Electrocortical desynchronization after microinfusion of kainic acid into the locus coeruleus in rats

CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system. We successfully cultivated CD-1 cells to a very high density (over 1 x 10(7) cells/ml). Prourokinase was stably secreted at about 180 IU/10(6) cells/24 h. Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers, then reattach and proliferate on fresh microcarriers. This makes it very easy to scale up production. The microcarriers could be reused several times without affecting adhesion, proliferation and prourokinase secretion. With CM-PECC membrane radial flow chromatography and MPG chromatography, the prourokinase in conditioned medium could be purified to a specific activity of 1 x 10(5) IU/mg of protein. The purification factor was about 600 fold, and approximately 90% of the biological activity was recovered.     

The role of the locus coeruleus and N-methyl-D-aspartic acid (NMDA) and AMPA receptors in opiate withdrawal

The turnover rates of plasma lactate, normalized for O2 consumption rate, are higher in the fetus than in the adult. This occurs despite very low rates of fetal gluconeogenesis which preclude the recycling of lactate carbon into glucose. In an effort to establish the main routes of disposal of fetal plasma lactate, 12 midgestation ovine fetuses (age 74 +/- 1 days) were infused intravenously at constant rate with L-[U-14C]lactate for a 4-hour period. At the end of the infusion, the amounts of 14C retained by the fetus and by the placenta, and the distribution of the retained 14C in free and protein-bound amino acids and in lipids were measured. Of the total 14C infused, 17.0 +/- 1.4% was recovered in the placenta, 4.0 +/- 0.3% in the fetal liver, and 15.0 +/- 0.8% in the extrahepatic fetal tissues. Of the retained radioactive carbon, 45-57% was recovered in the free and protein-bound amino acid fractions and 11-17% in the lipid fractions. Approximately 90% of the 14C in the free amino acid fractions was present as glutamate/glutamine, serine, glycine, and alanine carbon. In conjunction with data on fetal CO2 production from lactate carbon, these results demonstrate that the main routes of fetal lactate disposal are oxidation and synthesis of nonessential amino acids and lipids.     

Injection of corticotropin-releasing hormone into the locus coeruleus or foot shock increases neuronal Fos expression

Previous research suggests that corticotropin-releasing hormone can act in the locus coeruleus to increase the firing of locus coeruleus neurons and elicit physiological responses resembling those associated with stress. The present study used immunocytochemical detection of Fos as a measure of neuronal activation to identify brain areas that were activated by bilateral injections of corticotropin-releasing hormone into the locus coeruleus of rats. Injection of corticotropin-releasing hormone into the locus coeruleus increased the expression of Fos in the locus coeruleus as compared with injection of vehicle into the locus coeruleus or injection of corticotropin-releasing hormone into neighbouring pontine sites. The pattern of Fos expression throughout the brain after injections of corticotropin-releasing hormone into the locus coeruleus was generally consistent with the anatomical organization of efferent projections arising from the locus coeruleus; increased Fos expression was observed in many brain areas including the ventral lateral septum, septohypothalamic nucleus, bed nucleus of the stria terminalis, the central amygdaloid nucleus, the dorsomedial nuclei of the hypothalamus, and the thalamic paraventricular and rhomboid nuclei. Foot shock also increased Fos expression in the locus coeruleus and the other brain regions that expressed Fos after corticotropin-releasing hormone injections into the locus coeruleus. A few brain regions, most notably the hypothalamic paraventricular nucleus, expressed Fos in response to foot shock but not corticotropin-releasing hormone. These results indicate that local injection of corticotropin-releasing hormone into the locus coeruleus stimulates the activity of the locus coeruleus neurons. However, the pattern of Fos expression throughout the brain evoked by injection of corticotropin-releasing hormone into the locus coeruleus does not fully replicate the effects of foot shock.     

The midbrain periaqueductal gray and fear extinction: Opioid receptor subtype and roles of cyclic AMP, protein kinase A, and mitogen-activated protein kinase.

