bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKCζ
Authors: Clément-Chomienne Odile , Walsh Michael P.
Published: December 2009

The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: α, δ, ε, η, and ζ isoenzymes were detected. PKCα associated with the particulate fraction in the presence of Ca2+and was extracted by divalent cation chelators. PKCδ required detergent treatment for extraction from the EDTA – EGTA-washed particulate fraction. PKCε, η, and ζ were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKCζ, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKCζ-immunoreactive protein (Mr76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKCζ with peptide ε (a synthetic peptide based on the pseudosubstrate domain of PKCε) as substrate was 2.1 μmol Pi∙min−1∙(mg PKCζ)−1and, with peptide ζ as substrate, was 1.2 μmol Pi min−1∙(mg PKCζ)−1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKCζ (specific activity, 0.1–0.3 μmol Pi∙min−1∙mg−1). Two proteins, calponin and caldesmon, which have been implicated in the regulation of smooth muscle contraction and are phosphorylated by cPKC (a mixture of α, β, and γ isoenzymes), were also poor substrates of PKCζ (specific activities, 0.04 and 0.02 μmol Pi∙min−1∙mg−1, respectively). Chicken gizzard PKCζ was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKCζ are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.Key words: protein kinase C, isoenzymes, smooth muscle.
pH and temperature dependence of the rate of compound I formation from the reaction of prostaglandin endoperoxide synthase with hydrogen peroxide
Authors: Bakovic Marica , Dunford H. Brian
Published: December 2009

The formation of primary oxidized compound of prostaglandin endoperoxide synthase, compound I, was studied as a function of pH and temperature using hydrogen peroxide as a substrate. Analysis of the results indicates that compound I formation is influenced by an ionizable group with a pKaof 4.06 ± 0.04. The protonated form of hydrogen peroxide preferentially reacts with the unprotonated form of the enzyme over the pH range of 3.5 to 9.1, suggesting the importance of acid–base catalysis for compound I formation. The second-order rate constant for the reaction of the enzyme with hydrogen peroxide in the pH-independent region is (4.6 ± 0.2) × 105 M−1 s−1at an ionic strength of 0.1 M and temperature of 4.0 ± 0.2 °C. The effect of temperature on the rate of compound I formation was studied from 3.4 to 24.1 °C in the pH-independent region (pH 6.98) and at a constant ionic strength of 0.1 M. The kinetic parameters obtained from the temperature dependence are the following: Arrhenius activation energy, Ea = 102 ± 5 kJ/mol; free energy of activation, ΔG≠, 36 ± 3 kJ/mol; enthalpy of activation, ΔH≠, 100 ± 5 kJ/mol; entropy of activation, ΔS≠, 215 ± 9 J/mol K. These activation values are very different from those obtained for the reactions of other peroxidases and catalases with hydrogen peroxide, indicating profound differences in active site structure.Key words: kinetics, prostaglandin endoperoxide synthase, hydrogen peroxide, compound I, peroxidase, cyclooxygenase.
ATP- and EGF-stimulated phosphatidylinositol synthesis by two different pathways, phospholipase D and diacylglycerol kinase, in A-431 epidermoid carcinoma cells
Authors: Hosoi Kazuo , Shioda Yoshimi , Ueha Takao , Atsumi Toshiko , Sugita Kenji , Kurihara Kinji
Published: December 2009

The [3H]inositol incorporation into the membrane fraction of A-431 human epidermoid carcinoma cells was markedly increased by stimulation of the cells with either epidermal growth factor (EGF), ATP, bradykinin, or a calcium ionophore A23187 in the presence of 1 mM extracellular calcium ions; most incorporated [3H]inositol was found to have accumulated as phosphatidylinositol (PI). The EGF- and ATP-stimulated PI synthesis was inhibited by two protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM), but not by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Pretreatment of cells with pertussis toxin (IAP, islet-activating protein) inhibited the PI synthesis, [Ca2+]ielevation, and inositol trisphosphate (IP3) production by ATP, suggesting that the phospholipase C (PLC) system coupled with IAP-sensitive G protein is involved in the ATP-stimulated PI synthesis. On the other hand, the ATP stimulation increased the release of [3H]choline and [32P]phosphatidic acid (PA) from radiolabeled cells, and such release was not inhibited by IAP. In the presence of n-butyl alcohol, which prevents the production of PA by generation of phosphatidylbutanol, the ATP-stimulated PI synthesis was reduced. Because n-butyl alcohol did not inhibit IP3production and [Ca2+]ielevation, this fact suggests that the IAP-insensitive PLD system is involved in the ATP-stimulated PI synthesis. In A-431 cells, the stimulation of P2-purinergic receptors appears to activate the IAP-sensitive PLC system and IAP-insensitive PLD system, both of which are essential for the stimulation of PI synthesis. The present results imply the general prospect that ligand stimulation, which mobilizes second messengers and consumes their precursors, simultaneously provokes the pathway to synthesize and salvage the second messenger precursors as well.Key words: P2-purinergic receptors, phosphatidylinositol, EGF receptors, phosphatidic acid, A-431 cells.
Structural characterization of the O-antigenic polysaccharide of Escherichia coli serotype O17 lipopolysaccharide
Authors: Masoud Hussein , Perry Malcolm B.
Published: December 2009

