CH310T1/2 cells that were stably transfected with MyoD. Three of the E box motifs in the MGP proximal promoter interacted with C2C12 nuclear proteins. However, cotransfection of the MGP promoter with myogenic regulatory factors, including MyoD and myogenin, produced less than 2-fold activation compared with 20-fold activation of the desmin promoter. Mutational analyses of the MGP promoter demonstrated that increased expression in C2C12 myotubes did not require any of the E box motifs or the CArG-like element. A small region (-76 to -68) upstream of GC-rich domain (-64 to -51) significantly reduced promoter activities in both myoblasts and myotubes. The functional studies suggest that MGP is developmentally regulated during myogenesis by alternative pathways that utilize unidentified regulatory elements or ancillary factors.

Botulinum-induced muscle paralysis alters metabolic gene expression and fatigue recovery.

We evaluated the physiological, histochemical, and biochemical consequences of inhibiting contractile activity in rat skeletal muscles with botulinum toxin A (BTX). Contractile activity was entirely eliminated 12-18 h after a single, focal, intramuscular injection of BTX into the rat tibialis anterior muscle (TA). Neuromuscular transmission remained completely inhibited for 10-12 days, then slowly recovered. BTX-treated muscles exhibited a lower resistance to both high- and low-frequency fatigue at 7 and 14 days after injection, but contractile force recovered more rapidly in treated TA after fatigue. Treated TA showed a twofold increase in the activity of the triglyceride hydrolase enzyme lipoprotein lipase (LPL) and a comparable increase in the relative abundance of LPL steady-state mRNA. In contrast, there was a 28% reduction in protein levels of the muscle isozyme of glycogen phosphorylase (MGP) and a 70% decrease in relative MGP transcript levels. Similar changes in relative transcript levels of LPL and MGP were observed in the predominantly fast-twitch extensor digitorum longus after BTX injection, but relative LPL and MGP mRNA levels were not altered in predominantly slow-twitch soleus. Histochemical evidence indicated that fast-twitch glycolytic fibers had increased lipid content. These biochemical alterations were reversed 120 days after BTX treatment despite persistent atrophy.

Prolonged recovery and reduced adaptation in aged rat muscle following eccentric exercise.

We tested the hypothesis that exposure to eccentric (lengthening) contractions results in greater damage and more prolonged recovery in aged rat muscle (32 months) than in adult muscle (6 months), and that the adaptation usually associated with a single exposure to eccentric exercise is reduced in the aged muscle. Experiments were performed using a new rat model for aging studies. Fisher 344/Brown Norway F1 Hybrid. An ankle flexor, the tibialis anterior (TA), was subjected to a series of 24 eccentric contractions in situ and contractile function was assessed 1, 2, 5 and 14 days following. Eccentric exercise produced a similar reduction in maximum specific twitch and tetanic tension in the aged and adult muscles at 1 and 2 days postexercise. Adult muscles recovered by 5 days, while aged TA remained significantly impaired. Aged TA was fully restored by 14 days. Exercise adaptation was tested by subjecting the TA to a second exercise 14 days following the first. Contractile function was determined 2 days following the second exercise. Adult TA maintained its pre-exercise specific force following the second exercise, while aged TA again showed a significant reduction. Thus, a single exposure to eccentric exercise produced complete adaptation in the adult TA, but not in the aged muscles.

Primate rod and cone photoreceptors may differ in glucose accessibility.

PURPOSE: Glucose is crucial for the function of retinal photoreceptors, other retinal neurons, and glial cells. Exogenous glucose can be extracted from the retinal and choroidal circulation, and endogenous glucose may be generated from breakdown of intracellular glycogen stores. Because glucose deprivation is a critical component of retinal ischemia, the authors sought to determine the sites of glucose entry into and generation within the retina. METHODS: The localization of the glucose transporter, GluT-1, and the brain and muscle isozymes of glycogen phosphorylase, GlyP, was studied by immunohistochemistry of adult human and monkey retinas. RESULTS: Brain glycogen phosphorylase (B-GlyP) immunoreactivity was found in cone, but not rod, photoreceptors. There was immunostaining of foveal and peripheral cones throughout the cytoplasm from the outer segment to the synaptic pedicle. Short wavelength ("blue") cones were positive for B-GlyP. Diffuse staining of the inner and outer plexiform and the nerve fiber layers did not resemble the distinct morphology of Müller cells. Immunoreactivity to muscle GlyP (M-GlyP) was confined to selected synaptic layers of the inner plexiform layer in monkey retina. Staining with antibody to GluT-1 demonstrated diffuse reactivity throughout the retina, including the blood-retinal barrier cells, retinal pigment epithelium, and vascular endothelium. Ultrastructural immunohistochemistry showed staining of rod and cone inner and outer segments. CONCLUSIONS: These immunohistochemical studies indicate that rod and cone photoreceptors have the biochemical capability to transport exogenous glucose from the circulation. Only cones appear capable of using endogenous glycogen stores. These findings imply that cones could be more resistant to acute reductions in circulating glucose during hypoglycemia. However, during hypoxic insult, glycogenolysis and anaerobic glycolysis could result in increased production of intracellular lactic acid, potentially predisposing the cone to acidotic damage.

