Endothelial nitric oxide synthase expression in neurogenic urinary bladders treated with intravesical resiniferatoxin.

OBJECTIVE: To investigate endothelial nitric oxide synthase (eNOS) immunoreactivity in bladder biopsies from patients with neurogenic detrusor overactivity (NDO) before and after treatment with intravesical resiniferatoxin, and compare this with control material; the distribution of two other vascular markers, von Willebrand Factor (vWF) and the vascular endothelial growth factor (VEGF), was also studied. PATIENTS AND METHODS: Flexible cystoscopic bladder biopsies from eight controls investigated for asymptomatic microhaematuria and 19 patients with refractory spinal NDO enrolled in a clinical trial of intravesical treatment with escalating doses of resiniferatoxin were immunostained with polyclonal rabbit antibodies for eNOS, vWF and VEGF. Fewer baseline NDO specimens (eight) were available for vWF and VEGF staining. Computerized image analysis was used to quantify immunoreactivity, and the Mann-Whitney test for statistical analysis. RESULTS: eNOS immunoreactivity was found in the suburothelium and less often in the urothelium, with a distribution indicating a location in small blood vessels at the urothelium-suburothelium junction. Immunostaining for vWF showed a similar location. There was a trend to higher eNOS values before treatment in those responding than in those not responding to resiniferatoxin (P = 0.059), and a significant reduction in eNOS immunoreactivity after successful treatment (P = 0.016). VEGF staining was weaker but there was a significant increase in pretreatment biopsies of responders to resiniferatoxin (P = 0.048). Clinical and histopathology features were similar in both groups. CONCLUSIONS: The trend for higher eNOS expression in patients with NDO who responded to resiniferatoxin suggests that increased vasculature or vasodilatation in the suburothelium may be necessary for successful intravesical treatment. Further studies with more patients are required to confirm this relationship and to examine the mechanisms underlying changes in vasculature with levels of bladder overactivity.

Novel capsaicin (VR1) and purinergic (P2X3) receptors in Hirschsprung's intestine.

BACKGROUND/PURPOSE: Studies of Hirschsprung's disease (HSCR) have shown that hypertrophic nerves in aganglionic bowel are mainly of extrinsic origin and may contain sensory elements. Recent advances have shown a specific capsaicin receptor VR1 (vanilloid receptor-1), and an ATP-gated ion channel P2X(3), which are expressed by sensory neurons. METHODS: This study investigated, for the first time, the distribution of VR1- and P2X(3)-immunoreactivity in normal adult, infant, and HSCR large intestine, using specific antibodies for immunohistochemistry. RESULTS: VR1-immunoreactive fibers and nerve fascicles, but not somata, were detected in all regions of the bowel in controls with few weakly immunostained fibers in the mucosa/lamina propria. Hypertrophic nerve bundles in hypoganglionic and aganglionic bowel showed intense VR1-immunoreactivity, whereas normoganglionic regions of HSCR were similar to controls. P2X(3)-immunoreactive neuronal cell bodies, in some instances with long axonal processes, were detected in the myenteric and submucous plexuses in control infant, adult, and ganglionic HSCR samples. Aganglionic samples showed weak P2X(3)-immunoreactivity in hypertrophic nerve fasciculi in the submucous and myenteric plexuses. CONCLUSIONS: The presence of VR1- and P2X(3)-immunoreactivities in aganglionic HSCR bowel indicates that sensory nerves may form a significant proportion of its hypertrophic innervation. The functional significance of P2X(3) and VR1 receptors in enteric nerves deserves further investigation.

ATP-gated ion channel P2X(3) is increased in human inflammatory bowel disease.