The structure of the O-poly saccharide component of the lipopolysaccharide produced by Escherichia coli O17 (ATCC 23512) was determined by the use of methylation, periodate oxidation, one- and two-dimensional nuclear magnetic resonance spectroscopy, and mass spectrometric methods. The O-polysaccharide was found to be a high molecular weight polymer of repeating branched pentasaccharide units composed of D-mannose, D-glucose, and 2-acetamido-2-deoxy-D-glucose residues (3:1:1) and had the structure[Formula: see text]Key words: Escherichia coli, lipopolysaccharide, polysaccharide, NMR.
Accumulation of natural and synthetic betaines by a mammalian renal cell line
Authors: Randall K. , Lever M. , Peddie B. A. , Chambers S. T.
Published: December 2009

Intracellular accumulation of different betaines was compared in osmotically stressed Madin Darby canine kidney (MDCK) cells to model the betaine accumulation specificity of the mammalian inner medulla and to show how this accumulation differed from that of bacteria. All betaines accumulated less than glycine betaine. Arsenobetaine (the arsenic analogue of glycine betaine) accumulated to 12% of the glycine betaine levels and the sulphur analogue dimethylthetin accumulated to >80%. Most substituted glycine betaine analogues accumulated to 2–5% of intracellular glycine betaine concentrations, however, serine betaine accumulated to <0.5% of glycine betaine levels. Inhibition studies to distinguish the betaine ports were performed by the addition of proline. Butyrobetaine and carnitine accumulation was not proline sensitive, whereas that of omer betaines was. As with glycine betaine, the accumulation of propionobetaine and dimethylthetin was proline sensitive and osmoregulated. Pyridinium betaine was accumulated by both proline-sensitive and -insensitive systems, with a small increase under osmotic stress. High concentrations (10 times that of glycine betaine) of the dietary betaines proline betaine and trigonelline inhibited total betaine accumulation. Because α-substituted betaines are accumulated by bacteria and not by MDCK cells, these betaines may be the basis for design of antimicrobial agents.Key words: MDCK cells, betaine accumulation, osmolytes, betaine analogues.
The activation of β-galactosidase (E.coli) by Mg2+at lower pH values
Authors: Martinez-Bilbao Mercedes , Huber Reuben E.
Published: December 2009

The activation of β-galactosidase (E. coli) by Mg2+at pH values below 7.6 was studied. If the Mg2+concentration was high enough, the kcatvalues at pH values down to 5.0 remained at the same high level as at pH 7 and 7.6 (600–620 s−1with o-nitrophenyl-β-D-galactopyranoside as the substrate). Very high concentrations of Mg2+(greater than 100 mM at pH 5) were, however, needed to saturate the Mg2+site at lower pH values. The Kmvalues at low levels of Mg2+were high at every pH but they decreased and approached the same low value at every pH (about 0.13 mM) as the [Mg2+] was increased. These data indicate that it is difficult to bind Mg2+at lower pH values, but the kcatand Kmvalues of the enzyme, and therefore the rates of galactosylation (k2), degalactosylation (k3), and binding (Ks), do not change substantially as a function of pH provided that a Mg2+is bound to the enzyme. The data also showed that Mg2+and protons compete for the same site. Analysis by plotting log [Mg2+]midvs. pH showed that the binding of Mg2+to the free enzyme involves two groups with pKavalues in the vicinity of 7 and one group with a pKavalue near 5.5. (The values referred to as [Mg2+]midare the Mg2+concentrations that resulted in kcatvalues midway between basal and maximum.) The "apparent" pKavalues of the groups when a Mg2+was bound (at saturating [Mg2+]) all appeared to be below 5.0.Key words: β-galactosidase, magnesium, pH, activation, glutamic acid, histidine, binding.
Effects of ultraviolet light in vascular cells in vitro and in intact atherosclerotic explants: potential role of apoptosis in vascular biology
Authors: López-Candales Angel , Scott Michael J. , Wickline Samuel A. , Holmes Dennis R. , Thompson Robert W.
Published: December 2009

Complex cell-to-cell interactions are known to participate during vascular injury and remodeling, resulting in smooth muscle cell proliferation. Mechanical interventions have yielded little benefit in limiting this process and several site-specific genetic therapies are not yet clinically available. The aim of this study was to delineate the effect of very short wavelength ultraviolet (UVC) light therapy on the viability of macrophage and smooth muscle cells. Vascular cells were both treated in vitro and in intact explanted atherosclerotic aortic segments ex vivo with UVC light. Brief exposure to short wavelength UVC light in the absence of photosensitizers elicited a differential temporal and functional response among treated cells. However, dramatic reduction in both cellular viability and proliferative capacity with eventual cell demise was observed in all UVC-treated cells. Flow cytometry and immunohistochemical analyses revealed the presence of extensive DNA fragmentation, suggestive of apoptosis as a predominant pathway of cell death in these cells exposed to UVC light. We hypothesize that selective induction of apoptosis, in contrast to necrosis, with UVC light may represent a beneficial approach to interdict the complex biologic cascade of messengers that participate in the restenotic response to vascular injury.Key words: apoptosis, macrophage, smooth muscle cell, atherosclerotic aorta, New Zealand White rabbit.
Prevention of early glomerulopathy with tolrestat in the streptozotocin-induced diabetic rat
Authors: Donnelly Sandra M. , Huang Jannet T. , Zhou Xiaopeng P. , Whiteside Catharine I.
Published: December 2009