Four experiments studied the opioid receptor subtype and signal transduction mechanisms mediating fear extinction in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG). Microinjection of a μ- but not a δ- or κ-opioid receptor antagonist into the vlPAG retarded extinction. Extinction was also dose-dependently retarded by vlPAG infusions of a cyclic AMP (cAMP) analog but was unaffected by infusions of a protein kinase A activator or a mitogen-activated protein kinase inhibitor across wide dose ranges. The results show that fear extinction occurs via activation of vlPAG μ-opioid receptors and involves reductions in cAMP. These mechanisms are different from the cellular mechanisms for extinction in the amygdala and from the known cellular mechanisms for opioid analgesia in the vlPAG. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effects of benzodiazepine microinjection into the amygdala or periaqueductal gray on the expression of conditioned fear and hypoalgesia in rats.

Four experiments studied the effects of an intracranial microinjection of a benzodiazepine (midazolam) on the expression of conditioned fear (measured as passive avoidance) and conditioned hypoalgesia in rats. Unilateral microinjection of midazolam into the basolateral amygdala reduced both hypoalgesic and avoidance responses, whereas unilateral microinjection of midazolam into the ventrolateral region of the periaqueductal gray (vPAG) reduced the hypoalgesic response but not the avoidance response. The results are discussed in terms of gamma-aminobutyric acid–ergic inhibition of antinociceptive mechanisms in the vPAG and of the activation of these mechanisms by amygdala-based fear processes. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Differential effects of neurokinin-1 receptor activation in subregions of the periaqueductal gray matter on conditional and unconditional fear behaviors in rats.

Central neurokinin-1 (NK-1) receptors are thought to modulate aversion, whereas the periaqueductal gray matter (PAG) is a common pathway for the integration of fear behaviors. The authors determined whether injection of an NK-1 agonist (GR73632) into subregions of the PAG would alter fear-related behaviors in rats. Behavioral inactivity was increased by GR73632 injected into the caudodorsal PAG or the dorsal raphe. Flight behavior induced by stimulation of the dorsal PAG or by a footshock was decreased after injection of GR73632 into the dorsal PAG. Rats that had 6 pairings of a tone with a footshock after injection of GR73632 into the dorsal PAG displayed more freezing behavior than controls at the beginning of the session. However, there was no change in the shock- or the tone-induced freezing because some GR73632-treated rats, but no controls, froze during the baseline period. It is concluded that NK-1 receptors in the dorsal PAG modulate the unconditional but not the mnemonic aspects of fear behaviors. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Tyrosine kinase inhibitor herbimycin A reduces the stability of cyclin-dependent kinase Cdk6 protein in T-cells

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-beta] and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-alpha] cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-alpha, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-beta was similar to that of AraLAM. Differences in induction of TNF-alpha mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-alpha and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-alpha, and IL-10) and the retained ability to induce TGF-beta. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.     

cAMP, an activator of protein kinase A, suppresses the expression of sonic hedgehog

In Drosophila, it has been shown that protein kinase A and hedgehog have antagonistic actions during the formation of imaginal disks. In vertebrate skin, sonic hedgehog is expressed specifically in the feather bud epithelia. using an in vitro explant culture model we showed that dibutyryl cAMP, a protein kinase A (PKA) activator, suppresses the expression of Sonic hedgehog, (Shh) and continuous feather growth. The results suggest that Shh and PKA also have antagonistic action during vertebrate skin morphogenesis.     

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.     

Evidence that the modulation of membrane-associated protein kinase C activity by an endogenous inhibitor plays a role in N1E-115 murine neuroblastoma cell differentiation

Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.     

Differential contributions of medullary, thalamic, and amygdaloid serotonin to the antinociceptive action of morphine administered into the periaqueductal gray: A model of morphine analgesia.