P2X(3) is a novel ATP-gated cation channel that is selectively expressed by small-diameter sensory neurones in rodents, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. We have studied, for the first time, P2X(3) immunoreactivity in human inflammatory bowel disease, using Western blotting and immunohistochemistry. A major 66-kDa specific protein was found by Western blotting in all colon extracts. In the inflamed group there was a significant two-fold increase in the relative optical density of the 66-kDa band (21.2 +/- 3.1; n=8) compared to controls (11.4 +/- 3.7; n=8; P=0.009). In the control colon, P2X(3)-immunoreactive neurones were scattered throughout the myenteric and submucosal plexuses, with some neurones showing immunopositive axons/dendrites. The pattern of immunostaining was similar to the neuronal marker peripherin. In general, the intensity of the staining was greater in myenteric than submucosal neurones. The number of P2X(3)-immunoreactive neurones was significantly increased in the myenteric plexus of inflamed colon compared to controls (n=13; P=0.01). In humans, unlike rodents, P2X(3) is thus not restricted to sensory neurones. Increased P2X(3) in inflamed intestine suggests a potential role in dysmotility and pain, for which it represents a new therapeutic target.

Characterization of a promoter for the human glial cell line-derived neurotrophic factor gene.

To address the regulation of glial cell line-derived neurotrophic factor (GDNF) gene expression, we have isolated 5' extended cDNAs, cloned the human GDNF gene, and characterized the promoter. GDNF-encoding 5' extended cDNAs containing a novel exon were isolated via reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from human fetal kidney and adult skeletal muscle. The GDNF gene was isolated from a human genomic library in a P1 bacteriophage vector. Analysis of the 5' flanking sequence revealed a promoter that lacks a CCAAT-box motif and is GC rich. Consensus binding sites for a variety of transcription factors have been identified in the promoter. Promoter/reporter plasmids have been constructed by fusion of the promoter and a portion of exon I to a luciferase gene. The promoter/reporter construct and a number of promoter deletions were transiently transfected into two human cell lines known to express GDNF. Multiple enhancer and silencer regions were revealed as well as a minimal promoter sufficient for basal transcription. Finally, a RT-PCR assay, specific for transcripts originating from this GDNF promoter, was developed and used to show that this promoter is active in vivo. The results suggest GDNF is regulated in a complex manner.

Differential regulation of glial cell line-derived neurotrophic factor (GDNF) expression in human neuroblastoma and glioblastoma cell lines.

Human SK-N-AS neuroblastoma and U-87MG glioblastoma cell lines were found to secrete relatively high levels of glial cell line-derived neurotrophic factor (GDNF). In response to growth factors, cytokines, and pharmacophores, the two cell lines differentially regulated GDNF release. A 24-hr exposure to tumor necrosis factor-alpha (TNFalpha; 10 ng/ml) or interleukin-1beta (IL-1,; 10 ng/ml) induced GDNF release in U-87MG cells, but repressed GDNF release from SK-N-AS cells. Fibroblast growth factors (FGF)-1, -2, and -9 (50 ng/ml), the prostaglandins PGA2, PGE2, and PGI2 (10 microM), phorbol 12,13-didecanoate (PDD; 10 nM), okadaic acid (10 nM), dexamethasone (1 microM), and vitamin D3 (1 microm) also differentially effected GDNF release from U-87MG and SK-N-AS cells. A result shared by both cell lines, was a two- to threefold increase in GDNF release by db-cAMP (1 mM), or forskolin (10 microM). In general, analysis of steady-state GDNF mRNA levels correlated with changes in extracellular GDNF levels in U-87MG cells but remained static in SK-N-AS cells. The data suggest that human GDNF synthesis/release can be regulated by numerous factors, signaling through multiple and diverse secondary messenger systems. Furthermore, we provide evidence of differential regulation of human GDNF synthesis/release in cells of glial (U-87MG) and neuronal (SK-N-AS) origin.

Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors.