Hyperglycemia is of central importance in the pathogenesis of the complications of diabetes mellitus. Glucose activation of the polyol pathway may lead to renal arteriolar smooth muscle and glomerular mesangial cell hypocontractility. In the streptozotocin-induced diabetic rat, the effect of the aldose reductase inhibitor, tolrestat, in preventing glomerular hyperfiltration, renal hypertrophy, extracellular matrix accumulation, and mesangial cell hypocontractility was addressed. Streptozotocin-induced diabetic rats were followed for 12 weeks and half received tolrestat (25 mg/kg per day). Increased glomerular filtration rate was prevented by tolrestat (3.1 ± 0.3 vs. 1.8 ± 0.2 mL/min, diabetes vs. diabetes + tolrestat, p < 0.01), in part by reduction of the filtration fraction (0.39 ± 0.03 vs. 0.29 ± 0.01, diabetes vs. diabetes + tolrestat, p < 0.01). Tolrestat prevented the raised albumin excretion rates (3594 ± 1154 vs. 713 ± 161 mg/24 h, diabetes vs. diabetes + tolrestat, p < 0.01). Endothelin-1-induced contraction of isolated glomeruli was normal in tolrestat-treated diabetic animals compared with the hypocontractile diabetic glomeruli. Tolrestat prevented glomerular hypertrophy (1.86 ± 0.10 vs. 1.49 ± 0.03 μm2 × 105, diabetes vs. diabetes + tolrestat, p < 0.001) and attenuated the accumulation of basement-membrane-like material (50.2 ± 0.4% vs. 46.4 ± 0.8%, diabetes vs. diabetes + tolrestat, p < 0.001). Fractional mesangial expansion was unchanged in tolrestat-treated diabetic rats compared with untreated animals. Tolrestat prevents the functional changes of glomerular hyperfiltration, mesangial cell hypocontractility, and increased glomerular permeability to albumin. Polyol accumulation may have differential effects on glomerular growth and extracellular matrix accumulation in early diabetic nephropathy.Key words: aldose reductase, nephropathy, renal hemodynamics, renal morphometry, mesangium.
Role of oxidative mixed-disulfide formation in elastase–serine proteinase inhibitor (serpin) complex
Authors: Tyagi Suresh C.
Published: December 2009

To understand the role of thiol and oxidative mixed-disulfide exchange reaction in serpins, we analyzed the conformation of native and mixed-disulfide forms of α1-proteinase inhibitor (α1PI), α1-antichymotrypsin (α1-ACT), α2-antiplasmin (α2-AP), angiotensinogen, and ovalbumin. The conformation of native and oxidized mixed-disulfide serpins was measured by transverse urea gradient (TUG) gels. The results suggest that the acute phase proteins α1-PI and α1-ACT undergo conformational changes following oxidative mixed-disulfide formation and that α2-AP and angiotensinogen do not. The kinetics of disulfide formation was followed by measuring changes in absorbance at 412 nm resulting from Ellman's reaction of disulfide exchange. The rate of mixed-disulfide formation in albumin was 10-fold faster than in the serpin tested. The rate of disulfide exchange in α1-PI was 2-fold faster than that of α1-ACT. However, disulfide formation in α1-PI and α1-ACT was much slower than for any other serpin, e.g., α2-AP and angiotensinogen. We present evidence that α1-PI forms a dimer sensitive to thiol reduction, suggesting cysteinyl-mediated dimerization of α1-PI. The α1-PI also demonstrated two types of inter-protein disulfide linkages: one resulting in homodimer and other involving heterodimer formation. TUG–Western immunoblot methodology was developed to identify the conformational changes in serpins. We found that the conformational changes in serpins by mixed-disulfide formation are due to unfolding and not to decomposition or degradation in TUG gels. Using fluorescence measurements with isolated tryptic fragments of fluorescence-labelled elastase, we observed that the cysteinyl232in α1-PI interacted with the cysteinyl168of elastase in the proteinase–inhibitor complex. Our data suggests that serpin thiols may play an important role in forming a stable serpin–proteinase complex.Key words: serpin, extracellular matrix, proteinase, reduction, oxidation, inhibitor.
KATPchannel-dependent and -independent pathways of insulin secretion in isolated islets fromfa/faZucker rats
Authors: Chan Catherine B. , MacPhail Ruth M.
Published: December 2009

