Brain Creatine Kinase Research Articles

Community of caring: A continuum of normalized evidence-based programs to maintain function

ization time of flight MS, and conclusive protein identifications accomplished by liquid chromatography-tandem MS. Conclusions: Several proteins were reproducibly quantitatively affected by GSE in rat brain, either in abundance, or in isoform complexity. The majority were “old” proteins, i.e. those that had been shown to be differentially expressed in AD or transgenic mouse models of dementia (i.e. creatine kinase brain beta chain, glial fibrillary acidic protein). Because the direction of change for most proteins affected by GSE in the normal healthy rats was opposite to that in AD or transgenic demented mouse brain, administration of GSE to healthy adults to protect critical proteins may be a strategy for prevention of AD.

Rapid Discovery of Putative Protein Biomarkers of Traumatic Brain Injury by SDS–PAGE–Capillary Liquid Chromatography–Tandem Mass Spectrometry

We report the rapid discovery of putative protein biomarkers of traumatic brain injury (TBI) by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry (SDS-PAGE-Capillary LC-MS(2)). Ipsilateral hippocampus (IH) samples were collected from naive rats and rats subjected to controlled cortical impact (a rodent model of TBI). Protein database searching with 15,558 uninterpreted MS(2) spectra, collected in 3 days via data-dependent capillary LC-MS(2) of pooled cyanine dye-labeled samples separated by SDS-PAGE, identified more than 306 unique proteins. Differential proteomic analysis revealed differences in protein sequence coverage for 170 mammalian proteins (57 in naive only, 74 in injured only, and 39 of 64 in both), suggesting these are putative biomarkers of TBI. Confidence in our results was obtained by the presence of several known biomarkers of TBI (including alphaII-spectrin, brain creatine kinase, and neuron-specific enolase) in our data set. These results show that SDS-PAGE prior to in vitro proteolysis and capillary LC-MS(2) is a promising strategy for the rapid discovery of putative protein biomarkers associated with a specific physiological state (i.e., TBI) without a priori knowledge of the molecules involved.

Identification of mouse brain proteins associated with isoform 3 of metallothionein

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.

Transcription of brain creatine kinase in U87-MG glioblastoma is modulated by factor AP2

Our previous studies established in U87-MG glioblastoma cells that elevated cAMP increased transcription of the endogenous as well as a transiently-transfected brain creatine kinase (CKB) gene, despite the absence of a cAMP response element (CRE) in the CKB proximal promoter. This report employed transfection to show that the transcription of CKB in U87 cells is induced by transcription factor AP2α, which is known to be activated by cAMP. Dominant-negative forms of AP2α not only prevented the AP2α-mediated activation of CKB but also blocked the cAMP-mediated increase in CKB transcription caused by forskolin treatment. The mutation of the four potential AP2 elements within the CKB proximal promoter showed that induction of CKB by AP2 was mediated principally through the AP2 element located at −50 bp in the promoter. Electromobility shift assays revealed a protein in U87 nuclear extracts that bound to a consensus AP2α element as well as to the (−50) AP2 element in CKB. Interestingly, the CKB (−50) AP2 element contains GCCAATGGG which also bound NF-Y, the CCAAT-binding protein, suggesting that interplay between AP2 and NF-Y may modulate CKB transcription. This is the first report of a role for AP2 in the regulation of CKB transcription and of an AP2 element within which an NF-Y site is located.

The top 10 most abundant transcripts are sufficient to characterize the organs functional specificity: evidences from the cortex, hypothalamus and pituitary gland