Four cyclic-nucleotide phosphodiesterase (PDE) genes belonging to the PDE4 family (PDE4A, 4B, 4C and 4D) have been identified. All four isogenes, including several deletions and alterations of the amino, carboxyl and central catalytic domains, were expressed in insect cells. Lysates were characterised for enzyme activity by using the Km for substrate and the EC50 for activation by the cofactor Mg2+. The catalytic domain alone appears to be sufficient for the normal enzymatic function of PDE4 proteins. Substrate affinity varied by less than 2-fold between catalytic-domain forms of the PDE4A, 4B and 4D isogenes and the long forms (PDE4A5, PDE4B1 and PDE4D3). The affinity for Mg2+ varied by less than 4-fold between long and catalytic-domain forms of PDE4A and 4B. The catalytic-domain form of PDE4D, however, had a 12-fold lower affinity for Mg2+ that was restored by including a portion of the amino-terminal domain, upstream conserved region-2 (UCR2). This result suggests that the Mg2+-binding site of PDE4D involves the UCR2 region. Inhibition of the PDE4 proteins by synthetic compounds is apparently affected differently by the domains. For PDE4B, the catalytic domain is sufficient for interactions with the inhibitors studied: IBMX, trequinsin, rolipram, TVX 2706, RP 73401 and RS-25344. For PDE4D the catalytic-domain form is less sensitive than the long form to inhibition by RS-25344, rolipram and TVX 2706, by 1463-, 11-and 12-fold, respectively. Addition of UCR2 to the catalytic-domain form of PDE4D restored all the lost sensitivities. The catalytic-domain form of PDE4A showed a reduced inhibitor affinity with RS-25344 and TVX 2706 by 77- and 90-fold, respectively. Both catalytic-domain and long forms of PDE4 isogenes interacted with equal affinity with the non-specific inhibitors IBMX and trequinsin, as well as the very potent PDE4-specific inhibitor RP 73401. Other potent and specific PDE4 inhibitors, such as rolipram, RS-25344 or TVX 2706, appear to utilize non-catalytic domain interactions with PDE4D and 4A to supplement those within the catalytic domains. These observations suggest a different relation between amino and catalytic domains in PDE4D relative to PDE4B. We therefore propose a model to illustrate these isogene-specific PDE4 domain interactions with substrate, inhibitors and the co-factor Mg2+. The model for PDE4D is also discussed in relation to changes in the activation curve for Mg2+ and sensitivity to RS-25344 that accompany phosphorylation of the long form by protein kinase A.

Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and -4C.

Individual isozymes of family four cyclic-nucleotide phosphodiesterases (PDE-4s) were characterized and compared in order to advance our understanding of how PDE-4s regulate cAMP levels in cells. Full-length and shorter clones containing various functional domains were constructed and overexpressed using a recombinant baculovirus-infected Sf9 insect cell system. One form each of PDE-4C and 4D was purified 125- and 534-fold, respectively, using anion-exchange and affi-gel blue chromatography. The purified material was unaltered in size on SDS-polyacrylamide gels during purification and nearly homogeneous (> 95%) as estimated by both staining and immunoblotting. Approximately 1 mg of PDE-4D (74.7 kDa) and 3.7 mg of PDE-4C (61.4 kDa) could be isolated from a 6-L culture of cells. The physical characteristics of Stokes' radius and sedimentation coefficient for PDE-4 enzymes cloned from each of the four isogenes were determined using size-exclusion chromatography and sedimentation in glycerol gradients. Calculations indicate that both long and short forms can form dimers, although evidence for monomers and higher-order subunit association was seen. Furthermore, the results clearly show that all long and short forms of PDE-4 are highly asymmetric molecules. This work has shown that large amounts of PDE-4 proteins can be purified and characterized physically and enzymatically to yield information that will enable a greater understanding of how PDE-4 enzymes function in cells.

Regulation of glial cell line-derived neurotrophic factor release from rat C6 glioblastoma cells.