We hypothesized that altered insulin secretory patterns in obese (fa/fa) Zucker rats might be caused by changes in downstream stimulus–secretion coupling events, such as ATP-dependent potassium (KATP) channel activity. The functions of KATP-dependent and -independent pathways of insulin secretion were therefore compared in lean and fa/fa Zucker rat isolated islets. KATPchannel function was normal in fa/fa rat islets, as assessed by responsiveness to direct channel inactivators glybenclamide and quinine and by the receptor-mediated response to epinephrine and somatostatin. Altered sensitivity to glucose and mannoheptulose were explained by upstream alterations in glucose metabolism documented earlier. Despite normal inactivation of KATPchannels by ATP depletion of fa/fa rat islets, glucose-stimulated insulin secretion was not inhibited, leading to studies of a putative KATP-independent pathway. When islets were depolarized by incubating with 30 mM potassium and 0.25 mM diazoxide to bypass KATPchannels, glucose elicited a concentration-dependent response in both phenotypes. This response required glucose metabolism and Ca2+, as proven by experiments with nonmetabolizable glucose analogs and calcium chelation, but was only partially inhibited by a glycolytic inhibitor. Intermediates or products of oxidative metabolism are likely involved because α-ketoisocaproate also elicited a KATP-independent insulin response. The pattern of responses was similar in lean and fa/fa rat islets, indicating that neither of these pathways explains the insulin secretion by fa/fa rat islets depleted of ATP. In conclusion, phenotype-related differences in KATPchannel function were consistent with upstream changes in glucose metabolism in fa/fa rat islets. Further studies are required to understand the basis of insulin secretion in ATP-depleted islets from fa/fa rats.Key words: ATP-dependent K channels, insulin secretion, obesity, pancreatic islet, Zucker rat.
Regulation of 4-aminopyridine-sensitive, delayed rectifier K+channels in vascular smooth muscle by phosphorylation
Authors: Cole W. C. , Clément-Chomienne O. , Aiello E. A.
Published: December 2009

Voltage-gated, delayed rectifier K+current (KV) that is sensitive to 4-aminopyridine (4AP) block has been identified in all vascular smooth muscle tissues studied to date. These channels conduct outward, hyperpolarizing K+current that influences resting membrane potential and contributes to repolarization of action potentials. Smooth muscle cells in most arterial resistance vessels regulate Ca2+influx and contractile tone by low amplitude, tonic changes in membrane potential. Block of KVwith 4-aminopyridine leads to contraction and an enhanced myogenic response to increased intravascular pressure. We investigated the modulation of KVcurrents in isolated, freshly dispersed smooth muscle cells from rabbit portal vein and coronary arteries in whole-cell voltage clamp experiments. Our findings indicate that KVchannels are regulated by signal transduction mechanisms involving vasoactive agonists that activate cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). In this paper, the properties and potential function of KVchannels in vascular smooth muscle are reviewed. Further, the regulation and potential role of alterations in KVdue to β-adrenoceptor agonists, adenylyl cyclase and PKA, as well as angiotensin II, diacylglycerol, and PKC are discussed.Key words: potassium channels, smooth muscle, protein kinase A, protein kinase C, membrane potential.
Small GTPase-regulated phospholipase D in granulocytes
Authors: Houle Martin G. , Bourgoin Sylvain
Published: December 2009

This review examines the functional role of phospholipase D in the neutrophil. Phospholipase D is emerging as an important component in the signal transduction pathways leading to granulocyte activation. Through the second messenger it produces, phosphatidic acid, phospholipase D plays an active role in the regulation of granulocyte NADPH oxidase activation and granular secretion. Many factors from both the cytosol and the membrane are necessary for maximal phospholipase D activation. This paper will focus on the regulation of phospholipase D by low molecular weight GTP-binding proteins, tyrosine kinases, and protein kinase C.Key words: phospholipase D, low molecular weight GTP-binding proteins, tyrosine kinases, protein kinase C, granulocytes.
"Cross talk" between the bioactive glycerolipids and sphingolipids in signal transduction
Authors: Brindley David N. , Abousalham Abdelkarim , Kikuchi Yutaka , Wang Chuen-Neu , Waggoner David W.
Published: December 2009

Hydrolysis of phosphatidylcholine via receptor-mediated stimulation of phospholipase D produces phosphatidate that can be converted to lysophosphatidate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas phosphatidate and lysophosphatidate stimulate tyrosine kinases and activate the Ras–Raf–mitogen-activated protein kinase pathway. These three lipids can stimulate cell division. Conversely, activation of sphingomyelinase by agonists (e.g., tumor necrosis factor-α) causes ceramide production that inhibits cell division and produces apoptosis. If ceramides are metabolized to sphingosine and sphingosine 1-phosphate, then these lipids can stimulate phospholipase D and are also mitogenic. By contrast, ceramides inhibit the activation of phospholipase D by decreasing its interaction with the G-proteins, ARF and Rho, which are necessary for its activation. In whole cells, ceramides also stimulate the degradation of phosphatidate, lysophosphatidate, ceramide 1-phosphate, and sphingosine 1-phosphate through a multifunctional phosphohydrolase (the Mg2+-independent phosphatidate phosphohydrolase), whereas sphingosine inhibits phosphatidate phosphohydrolase. Tumor necrosis factor-α causes insulin resistance, which may be partly explained by ceramide production. Cell-permeable ceramides decrease insulin-stimulated glucose uptake in 3T3-L1 adipocytes after 2–24 h, whereas they stimulate basal glucose uptake. These effects do not depend on decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 or the interaction of insulin receptor substrate-1 with phosphatidylinositol 3-kinase. They appear to rely on the differential effects of ceramides on the translocation of GLUT1- and GLUT4-containing vesicles. It is concluded that there is a significant interaction and "cross-talk" between the sphingolipid and glycerolipid pathways that modifies signal transduction to control vesicle movement, cell division, and cell death.Key words: ceramides, insulin resistance, phosphatidate, phospholipases, signal transduction.
The DNA-dependent protein kinase, DNA-PK: 10 years and no ends in sight
Authors: Lees-Miller S. P.
Published: December 2009