The relative contribution of serotonin (5HT) neurotransmission within the medulla (rostral ventromedial medulla) and forebrain (amygdaloid central nucleus and nucleus parafascicularis thalami) to the antinociceptive action of morphine microinjected into the ventrolateral periaqueductal gray (vPAG) was evaluated. The 5HT receptor antagonist methysergide was microinjected into the medulla, forebrain, (or both) after injection of morphine into the vPAG. The contribution of 5HT to the antinociceptive action of morphine was observed to depend on (a) the dose of morphine administered into the vPAG, (b) the site(s) at which methysergide was administered, and (c) the level of the neuraxis at which the behavioral assay was organized. Results of the present study were combined with those of previous studies from this laboratory and presented as a model of the mechanisms by which morphine administered into the vPAG generates its antinociceptive action. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effect of PD 098059, a specific inhibitor of mitogen-activated protein kinase kinase, on urokinase expression and in vitro invasion

The elevated expression of the urokinase-type plasminogen activator gene, which is necessary for the invasive phenotype of several types of cancers, is controlled by growth factors such as epidermal growth factor, transforming growth factor a, and fibroblast growth factor which bind to and activate protein tyrosine kinase transmembrane receptors. Since these activated receptors communicate with the nucleus via a signaling pathway in which c-Raf-1, mitogen-activated protein kinase kinase 1 (MEK1), and the extracellular signal-regulated kinases are sequentially activated, we determined the effect of a specific MEK1 inhibitor (PD 098059) on urokinase expression in two squamous cell carcinoma cell lines (UM-SCC-1 and MDA-TU-138) characterized as avid secretors of the plasminogen activator. PD 098059 treatment of either cell line reduced the amount of secreted urokinase in a dose-dependent manner. In contrast, a compound (daidzein) chemically unrelated to PD 098059 had little effect on urokinase secretion. The effect of PD 098059 on urokinase secretion in UM-SCC-1 cells was reversible and correlated with decreased extracellular signal-regulated kinase 1 activity. PD 098059 caused a dose-dependent reduction in the in vitro invasiveness of UM-SCC-1 cells whereas it had little effect on proliferation rates. Transient transfection assays with a chloramphenicol acetyl transferase reporter driven by the urokinase promoter indicated that diminished secretion of the protease was largely a consequence of reduced promoter activity. These findings suggest that interfering with MEK1 may provide a novel means of controlling the invasiveness of tumors in which this signaling cascade is activated by autocrine and/or paracrine growth factors.     

The 58-kilodalton inhibitor of the interferon-induced double-stranded RNA-activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties

The interferon-induced RNA-dependent protein kinase (PKR) is considered to play an important role in the cellular defense against viral infection and, in addition, has been suggested to be a tumor suppressor gene because of its growth-suppressive properties. Activation of PKR by double-stranded RNAs leads to the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) and a resultant block to protein synthesis initiation. To avoid the consequences of kinase activation, many viruses have developed strategies to down-regulate PKR. Recently, we reported on the purification and characterization of a cellular inhibitor of PKR (referred to as p58), which is activated during influenza virus infection. Subsequent cloning and sequencing has revealed that p58 is a member of the tetratricopeptide repeat (TPR) family of proteins. To further examine the physiological role of this PKR inhibitor, we stably transfected NIH 3T3 cells with a eukaryotic expression plasmid containing p58 cDNA under control of the cytomegalovirus early promoter. By taking advantage of a recently characterized p58 species-specific monoclonal antibody, we isolated cell lines that overexpressed p58. These cells exhibited a transformed phenotype, growing at faster rates and higher saturation densities and exhibiting anchorage-independent growth. Most importantly, inoculation of nude mice with p58-overexpressing cells gave rise to the production of tumors. Finally, murine PKR activity and endogenous levels of eIF-2 alpha phosphorylation were reduced in the p58-expressing cell lines compared with control cells. These data, taken together, suggest that p58 functions as an oncogene and that one mechanism by which the protein induces malignant transformation is through the down-regulation of PKR and subsequent deregulation of protein synthesis.     

Epidermal growth factor (EGF) receptor blockade inhibits the action of EGF, insulin-like growth factor I, and a protein kinase A activator on the mitogen-activated protein kinase pathway in prostate cancer cell lines