Using serial analysis of gene expression, we have identified the most abundant mRNA transcripts in parietal cortex, hypothalamus and pituitary gland in adult male mice. High mRNA abundance of neurogranin (cell signalling and communication) was characteristic of the cortex. The common molecular features of cortex and hypothalamus were high abundance of mRNA encoding mitochondrial enzymes such as reduced form of nicotinamide adenine dehydrogenase (NADH) 4 and cytochrome c oxidase 2 (energy metabolism), brain creatine kinase (energy metabolism) and myelin basic protein (cell structure). In the hypothalamus, mRNA levels of apolipoprotein E (lipid metabolism), prostaglandin D2 (cell signalling and communication) and secreted acidic cysteine-rich glycoprotein (extracellular matrix) were especially high. A common molecular feature of the hypothalamus and pituitary was high mRNA abundance of guanine nucleotide binding protein alpha stimulating complex locus (cell signalling and cell communication). The pituitary gland was characterized by high expression of genes encoding hormones such as growth hormone, pro-opiomelanocortin and prolactin, as well as neuronatin (cell differentiation) and four potential novel transcripts. Thus, these results show that the cortex, hypothalamus and pituitary gland can be specifically characterized according to their 10 most abundant transcripts. In addition, the current study serves as a basis for future studies on the potential novel transcripts and the transcripts with unclear functions despite their extremely high abundance, as well as studies on physiology and pathology of the two brain regions and pituitary gland.

Expression of Creatine Kinase Isoenzyme Genes during Postnatal Development of Rat Brain Cerebrum: Evidence for Posttranscriptional Regulation

Brain creatine kinase (CKB) has a central role in the regeneration of ATP in the brain. During postnatal development of rat brain cerebrum, the CKB protein level was very low at postnatal day 1 and week 1 but by week 4 had increased 6- to 7-fold and remained constant through week 10. Surprisingly, CKB mRNA levels were already maximal at postnatal day 1 and week 1, indicating that CKB protein expression does not simply reflect the levels of CKB mRNA and is likely regulated posttranscriptionally during early postnatal times. Interestingly, the majority of cytoplasmic CKB mRNA was found to be associated with polyribosomes both at postnatal day 3 and week 6. Therefore, low CKB protein levels at early postnatal times could either be due to (1) normal translation initiation of CKB mRNA followed by a subsequent arrest during elongation or termination and/or (2) normal translation of CKB mRNA followed by rapid degradation of CKB protein. However, CKB protein increased coincidently with ubiquitous mitochondrial CK protein, suggesting that a functional phosphocreatine energy shuttle is formed in the cerebrum during postnatal development. The apparent posttranscriptional regulation of CKB in early postnatal cerebrum contrasts with the transcriptional regulation controlling accumulation of CKB protein in postnatal developing cerebellum.

Identification of an I(2) binding protein from rabbit brain.

Imidazoline-2 binding proteins exist as a heterogeneous population. The aim of this study was to isolate and identify I(2) binding proteins from rabbit brain using an affinity column synthesized with a highly selective I(2) ligand, 2-(2-benzofuranyl)2-imidazoline (2BFI). The results revealed an approximately 45-kD protein to be brain creatine kinase (EC 2.7.3.2). [(3)H]-2BFI (5nM) was able to bind specifically to the purified enzyme. This study has identified brain creatine kinase as a novel I(2) binding protein.

The study of the creatine kinase in rat brain during ischemia by magnetization transfer and biochemical analysis

Various methods are used to study the biochemical changes in the central nervous system under normal and pathological conditions. The magnetization transfer 31P magnetic resonance technique was used here to measure the creatine kinase (CK) reaction rate constant in vivo in rats with cerebral ischemia. The measurements indicated that the rate constant of the CK reaction was significantly reduced in the case of chronic brain ischemia in aged rats. The similar reduction of the creatine kinase activity was found in the ischemic rat brain homogenate measured by biochemical analysis. At the same time, corresponding conventional phosphorus magnetic resonance spectra showed negligible or no change in signal intensities of compounds containing macroergic phosphates.Acknowledgements: This work was supported by the Grant Category C and Comenius University Grant No. X/2003.