The phenomenon of DNA-dependent protein phosphorylation was first described little over 10 years ago. Since then a DNA-dependent protein kinase, DNA-PK, has been purified from human cells and many of its biochemical properties have been characterized. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a DNA-targeting protein, Ku. When assembled on a suitable DNA molecule, the DNA-PK holoenzyme acts as a serine/threonine protein kinase that in vitro phosphorylates many DNA binding and non-binding proteins and transcription factors. Recent genetic studies point strongly to functions in DNA double-strand break repair and V(D)J recombination. In addition, biochemical studies suggest a role in the regulation of transcription. Here we discuss, from a historical perspective, the events leading up to our current understanding of the function of DNA-PK, including recent results from our own studies suggesting the involvement of DNA-PK in apoptosis and in viral infection of human cells.Key words: DNA-PK, DNA-dependent protein kinase, Ku, protein kinase, DNA repair, phosphatidyl inositol-3-kinase.
Molecular mechanisms underlying the interaction of motuporin and microcystins with type-1 and type-2A protein phosphatases
Authors: Craig Marcia , Luu Hue Anh , McCready Tara L. , Holmes Charles F. B. , Williams David , Andersen Raymond J.
Published: December 2009

Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA, and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase–toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and noduIarin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.Key words: microcystins, nodularin, motuporin, protein phosphatases, protein phosphorylation.
Expression of Oct-4 during differentiation of murine F9 cells
Authors: Wang Liangsu , Schultz Gilbert A.
Published: December 2009

Oct-4 is a transcription factor that shares a common structural motif with members of the POU family. The mRNA for Oct-4 is found in growing oocytes and in totipotent or pluripotent cells of the early mouse embryo. Oct-4 is down-regulated in embryos during differentiation events associated with blastocyst implantation and gastrulation. Oct-4 gene expression is also down-regulated when murine embryonic stem cells or embryonal carcinoma cells are induced to differentiate in the presence of retinoic acid. A polyclonal antibody that can recognize a unique peptide sequence in the C-terminus of mouse Oct-4 has been prepared. It specifically recognizes Oct-4 protein as tested by Western blots and gel mobility shift assays. This antibody has been used to measure Oct-4 protein levels during retinoic acid induced differentiation of F9 embryonal carcinoma cells. It was observed that Oct-4 protein was abundant in undifferentiated F9 cells but decreased to levels below detection as the cells differentiated, consistent with changes in levels of expression in early embryos.Key words: octamer, DNA-binding protein, transcription factor, embryonal carcinoma cells.
Chromatin structure and function: the heretical path to an RNA transcription factor
Authors: Krause Margarida O.
Published: December 2009

This review represents a synthesis of the work of the author and her collaborators through 40 years of research aimed at an understanding of chromatin composition and functional arrangement. It describes the progressive experimental stages, starting with autoradiography and protein analysis and continuing on to a more functional approach testing the template properties of intact nuclei, as well as nuclei depleted of, or reconstituted with, defined fractions extracted from the chromatin of other cell lines or tissues. As new questions were raised at each phase of these studies, the investigation was shifted from chromosomal proteins to the role of a small RNA that coextracted with one protein fraction and whose properties suggested a transcription-activating function. The active RNA was identified as a class in RNA, designated as 7 SK. Its properties suggested a role in the activation of two oncogenes, the SV 40 T-antigen and the mammalian c-myc gene. A detailed analysis of the c-myc gene expression during transformation induction in temperature-sensitive mammalian cells finally culminated in in vivo evidence for a role of 7 SK in c-myc deregulation, using cells transfected with antisense oligonucleotides to block 7 SK activity. This was followed by an investigation of promoter targeting by 7 SK RNP using electrophoretic mobility shift assays with whole or 7 SK-depleted cell extracts. Taken together, these studies indicate that 7 SK RNP participates in transformation-dependent deregulation of the c-myc gene by activation of two c-myc minor promoters. The implications of these findings are discussed.Key words: chromatin structure, histones, nonhistones, 7 SK RNA, the c-myc gene, transcription regulation, SV 40, transformation.
Influence of testosterone on chondroitin sulphate proteoglycan in the rat prostate
Authors: Terry Doris E. , Clark Albert F.
Published: December 2009

There are recognized interactions between prostatic stromal and epithelial cells. These interactions may be influenced by the composition of the extracellular matrix, which is composed of proteins such as collagen, laminin, fibronectin, and proteoglycans (PGs) such as chondroitin sulphate proteoglycan (CSPG). In our continuing studies on prostate biology, we examined the three lobes of the normal adult rat prostate, i.e., ventral, dorsal, and lateral, for CSPG by indirect immunofluorescence, using an immunospecific monoclonal antibody (CS-56) for the chondroitin sulphate (CS) moiety of the PG. Staining of the prostate sections with the CS-56 antibody followed by labelling with IgG fluorescein isothiocyanate conjugate indicated strong fluorescent signals associated with the ventral lobe basement membrane. The signal was stronger and more continuous in the distal acini than in the proximal acini. The staining of the dorsal and lateral lobes was less intense than that of the ventral lobe. Following castration of the rats, the basement membrane staining became discontinuous. Androgen replacement by administration of testosterone propionate (TP) reversed the effects of castration. Quantification of the total CS content showed decreases of about 60% in the ventral and lateral lobes after castration. TP administration for 14 days increased the total CS content several fold above the values for castrated rats in all the lobes. The results demonstrated that CS content was significantly higher for TP-treated animals, suggesting mat the expression of prostate CSPG is regulated by androgens. This approach should be useful in the study of the extracellular matrix in prostate biology.Key words: androgen, basement membrane, extracellular matrix, glycosaminoglycan, prostate.
Presence of two new fatty acid binding proteins in catfish liver
Authors: Pietro Santiago M. Di , Santomé José A.
Published: December 2009