For assays involving glycosyltransferases or transporters, several GDP-sugars are either commercially unavailable or expensive. We describe an enzymatic synthesis of GDP-d-[3H]arabinosep and GDP-l-[3H]fucose that yields 66-95% nucleotide-sugar from the appropriate radiolabeled sugar in less than 30 min. The coupled reaction requires Mg2+, ATP, and GTP along with the appropriate radioactive monosaccharide, sugar-1-kinase, and pyrophosphorylase. The latter two activities are present in a cytosolic fraction of Crithidia fasciculata, which is easily grown at room temperature in simple culture medium without serum or added CO2. Addition of commercial yeast inorganic pyrophosphatase shifts the equilibrium of the pyrophosphorylase reaction toward nucleotide-sugar formation. To verify that these nucleotide-sugars are biologically active, we tested their ability to serve as substrates for glycosyltransferases. GDP-l-[3H]fucose functions as the donor substrate for recombinant human fucosyltransferase V, and GDP-d-[3H]arabinosep serves as the donor substrate for the arabinosyltransferase activities present in Leishmania major microsomes.     

The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K+ secretory channels in the kidney. Addition of 1 microM forskolin or 100 microM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 microM forskolin, within 3 min, significantly increased whole-cell K+ current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (i) Addition of 100 microM 8-bromo-cAMP mimics the effect of forskolin and (ii) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 microM) and cAMP (100 microM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.     

Structure of malhamensilipin A, an inhibitor of protein tyrosine kinase, from the cultured chrysophyte Poterioochromonas malhamensis

A new chlorosulfolipid, malhamensilpin A [1] was isolated from the cultured chrysophyte Poterioochromonas malhamensis. Malhamensilipin A was demonstrated to be a modest inhibitor of pp60v-src protein tyrosine kinase. The structure was determined by detailed spectral analysis to be a novel C24 hexachloro lipid containing a vinyl sulfate ester (2,11,12,13,15,16-hexachloro-14-hydroxy-n-tetracos-1E-enol-1-sulfa te).     

KARP-1: a novel leucine zipper protein expressed from the Ku86 autoantigen locus is implicated in the control of DNA-dependent protein kinase activity

The Ku autoantigen plays an integral role in mammalian DNA double-strand break repair as the DNA binding component of the DNA-dependent protein kinase (DNA-PK) complex. Here, we demonstrate that a second gene, KARP-1 (Ku86 Autoantigen Related Protein-1), is expressed from the Ku86 locus. The KARP-1 gene utilizes an upstream promoter and additional exons which results in an extra 9 kDa of protein appended onto the normal Ku86 polypeptide. The KARP-1-specific domain encodes interdigitating hexa- and penta-heptad repeats of leucine residues flanked by a very basic region. Intriguingly, the catalytic subunit of DNA-PK also contains a hexa-heptad repeat of leucines. Consistent with this observation, we observed that human cell lines stably expressing dominant-negative constructs of KARP-1 resulted in diminished DNA-PK activity and X-ray hypersensitivity and that a KARP-1 antibody significantly neutralized DNA-PK activity in vitro. Finally, we present data which suggests that KARP-1 may be primate-specific. These observations have important repercussions for mammalian DNA double-strand break repair.     

Effects of a protein kinase C inhibitor, Ro 31-7549, on the activation of human leukocytes by particulate stimuli

1. A new specific protein kinase C (PKC) inhibitor, Ro 31-7549, was used to explore the mechanisms by which particulate stimuli, quartz and chrysotile, stimulate human polymorphonuclear leukocytes (PMNL) to produce reactive oxygen metabolites (ROM). Also soluble stimuli, formyl-Methionyl-Leucyl-Phenylalanine (fMLP) and phorbol myristate acetate (PMA) were used. 2. Ro 31-7549 inhibited chrysotile-induced free intracellular calcium ([Ca2+]i) elevations but did not have an effect on quartz-induced elevations of [Ca2+]i. Both quartz and chrysotile induced production of ROM were partially inhibited by Ro 31-7549. fMLP-induced elevation of [Ca2+]i was inhibited by Ro 31-7549 whereas PMA did not affect [Ca2+]i. Ro 31-7549 strongly inhibited fMLP-induced ROM production, and completely abolished that induced by PMA. 3. These result suggest that PKC may have an important role in the activation of PMNL to produce ROM by particulate and soluble stimuli. However, the inhibition of chrysotile-, but not of quartz-induced [Ca2+]i elevations by Ro 31-7549 provides evidence that both PKC-dependent and -independent mechanisms may play a role in the activation of human leukocytes to produce ROM.