Sexual dimorphism in rat cerebrum and cerebellum: different patterns of catalytically active creatine kinase isoenzymes during postnatal development and aging

During postnatal development, maturation and aging the Wistar rat cerebrum and cerebellum synthesize, in a different sex-dependent manner, catalytically active dimeric cytosolic (c) muscle-type (MM) and heart-type (MB) creatine kinase (CK), besides the supposedly sole type brain-specific (BB) CK. In both sexes, typical and atypical neuromuscular cCK isoenzymes were present during the study for 26 months. As in rat heart, females showed more cerebral cCK variants (41%) in comparison to males. Female rats exhibited about 93% more cerebellar variants of cCK isoenzymes as compared to males. The male cerebellum showed predominantly BB- and MB-CK during the whole study in comparison to the female one that contained all neuromuscular cCK variants. Only female rats showed decreases and increases of cerebral CK specific activity. In contrast to males, coinciding with the weaning period, cerebral female CK activity decreased 45% from 14 to 21 days and increased about 3-fold in female rats and only 1.3-fold in males from 21 to 45 days of age. Contrary to the remarkable 4-fold increase of chicken brain CK specific activity exhibited at old age, the rat did not show another cerebral CK activity increase during senescence in either sex. However, sex differences of CK specific activity appeared in the cerebellum at all ages. From the sex-specific plateau phase at 45–60 days until 2.2 years of age, about a 41% independent increase of cerebellar CK specific activity was observed in both sexes. After puberty, the differential cerebellum–cerebrum values of CK specific activity were higher for female rats than males during youth, adulthood and senescence. The present work shows that in rat cerebrum and cerebellum, production of ATP through anaerobic transphosphorylation by the CK/PC system is sex-and age-specific, especially in the cerebellum, when glycolysis and the Krebs cycle lose capacity. As in rat heart, under physiological conditions at all ages the several cCK isoenzymes do participate in a gender-specific manner, in favor of females, in diverse functions of the different cell compartments of glial and neuronal cells with regard to their high and fluctuating energy demands not completely covered by anaerobic and aerobic glycolysis.

Transgenic livers expressing mitochondrial and cytosolic CK: mitochondrial CK modulates free ADP levels.

The function of creatine kinase (CK) and its effect on phosphorus metabolites was studied in livers of transgenic mice expressing human ubiquitous mitochondrial CK (CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination. (31)P NMR spectroscopy and saturation transfer were recorded in livers of anesthetized mice to measure high-energy phosphates and hepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxes mediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28-560 micromol. g wet wt(-1). min(-1)), the fraction of phosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrations calculated by assuming equilibrium of the CK-catalyzed reaction in vivo decreased from 84 +/- 9 to 38 +/- 4 nmol/g wet wt. Calculated free ADP levels in mice expressing high levels of CK-B (920-1,635 micromol. g wet wt(-1). min(-1)) were 52 +/- 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 +/- 4 nmol/g wet wt. These findings indicate that CK-Mit catalyzes its reaction equally well in both directions and can lower hepatic apparent free ADP concentrations.

Inhibition of creatine kinase activity in vitro by ethylmalonic acid in cerebral cortex of young rats.

Short-chain acyl-CoA dehydrogenase deficiency is an inherited metabolic disorder biochemically characterized by tissue accumulation of ethylmalonic (EMA) and methylsuccinic (MSA) acids and clinically by severe neurological symptoms. In the present study we investigated the in vitro effects of EMA and MSA on the activity of creatine kinase (CK) in homogenates from cerebral cortex, skeletal and cardiac muscle of rats. EMA significantly inhibited CK activity from cerebral cortex, but did not affect this activity in skeletal and cardiac muscle. Furthermore, MSA had no effect on this enzyme in all tested tissues. Glutathione (GSH), ascorbic acid and alpha-tocopherol, and the nitric oxide synthase inhibitor L-NAME, did not affect the enzyme activity per se, but GSH fully prevented the inhibitory effect of EMA when co-incubated with EMA. In contrast, alpha-tocopherol, ascorbic acid and L-NAME did not influence the inhibitory effect of the acid. The data suggest that inhibition of brain CK activity by EMA is possibly mediated by oxidation of essential groups of the enzyme, which are protected by the potent intracellular, endogenous, naturally occurring antioxidant GSH.