A basic fatty acid binding protein (FABP), closely related to that of chicken liver, was isolated and characterized from catfish (Rhamdia sapo) liver in a previous work. Results herein show the presence of another two FABPs in which partial amino acid sequences reveal great similarity with the corresponding sequences of other already known FABPs belonging to the heart type. The purification procedures for both proteins involve gel filtration, anion-exchange chromatography, and sodium dodecyl sulfate – polyacrylamide gel electrophoresis (as a last step). Because both FABP N-termini were blocked, they were submitted to in-gel tryptic digestion and the resulting peptides were separated by high performance liquid chromatography, and sequenced by Edman degradation. One of these proteins presented the highest identity percentage when compared with those of the human and bovine heart and bovine brain (81%), and the other when compared with those of chicken retina (75%) and mouse and bovine heart FABP (70%). The presence of several FABPs plus the fact that they belong to different types, as found in the Rhamdia sapo liver, is unusual in mammals, which express a characteristic liver-type member of this protein family.Key words: fatty acid binding protein, liver, catfish, Rhamdia sapo.
Characterization of the Ca2+-binding properties of calgizzarin (S100C) isolated from chicken gizzard smooth muscle
Authors: Allen Bruce G. , Walsh Michael P. , Durussel Isabelle , Cox Jos A.
Published: December 2009

Calgizzarin is a Ca2+-binding protein of the S100 family that has been implicated in the regulation of cytoskeletal function through its Ca2+-dependent interaction with annexin I. The Ca2+-binding properties of calgizzarin (S100C) have not previously been thoroughly characterized. Calgizzarin, therefore, was purified from chicken gizzard smooth muscle by exploiting its Ca2+-dependent interaction with the hydrophobic matrix phenyl-Sepharose and is shown by45Ca2+overlay to bind Ca2+more weakly than does calmodulin. Gel filtration in the absence and presence of Ca2+suggested a dimeric structure of calgizzarin and indicated a more compact structure in the presence of Ca2+. Flow dialysis experiments indicated that, at physiological ionic strength, calgizzarin binds two Ca2+ions per monomer (four per native dimer), as predicted from the deduced amino acid sequence which contains two putative EF-hands, with [Ca2+]0.5of 0.52 mM and nHof 1.4 in the absence of Mg2+and [Ca2+]0.5of 0.3 mM and nHof 1.2 in the presence of 10 mM MgCl2. The hydrophobic fluorescent probe 2-p-toluidinylnaphthalene-6-sulphonate was used to demonstrate Ca2+-dependent exposure of a hydrophobic site(s) in calgizzarin. This approach also indicated the ability of calgizzarin to bind Zn2+. Interestingly, the affinity of calgizzarin for Ca2+was enhanced approximately 10-fold in the presence of the hydrophobic probe, possibly reflecting an increased affinity for Ca2+when calgizzarin binds to a target protein. Finally, the distribution of calgizzarin among chicken tissues was examined by immunoblotting: calgizzarin was expressed at its highest levels in lung tissue, followed by smooth muscle tissues (oesophagus, large intestine, trachea, and gizzard), kidney, liver, brain, and heart; it was not detected in small intestine or skeletal muscle.Key words: calcium-binding protein, calgizzarin, S100C, smooth muscle, zinc.
Purification and partial characterization of bovine kidney aldehyde dehydrogenase able to oxidize retinal to retinoic acid
Authors: Bhat Pangala V. , Poissant Lyne , Wang Xiao Ling
Published: December 2009

A NAD-dependent enzyme that catalyzes the oxidation of retinal to retinoic acid has been purified to homogeneity from bovine kidney. The procedures used in the purification included ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Affi-gel blue and chromatography on a Mono-Q anion-exchange column. On the Mono-Q column, the enzyme aldehyde dehydrogenase (ALDH) resolved into two activity peaks designated as ALDH1and ALDH2. The enzymes ALDH1and ALDH2were purified about 114- and 65-fold, respectively. Gel filtration chromatography of the partially purified native enzyme on Sephacryl S-200 HR exhibited a molecular mass of about 108 kDa. Electrophoresis of the purified enzymes under nondenaturing conditions showed a single protein band. However, sodium dodecyl sulfate – polyacrylamide gel electrophorsis indicated three protein bands in the 55, 30, and 22 kDa molecular mass regions. Both enzymes exhibited a broad substrate specificity oxidizing a wide variety of aliphatic and aromatic aldehydes. The ALDH1enzyme had a pI of 7.45 and exhibited a low Km(6.37 μM) for retinal, while the ALDH2enzyme was found to have very low Kmfor acetaldehyde (0.98 μM). Based on its kinetic properties, it is suggested that the ALDH1enzyme may be the primary enzyme for oxidizing retinal to retinoic acid in bovine kidney.Key words: aldehyde dehydrogenase, vitamin A, retinal oxidation, retinoic acid.
Modulation of the cleavage of glycosylphosphatidylinositol-anchored proteins by specific bacterial phospholipases
Authors: Sharom Frances J. , McNeil Gary L. , Glover John R. , Seier Sandra
Published: December 2009