A subgroup of human VH3 germline genes that encode a high-avidity synovial rheumatoid factor.

We have previously derived and identified a highly avid monoclonal IgM rheumatoid factor (mRF), C6, from unstimulated rheumatoid synovial cells (RSC). At the time, the closet VH germline gene, VH26, demonstrated only 88% homology with C6. To identify the germline counterpart of C6, genomic DNA from the same rheumatoid arthritis (RA) patient from whom C6 was derived was used in the polymerase chain reaction (PCR). Four of the six closely related germline genes that we sequenced had exonic regions that were identical with the VH region of C6 cDNA. These six germline sequences differed in their intronic regions, suggesting that they were distinct, but closely related genomic sequences. To further evaluate the extent of these related genes we identified nine additional germline genes having VH-encoding exons that were 86-97% identical to the C6 cDNA sequence. Furthermore, we examined the polymorphic nature of the C6 VH gene using single strand conformation polymorphism (SSCP), and identified two peaks, confirming the existence of highly homologous genes. The sequence and polymorphism data suggest that: (1) the VH region of the high avidity mRF C6 was derived from an unmutated germline gene; (2) C6 was encoded by a VH gene belonging to a set of homologous genes within the larger VH3 family; and (3) in addition to somatic rearrangements of B-cell genes and antigen-driven somatic mutation, gene duplication and conversion events of germline genes could be important in generating diversity and polyclonality among high-affinity pathogenic autoantibodies.

Regulation of the rat muscle glycogen phosphorylase-encoding gene during muscle cell development.

The muscle isozyme of glycogen phosphorylase (MGP) catalyzes the hydrolysis hydrolysis of intracellular glycogen in mammalian tissues and is produced in skeletal muscle, brain and heart. The MGP gene is developmentally and neutrally regulated in skeletal muscle, but little is known about the gene's transcriptional regulation. We have isolated and characterized the 5' flanking region of rat MGP. Truncated portions of the MGP 5' flanking region were coupled to the bacterial cat reporter gene and used in transient transfection assays in the mouse muscle C2C12 cell line. The region between -211 and +62 contained the smallest regulatory domain capable of demonstrating developmentally regulated myogenic expression in C2C12 cells. This was in contrast with findings from another investigation that transfected this cell line with human MGP [Lockyer and McCracken, J. Biol. Chem. 266 (1991) 20262-20269]. A 172-nucleotide (nt) region between -839 and -666 functioned as a potent enhancer in C2C12 cells when coupled to its cognate promoter, but not when coupled to a simian virus 40 promoter. This rat MGP enhancer region is 78% identical to a comparable region of the human MGP 5' flanking region, but contains only one putative regulatory element that has been previously identified in other muscle genes. These data suggest that rat MGP transcription in C2C12 muscle cells is modulated by a potent enhancer that utilizes novel regulatory elements.

Characterization of the 5' flanking region of the gene encoding rat liver glycogen phosphorylase.

A genomic region encompassing 800 bp of the promoter-regulatory region and exon 1 of the gene (LGP) encoding rat liver glycogen phosphorylase has been isolated and characterized. Transcripts of the LGP gene initiate predominantly within an 8-bp region 48-bp upstream from the start codon. Additional transcripts were detected that initiate as far as 95 bp upstream from the start codon. To identify cis-acting sequences involved in regulating transcription, HepG2 cells were transfected with vectors containing serial deletions of the promoter-regulatory region of LGP ligated to the cat reporter gene. Two upstream regions were found to enhance transcription. One of these regions contains an alternating purine-pyrimidine sequence. LGP, which lacks a consensus TATA sequence, is like TATA-less and CAAT-less housekeeping genes in that it contains G + C-rich domains upstream from multiple transcription start points. Nuclear proteins from adult rat tissues bound in a tissue-specific fashion to one of these G + C-rich regions.