Bioenergetics of Adaptation to a Salinity Transition in Euryhaline Teleost (Oreochromis mossambicus) Brain

Freshwater (FW) teleosts are capable of acclimating to seawater (SW) following such a transfer from FW. However, their osmoregulating mechanisms are still unclear, particularly those in the brain. The present study was conducted to examine acute changes that occur in brain Na(+)-K(+)-ATPase activity, creatine kinase (CK) activity, creatine, creatinine contents, and ATP levels of tilapia (Oreochromis mossambicus) in response to this transition. After transfer to SW (25 ppt), the Na(+)-K(+)-ATPase activity was maintained for 8 hr at higher levels than that in FW. In contrast, in 35 ppt SW, Na(+)-K(+)-ATPase was maintained at a even higher level than in FW for the first 2 hr. Brain Na(+)-K(+)-ATPase contents in both the 25 and 35 ppt SW groups were significantly elevated within 1 and 0.5 hr after transfer from FW, respectively. Interestingly, brain CK activities and content (homodimer of the B subunit [BB] form) in both the 25 and 35 ppt SW groups were significantly elevated within 1 hr after transfer from FW. The ATP contents in 35 ppt SW increased abruptly within 0.5 hr, and then gradually decreased during the next 2 hr. Unlike the 35 ppt group that declined in ATP contents, the 25 ppt group leveled off within 24 hr. The elevations in CK activity and creatine levels after transfer from FW to SW imply that abrupt salinity changes alter phosphocreatine/CK ratio. Such changes are needed to satisfy the increases in the energetic requirement of the cotransport mechanisms mediating osmoregulation.

511 Normal range of creatine kinase brain isoenzyme (BB-CK) activity in plasma of fetuses during the second trimester

511 Normal range of creatine kinase brain isoenzyme (BB-CK) activity in plasma of fetuses during the second trimester

Altered brain phosphocreatine and ATP regulation when mitochondrial creatine kinase is absent.

In cerebral gray matter, ATP concentration is closely maintained despite rapid, large increases in turnover and low substrate reserves. As seen in vivo by (31)P nuclear magnetic resonance (NMR) spectroscopy, brain ATP is stable early in seizures, a state of high energy demand, and in mild hypoxia, a state of substrate deficiency. Like other tissues with high and variable ATP turnover, cerebral gray matter has high phosphocreatine (PCr) concentration and both cytosolic and mitochondrial creatine kinase (UbMi-CK) isoenzymes. To understand the physiology of brain creatine kinases, we used (31)P NMR to study PCr and ATP regulation during seizures and hypoxia in mice with targeted deletion of the UbMi-CK gene. The baseline CK reaction rate constant (k) was higher in mutants than wild-types. During seizures, PCr and ATP decreased in mutants but not in wild-types. The k-value for the CK catalyzed reaction rate increased in wild-types but not in the mutants. Hypoxic mutants and wild-types showed similar PCr losses and stable ATP. During recovery from hypoxia, brain PCr and ATP concentrations returned to baseline in wild-types but were 20% higher than baseline in the mutants. We propose that UbMi-CK couples ATP turnover to the CK catalyzed reaction rate and regulates ATP concentration when synthesis is increased.

Brain Creatine Kinase and Creatine Transporter Proteins in Normal and Creatine-Treated Rabbit Pups

Systemic creatine (Cr) supplementation increases brain phosphocreatine (PCr) and prevents hypoxic seizures in 15-day-old rabbits . Between 5 and 30 days of age during normal development, rabbit gray matter mitochondrial creatine kinase (Mi-CK) increases 400% while cytosolic CK (BB-CK) increases 60%. In white matter, both isoenzymes show smaller, similar increases (40%) during this period. The Cr transporter protein decreases 60% between 5 and 15 days in both regions. In vivo CK rate constants measured by ³¹P nuclear magnetic resonance increase 30% between 10 and 20 days, and then fall 50% between 20 and 30 days in predominantly gray matter slices. Similar maturational changes are seen in predominantly white matter slices. Injecting Cr at 15 days does not significantly change BB-CK or Mi-CK isoenzymes or the in vivo CK reaction rate constants. Thus, the largest change in the CK system associated with suppression of hypoxic seizures in Cr-treated rabbits is increased PCr in gray and white matter.