Many enzymes are tethered to the extracellular face of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. These proteins can be released in soluble form by the action of GPI-specific phospholipases. Little is currently known about the factors modulating this release. We investigated the effects of several experimental variables on the cleavage of the GPI-anchored proteins 5′-nucleotidase, acetylcholinesterase, and alkaline phosphatase by phospholipases from Bacillus thuringiensis and Staphylococcus aureus. Phospholipase activity was not inhibited by isotonic salt and was relatively unaffected by buffer type and concentration. In both cases, the optimum pH for cleavage was ~ 6.5. Over 80% of 5′-nucleotidase activity present in the lymphocyte plasma membrane was cleaved by the B. thuringiensis enzyme, and the initial rate of release was linear with phospholipase concentration. All three GPI-anchored proteins were released from lymphocyte plasma membrane at comparable phospholipase concentrations, suggesting that they have similar anchor structures. The catalytic activity of 5′-nucleotidase appeared to increase following conversion to the soluble form. The relative surface charge of the host plasma membrane modulated catalytic activity towards GPI-anchored proteins, depending on the net charge of the phospholipase. Studies on purified lymphocyte 5′-nucleotidase reconstituted into bilayers of dimyristoylphosphatidylcholine indicated that the efficiency of phospholipase cleavage was 12- to 50-fold lower when compared with the native plasma membrane. The ability of the phospholipase to cleave the GPI anchor was further reduced when the bilayer was in the gel phase.Key words: glycosylphosphatidylinositol anchor, phospholipase C, 5′-nucleotidase, acetylcholinesterase, alkaline phosphatase.
Heterogeneity of myosin heavy-chain expression in fast-twitch fiber types of mature avian pectoralis muscle
Authors: Rosser B. W. C. , Waldbillig D. M. , Wick M. , Bandman E.
Published: December 2009

The aims of this study are to investigate the diversity of myosin heavy-chain (MyHC) expression among avian fast-twitch fibers, and to test the hypothesis that dissimilar MyHC isoforms are found in each of the principal avian fast-twitch fiber types. MyHCs within the muscle fibers of the pectoralis of 31 species of bird are characterized using immunocytochemical methods. A library of 11 monoclonal antibodies previously produced against chicken MyHCs is used. The specificity of these antibodies for MyHCs in each of the muscles studied is confirmed by Western blots. The results show that avian fast-twitch glycolytic fibers and fast-twitch oxidative-gylcolytic fibers can contain different MyHCs. Among the species studied, there is also a conspicuous variety of MyHC isoforms expressed. In addition, the results suggest that two epitopes are restricted to chickens and closely allied gallinaceous birds. There are no apparent correlations between MyHC epitope and presupposed contractile properties. However, the presence of different isoforms in different fast-twitch fiber types suggests a correlation between isoform and contractile function.Key words: muscle, fiber, myosin, avian.
Blistering skin diseases: models for studies on epidermal–dermal adhesion
Authors: Bruckner-Tuderman Leena
Published: December 2009

Blistering skin diseases represent a genetically and biologically heterogeneous group of pathologic conditions with attenuated epithelial adhesion in the skin or in mucosal surfaces. Investigations on genetic blistering disorders with ultrastructural analysis, determination of candidate genes, and isolation and characterization of the target proteins have had a major impact on our understanding of normal epithelial–mesenchymal interactions and epithelial cell adhesion in general. A high degree of molecular heterogeneity underlies the different pathological phenotypes, and certain genotype–phenotype correlations are starting to emerge. Accumulating data on the molecular basis of different blistering diseases have already disclosed ample new information on the physiological functions of many of the molecular components of the dermal–epidermal junction, and we expect a rapid expansion of knowledge on the field when genotype–phenotype correlations of more patients are discerned.Key words: anchoring fibrils, basement membrane, blistering disorders, COL7A1 mutations, recessive dystrophic epidermolysis bullosa.
Heparan sulfate proteoglycans function in the binding and degradation of vitronectin by fibroblast monolayers
Authors: Wilkins-Port Cynthia E. , McKeown-Longo Paula J.
Published: December 2009

Vitronectin, a 75-kDa plasma protein is also found in the extracellular matrix, where it is believed to promote cell adhesion and migration. In addition to its role in adhesion, matrix vitronectin is also believed to function as an opsonin promoting the clearance of thrombin–serpin complexes from the matrix. Vitronectin is cleared from the matrix by receptor-mediated endocytosis followed by lysosomal degradation, suggesting that cells can regulate the levels of vitronectin present in the matrix. However, the mechanism by which plasma vitronectin associates with the extracellular matrix remains unclear. Studies were conducted to define the binding site(s) for vitronectin in fibroblast cell layers. Sodium chlorate, a competitive inhibitor of proteoglycan sulfation, produced a dose-dependent decrease in both binding and degradation of vitronectin. This inhibition was reversible in that removal of chlorate returned both binding and degradation of vitronectin to near control levels within 24 h. The binding of vitronectin to cell layers was not dependent on cells because vitronectin bound directly to isolated matrix. Isolated matrices prepared from cell layers treated with sodium chlorate also exhibited a dose-dependent decrease in vitronectin binding, consistent with the binding site for vitronectin in the matrix being sulfated proteoglycans. Binding and degradation of vitronectin were also sensitive to the addition of exogenous heparin, suggesting that the heparin binding domain of vitronectin was mediating binding to the matrix. Incubating fibroblast monolayers with heparinase III resulted in a 40% decrease in binding and degradation of vitronectin. Taken together, the above findings suggest that vitronectin's binding to the matrix and its subsequent degradation are dependent on heparan sulfate proteoglycans.Key words: endocytosis, vitronectin, proteoglycan, extracellular matrix.
An in vitro system for the study of matrix metalloproteases during decidualization in the mouse
Authors: Romagnano Linda , Afonso Suzanne , Babiarz Bruce
Published: December 2009