We have monitored glial cell line-derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release. Whereas most factors tested had minimal effect, a 24-h treatment with fibroblast growth factor-1, -2, or -9 elevated secreted GDNF protein levels five- to 10-fold. The proinflammatory cytokines interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and lipopolysaccharide elevated GDNF release 1.5- to twofold. Parallel studies aimed at elucidating intracellular events that may regulate GDNF synthesis/release demonstrated the involvement of multiple signaling pathways. GDNF levels were increased by phorbol 12,13-didecanoate (10 nM) activation of protein kinase C, the Ca2+ ionophore A23187 (1 microM), okadaic acid (10 nM) inhibition of type-2A protein phosphatases, nitric oxide donors (1 mM), and H2O2 (1 mM)-induced oxidative stress. Elevation of cyclic AMP levels by either forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) repressed GDNF secretion, as did treatment with the glucocorticoid dexamethasone (1 microM). Our results demonstrate that diverse biological factors are capable of modulating GDNF protein levels and that multiple signal transduction systems can regulate GDNF synthesis and/or release.

Multiple splice variants of phosphodiesterase PDE4C cloned from human lung and testis.

Four closely related cyclic-nucleotide specific phosphodiesterase (PDE4) genes have been identified in both humans and rats: PDE4A, 4B, 4C and 4D. We have now cloned cDNAs for multiple splice variants of human PDE4C. Two splice variants, PDE4C-791 and PDE4C-426, were isolated from a fetal lung library. The longest open reading frame (ORF) of 791 amino acids (aa) is encoded by PDE4C-791, which is similar to a recently described cDNA [Engels, P., Sullivan, M., Muller, T. and Lubbert, H. FEBS Lett. 358 (1995) 305-10], except that an alternative 5'-end sequence upstream of the first methionine extends the PDE4C-791 ORF by 79 aa. The PDE4C-426 variant contains 3 insertions that are located 5' to the catalytic domain and encode several in-frame stop codons. The predicted 426 aa protein initiates at a methionine 365 aa within PDE4C-791. A baculovirus clone starting at this methionine expressed an enzymatically active protein. Two additional splice variants, PDE4C-delta54 and PDE4C-delta109, were found in testis mRNA. PDE4C-delta54 contained a novel 5'-end region and a deletion of 162 nt; the predicted protein deletes 54 aa from the amino-terminal region. The PDE4C-delta54 protein produced in baculovirus-infected cells was enzymatically active and sensitive to PDE4-specific inhibitors. The PDE4C-delta109 protein is similar to PDE4C-delta54 but has an additional 55 aa deleted in the catalytic domain; it lacked enzymatic activity. Analysis of uncloned total mRNA from 4 tissue sources by polymerase chain reaction (PCR) confirmed the presence of mRNAs with the two deletions and three insertions that we observed in cDNA clones. The PDE4C-delta54 variant was found only in testis and the 5'-extended region of PDE4C-791 was seen only in lung and the melanoma cell line G361. Hence, tissue-specific expression of various PDE4C isoforms should be considered in understanding how these gene products modulate cellular responses to cAMP.

Isolation of a cDNA encoding a human rolipram-sensitive cyclic AMP phosphodiesterase (PDE IVD).

cDNAs encoding human family-IV phosphodiesterase, subtype D (hPDE IVD) were isolated from a human heart cDNA library. The overlapping cDNAs encode a polypeptide of 604 amino acids (aa) with a predicted M(r) of 68,502, which is 91.4% identical to the rat homolog, rPDE IVD. hPDE IVD produced in Escherichia coli was inhibited by rolipram. Expression of the hPDE IVD mRNA is widespread in human tissues and most abundant in skeletal muscle.

Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter.

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin.

Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-D-(1, 4-alpha-D-glucano)-transferase as deduced from the nucleotide sequence of the glg B gene.

The nucleotide sequence of the glg B gene, coding for branching enzyme (EC 2.4.1.18), was elucidated. It consists of 2181 base pairs specifying a protein of 727 amino acids. The deduced amino acid sequence was consistent with the amino acid analysis that was obtained with the pure protein as well as with the molecular weight determined from sodium dodecyl sulfate-gel electrophoresis. The deduced amino acid sequence was also consistent with the amino-terminal amino acid sequence and the amino acid sequence analysis of various peptides obtained from CNBr degradation of purified branching enzyme.

Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature alpha-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently.

Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose synthetase as deduced from the nucleotide sequence of the glg C gene.

The nucleotide sequence of the glg C gene of Escherichia coli K12, coding for ADP-glucose synthetase, has been determined. The structural gene consists of 1293 base pairs, which specify a protein of 431 amino acids. The amino acid sequence deduced from the DNA sequence is consistent with the known NH2-terminal amino acid sequence and the amino acid composition of ADP-glucose synthetase. The translation start of the structural gene of glycogen synthase, glg A, starts immediately after termination of the glg C gene.

Regulation of bacterial glycogen synthesis.

The formation of the alpha 1,4 glucosidic linkages of bacterial glycogen occurs first by synthesis of ADPglucose from ATP and alpha glucose 1-P and then transfer of the glucose moiety from the formed sugar nucleotide to a pre-existing glucan primer. Unlike mammalian glycogen synthesis, regulation occurs at the synthesis of the sugar nucleotide. Generally glycolytic intermediates activate ADPglucose synthesis while AMP, ADP and/or Pi inhibit ADPglucose synthesis. A variation of activator specificity is is seen when the enzyme is isolated from different bacteria and is thought to be related to the predominant type of carbon assimilation or dissimilation pathways present in the particular organism. Evidence indicating that the allosteric activation effects observed in vitro are physiologically pertinent for the regulation of glycogen synthesis is reviewed. The recent experiments in identifying the allosteric activator site of the Escherichia coli ADPglucose pyrophosphorylase as well as other chemical modification studies identifying amino acid residues essential for allosteric activation and for catalytic activity are discussed. Evidence is also presented for the covalent modification of the Rhodopseudomonas sphaeroides ADPglucose pyrophosphorylase by bromopyruvate at its allosteric activator site. Regulation of the biosynthesis of glycogen also occurs at the genetic level and the current evidence for the existence of a glycogen operon is presented. In addition the current studies concerning the cloning of the DNA region containing the Escherichia coli structural genes coding for the glycogen biosynthetic enzymes as well as the nucleotide sequence of the E. coli ADPglucose pyrophosphorylase are presented.

Periplasmic localization of nicotinate phosphoribosyltransferase in Escherichia coli.

Nicotinate phosphoribosyltransferase (NAPRTase) in Escherichia coli mediates the formation of nicotinate mononucleotide, a direct precursor of nicotinamide adenine dinucleotide (NAD), from nicotinate and 5-phosphoribosyl-1-pyrophosphate. Specifically, NAPRTase contributes to NAD synthesis by utilizing intracellular nicotinate formed from NAD degradation products, which are recycled by NAD cycle enzymes and exogenous nicotinate when it is available. In previous studies, it has been tacitly assumed that almost all NAD cycle enzymes are localized in the cytoplasm of E. coli. The results of this investigation provide evidence that NAPRTase is a periplasmic (extracytoplasmic) enzyme. The osmotic shock of exponential-phase cells of E. coli K-12 and ML 308-225 resulted in the release of 63 to 72% and 42 to 48%, respectively, of the NAPRTase into the shock medium. In addition, when exponential cells of strains K-12 and ML 308-225 were converted into spheroplasts, 75 to 84% and 54 to 68%, respectively, of the enzyme was released into the spheroplast medium. Since previous estimates of the effective levels of NAPRTase present in putative repressed and derepressed E. coli cells appeared to be very low, a more convenient and accurate alternative method for the evaluation of NAPRTase in whole cells was developed. The results show that NAPRTase is subject only to a modest degree of enzyme repression. In addition, no evidence was found for the presence of a protein or low-molecular-weight inhibitor of the enzyme in repressed cells.