Opposite transitions of chick brain catalytically active cytosolic creatine kinase isoenzymes during development

Postnatally the rat brain synthesizes catalytic forms of muscle type (MM) and heart type (MB) creatine kinase (CK), besides the supposedly sole type vertebrate brain-specific (BB) CK. We intended to demonstrate that in Rhode Island chicken brain, cytosolic (c) CK isoenzymatic transitions, (for example BB–CK is followed by the appearance of MB–CK and MM–CK during muscle differentiation), can also occur during development and aging. Cytosolic post 125 000×g, mitochondrial CK-free, brain samples were obtained for zone electrophoresis separation and identification of catalytically active cCK isoforms. BB–CK was never found during chicken brain ontogeny. Against the accepted view, an opposite isoenzyme transition pattern from MM through BB–CK was found in the chicken embryonic brain from the very early stages of development up to day 2 post-hatching. At very early stages of chicken brain ontogeny constitutive MM– and MB–CK isoenzymes were present before the advent of creatine. It seems to be that typical and atypical brain MM– and MB–CK could be working as ATPases in the absence of creatine before embryonic stage 28 (day 5.5) and/or such CK isoforms may begin to form part of the slow component b in developing early neurons and later in the nuclei of glial cells to be used by the CK/phosphocreatine (PC) system as the neural tissues mature. The post-hatching transition pattern showed simultaneous expression of more than one CK isoenzyme within the same neural sample as in post-natal rat brain, presumably due to regional differential transphosphorylation requirements. Strain-dependent enzymatic specific activities have been reported in several species. Since equivalent values of brain CK specific activity were obtained previously from the embryonic plateau phase of CK activity during White Leghorn development, and those from Rhode Island brain neurons cultured 11 days, we compared if, in vivo, a similar brain CK specific activity pattern was physiologically equivalent during Rhode Island and White Leghorn chicken ontogeny. We found quantitatively different strain-specific CK specific activity patterns during this period. Rhode Island brain CK activity values were approximately 4.5-fold those of White Leghorn ones. This indicates that production of energy from anaerobic metabolism and transphosphorylation by the CK/PC system to synthesize ATP more efficiently is strain-specific. In Rhode Islands, there was an age-dependent increase of CK specific activity, mostly in older animals (440% above the value found during the embryonic plateau), when the Krebs cycle and glycolysis lose capacity. During adult life and aging, under physiological conditions, the three CK isoenzymes may participate in diverse functions of the different cell compartments of brain glia and neurons with regard to their high and fluctuating energy demands that are not completely covered by anaerobic and aerobic glycolisis.

Creatine kinase isoenzymes specificities: histidine 65 in human CK-BB, a role in protein stability, not in catalysis

Creatine kinases (CK) play a prominent role in cell energy distribution through an energy shuttle between mitochondria and other organelles. Human brain CK was cloned and overexpressed in COS-7 cells. We then deleted His-65 and/or Pro-66 situated near the center of a flexible loop as shown by X-ray crystallography on mitochondrial and cytosolic CK. The ΔH65 mutant had nearly the same affinity for its substrates as wild isoenzyme, but its stability was very low. Unlike ΔH65, ΔH65P66 had a eightfold decreased affinity for creatine phosphate and was unable to dephosphorylate cyclocreatine phosphate. Our results demonstrate that, despite an overall similar shape of the proteins, this loop accounts for some subtle differences in isoenzyme functions.

Maturational changes in rabbit brain phosphocreatine and creatine kinase.