Decidualization results in the remodeling of the extracellular matrix with the loss of collagen type I and the appearance of basement membrane matrix components. We have developed an in vitro assay system to study matrix metalloproteases during mouse decidualization. Uterine stroma, or decidua isolated from day 7.5 pregnant mice, were grown on a three-dimensional collagen type I matrix (Vitrogen). Gelatin zymography of conditioned media from these cultures showed constitutive secretion of processed forms of gelatinase A at 65, 62, and 59 kDa with 62 kDa predominating. Similar patterns of gelatinase A expression were obtained from tissue lysates of decidualizing uteri from days 5.5 to 7.5 of development. Cells cultured on Vitrogen, but not on plastic or matrix-coated dishes, were able to process the proenzyme to the 59 kDa form as observed in vivo. Only stroma cells cultured on a coating of collagen type I displayed the same increase in the 59 kDa zymogen. Decidua cells grown on Vitrogen attached and then migrated into aggregates that eventually penetrated the gel and spread as differentiated decidua on the underlying plastic. These preliminary results suggested that the in vitro assay system can be used to study the role of metalloproteases in matrix remodeling during decidualization.Key words: matrix metalloproteases, uterine stroma, decidua, gelatinase A, Vitrogen.
Functional expression of Na–Ca exchanger clones measured with the fluorescent Ca2+-indicating dye fluo-3
Authors: Schnetkamp Paul P. M.
Published: December 2009

The process of Ca2+homeostasis is of prime importance to all cells because of the ubiquitous role of cytoplasmic Ca2+as an intracellular messenger and the cytotoxicity of sustained elevated cytosolic Ca2+concentrations. Two classes of plasma membrane proteins are responsible for maintaining cytosolic free Ca2+in the submicromolar range against a very large electrochemical Ca2+gradient across the plasma membrane, the ATP-driven Ca2+pump and Na–Ca exchangers. Two types of Na–Ca exchangers are known, the 3Na:1Ca exchangers found in heart, brain, kidney, and most other tissues and the 4Na:1Ca+1K exchanger found in retinal rod and cone photoreceptors. Functional expression of Na–Ca(/K) exchangers is most often measured as45Ca uptake in Na+-loaded cells or as Na–Ca exchange currents with the giant excised patch technique. In this study, two functional assays used to detect expression of the bovine heart Na–Ca exchanger in CHO cells are described. Both assays are based on measurements of cytosolic free Ca2+with the fluorescent Ca2+-indicating dye fluo-3 and should be equally applicable in the study of functional expression of both Na–Ca and Na–Ca/K exchanger clones.Key words: Na–Ca exchange, Ca homeostasis, fluo-3, ion transport, functional expression.
Phosphorylation and activation of smooth muscle myosin light chain kinase by MAP kinase and cyclin-dependent kinase-1
Authors: Morrison Donna L. , Sanghera Jasbinder S. , Stewart Justine , Pelech Steven L. , Sutherland Cindy , Walsh Michael P.
Published: December 2009

Smooth muscle myosin light chain kinase (MLCK) features several consensus sites of phosphorylation by proline-directed protein serine/threonine kinases. The phosphorylation of MLCK by two proline-directed kinases isolated from sea star oocytes, i.e., p44mpk(Mpk, a mitogen-activated protein kinase homologue) and cyclin-dependent kinase-1 (CDK1, also known as p34cdc2), was investigated. Chicken gizzard MLCK was phosphorylated on seryl and fhreonyl residues by both Mpk and CDK1. Phosphorylation of MLCK to 0.6 mol Pi/mol by Mpk increased the Vmaxof phosphotransferase activity towards a synthetic peptide corresponding to residues 11–23 of the 20-kDa light chain of myosin by 1.6-fold. Phosphorylation of MLCK to 1.0 mol Pi/mol by CDK1 increased the Vmaxby 2.3-fold. Phosphorylation by either kinase had no significant effect on the concentration of calmodulin required for half-maximal activation of MLCK. Analysis of the phosphorylation of synthetic peptides containing consensus phosphorylation sites for Mpk and CDK1 indicated that the major site of phosphorylation in MLCK by Mpk was Ser-834, and by CDK1 was Thr-283. Both of these sites are located outside the cafmodulin-binding site (residues 796–815), consistent with the observation that phosphorylation by Mpk or CDK1 was unaffected by the presence of bound Ca2+/calmodulin. These results indicate that MLCK activity may be regulated by phosphorylation catalyzed by proline-directed kinases, possibly directed at Thr-40 and Thr-43 at the amino terminus of MLCK.Key words: myosin light chain kinase, mitogen-activated protein kinase, cyclin-dependent kinase.