Annals of the New York Academy of SciencesVolume 893, Issue 1 p. 309-313 Maturational Changes in Rabbit Brain Phosphocreatine and Creatine Kinase T. KEKELIDZE, Corresponding Author T. KEKELIDZE Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA Corresponding author: Téa Kekelidze, Ph.D., Department of Radiology, Brigham and Women's Hospital, LMRC, 221 Longwood Avenue, Boston, Massachusetts 02115. Phone: 617- 732-5623; fax: 617- 278-0610. e-mail: [email protected]Search for more papers by this authorI. KHAIT, I. KHAIT Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USASearch for more papers by this authorA. TOGLIATTI, A. TOGLIATTI Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USASearch for more papers by this authorJ. BENZYCRY, J. BENZYCRY Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USASearch for more papers by this authorR. MULKERN, R. MULKERN Department of Radiology, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USASearch for more papers by this authorD. HOLTZMAN, D. HOLTZMAN Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USASearch for more papers by this author T. KEKELIDZE, Corresponding Author T. KEKELIDZE Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA Corresponding author: Téa Kekelidze, Ph.D., Department of Radiology, Brigham and Women's Hospital, LMRC, 221 Longwood Avenue, Boston, Massachusetts 02115. Phone: 617- 732-5623; fax: 617- 278-0610. e-mail: [email protected]Search for more papers by this authorI. KHAIT, I. KHAIT Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USASearch for more papers by this authorA. TOGLIATTI, A. TOGLIATTI Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USASearch for more papers by this authorJ. BENZYCRY, J. BENZYCRY Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USASearch for more papers by this authorR. MULKERN, R. MULKERN Department of Radiology, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USASearch for more papers by this authorD. HOLTZMAN, D. HOLTZMAN Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USASearch for more papers by this author First published: 06 February 2006 https://doi.org/10.1111/j.1749-6632.1999.tb07844.xCitations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume893, Issue1OXIDATIVE/ENERGY METABOLISM IN NEURODEGENERATIVE DISORDERSNovember 1999Pages 309-313 RelatedInformation

Conditions providing enhanced transfection efficiency in rat pheochromocytoma PC12 cells permit analysis of the activity of the far-upstream and proximal promoter of the brain creatine kinase gene

While brain creatine kinase (CKB) is expressed at highest levels in the brain, where it functions in regenerating ATP, the gene elements and protein factors regulating CKB transcription in neuronal and glial cells have not been identified. To investigate the regulation of CKB in neuronal cells, we examined the expression of the promoter proximal and 5′ far-upstream regions of the rat CKB gene transiently transfected into rat PC12 pheochromocytoma cells. Initially, these experiments were hampered by the extremely low transfection efficiency of PC12 cells. We increased efficiency by greater than 200-fold by employing CaPO 4-precipitated DNA transfection into PC12 cells which were optimized for transient transfection by: (i) culturing cells in polylysine-coated dishes to insure attachment throughout transfection; (ii) exposing cells to transfected DNA for an optimal time and employing a glycerol shock; and, most importantly, (iii) dissociating the characteristic self-adhesive clumps of PC12 into mostly single cells. Use of the plasmid expressing green fluorescent protein allowed identification of the transfected cells that averaged 10–20% of the total. Analyses of CKB promoter-CAT gene constructs showed that in PC12 cells expression of the proximal (0.2 kb) CKB promoter was low while expression of the 1.4 kb promoter was three fold higher and the 2.9 kb promoter was ten fold higher, suggesting the presence of at least two upstream cis-acting, positive regulatory elements. In agreement, the steady-state CKB mRNA level was higher in PC12 than in other neuronal cell lines examined, possibly reflecting the effects of positive upstream factors. The results are discussed in relation to how this economical and straightforward transfection procedure may be useful in identify factors regulating the transcription of CKB and other genes expressed in neuronal cells.

Proximal promoter of the rat brain creatine kinase gene lacks a consensus CRE element but is essential for the cAMP‐mediated increased transcription in glioblastoma cells

Proximal promoter of the rat brain creatine kinase gene lacks a consensus CRE element but is essential for the cAMP‐mediated increased transcription in glioblastoma cells