bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxytocin induced cAMP‐dependent protein kinase activation and urokinase‐type plasminogen activator production in LLC‐PK1 renal epithelial cells is mediated by the vasopressin V2‐receptor
Authors: Jans David A. , Pavo Imre , Fahrenholz Falk
Published: July 2002

Using a variety of peptide analogues of oxytocin (OT) and Arg8‐vasopressin (AVP), OT‐mediated induction of urokinase‐type plasminogen activator (uPA) was examined in LLC‐PK1 renal epithelial cells, which possess distinct high‐affinity receptors of both the OT‐ and vasopressin renal (V2‐) types. OT or OT‐receptor specific agonists induced concentration‐dependent cAMP synthesis, activation of the cAMP‐dependent protein kinase (cAMP‐PK) and uPA production consistent with their respective binding affinities for the V2‐ and not the OT‐receptor. OT‐mediated uPA induction could be inhibited in a concentration‐dependent fashion by coincubation with a V2/V1‐receptor specific antagonist, but not by an OT‐receptor specific antagonist. Results implied that stimulation of cAMP‐ and uPA responses in LLC‐PK1 cells by OT was V2‐receptor‐mediated.
Structure‐activity profiles of macrolactam immunosuppressant FK‐506 analogues
Authors: Kawai Megumi , Lane Benjamin C. , Hsieh Gin C. , Mollison Karl W. , Carter George W. , Luly Jay R.
Published: July 2002

The immunosuppressive agent FK‐506 has received much attention due to its efficacy and potency in the areas of transplant rejection and autoimmune disease. Calcineurin, a Ca2+‐calmodulin activated phosphatase, was recently implicated in the immunosuppressive mechanism of FK‐506. In our ongoing search for superior immunosuppressive agents, we have synthesized several analogues of FK‐506 and tested their mechanistic and immunosuppressive actions. It was found that C‐18 hydroxyl analogues of ascomycin, an analogue of FK‐506 also called FR900520, bound tightly to immunophilin FKBP‐12, but do not show any immunosuppressive activity in vitro or in vivo despite good bioavail‐ability. Further, they reverse the inhibition of calcineurin caused by FK‐506/FKBP‐12 complex.
Identification of domains of a cloned rat brain GABA transporter which are not required for its functional expression
Authors: Bendahan Annie , Kanner Baruch I.
Published: July 2002

The sodium and chloride coupled γ‐aminobutyric acid (GABA) transporter purified from rat brain, belongs to a superfamily of neurotransmitter transporters. They are involved in the termination of synaptic transmission and are predicted to have 12 membrane spanning α‐helices with both amino‐ and carboxyl‐termini oriented toward the cytoplasm. In order to define the domains not required for functional expression, we have constructed and expressed a series of deletion mutants in GAT‐1, the cDNA clone encoding for the transporter. Transporters truncated at either end until just a few amino‐acids distance from the beginning of helix 1 and the end of helix 12, retain their ability to catalyze sodium and chloride‐dependent GABA transport.
Deletion of NH2− and COOH‐terminal sequences destroys function of the Ca2+ ATPase of rabbit fast‐twitch skeletal muscle sarcoplasmic reticulum
Authors: Skerjanc Ilona S. , Clarke David M. , Loo Tip W. , MacLennan David H.
Published: July 2002

Deletion mutants of the Ca2+ ATPase of rabbit fast‐twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) were constructed and expressed in COS‐1 cells. The mutants were expressed at levels 7‐ to 15‐fold lower than the wild‐type and were inactive. In vitro transcription‐translation‐insertion experiments showed that deletion of transmembrane sequences M1 and M2, but not of M8, M9, M10 or the NH2− terminal 30 amino acids inhibited the stable insertion of the enzyme into the membrane. Thus there was no correlation between loss of function and membrane insertion. A signal sequence for membrane insertion may exist in M1 and M2.
Creatine kinase function in mitochondria isolated from gravid and non‐gravid guinea‐pig uteri
Authors: Clark Joseph F. , Kuznetsov Andrey V. , Khuchua Zaza , Veksler Vladimir , Ventura-Clapier Renée , Saks Valdur
Published: July 2002

Mitochondria from gravid and non‐gravid guinea pig uteri were isolated and respiratory rates examined to determine the responses to ATP, ADP and creatine. It was found that mitochondria isolated from gravid uterus had (i) a markedly higher respiration rate in state 3; (ii) a greater activation of respiration by creatine in the presence of 0.1 mM ATP and (iii) an elevated specific activity of mitochondrial creatine kinase. It was shown by a competitive enzyme method, using pyruvate kinase to trap ADP, that despite the presence of creatine kinase in the mitochondria, there is no functional coupling between mitochondrial creatine kinase and oxidative phosphorylation as has been shown for striated muscle. It is suggested that the function of uterine Mi‐CK is to favour high energy phosphate turnover in conditions of increased metabolic demand in gestating uterine smooth muscle.
Cloning a novel human brain inward rectifier potassium channel and its functional expression in Xenopus oocytes
Authors: Tang Weimin , Yang Xian-Cheng
Published: July 2002

We have cloned a novel inward rectifier K+ channel (hIRK2) from a human frontal cortex cDNA library. The amino acid sequence of hIRK2 shares 60% and 40% identity with the mouse IRK1 and the rat ROMK1 channels, respectively. Xenopus oocytes injected with hIRK2 cRNA showed an inwardly rectifying K+ current that had a prominent ‘N‐shape’ I‐V curve and was blocked by extracellular Ba2+. The hIRK2 channel has two unique features: (a) an 18 amino acid insertion between the first transmembrane region and the pore, and (b) restricted mRNA distribution found only in human brain and heart.
The cupredoxin fold is found in the soluble CuA and CyoA domains of two terminal oxidases
Authors: Wittung Pernilla , Källebring Bruno , Malmström Bo G.
Published: July 2002

The CD spectra of the CuA domain from subunit II of Paracoccus cytochrome c oxidase and the CyoA domain of subunit II from E. coli quinol oxidase have been recorded in the wavelength region 260‐185 nm. A computer program based on a set of CD spectra of proteins with known structures, and employing the stastistical method of variable selection, has been used to estimate the distribution of five forms of secondary structure. The analysis was improved by including the CD spectra of azurin and plastocyanin in the basis set. For the CuA domain, an estimate from the primary structure was also made. The results show that the soluble domains have the cupredoxin fold, with very little helical structure and a predominance of β‐strands. The CyoA domain is very similar to azurin, but the β‐structure in the CuA protein resembles that in plastocyanin.
Molecular cloning of cDNA encoding the 110 kDa and 21 kDa regulatory subunits of smooth muscle protein phosphatase 1M
Authors: Chen Yu Hua , Chen Mao Xiang , Alessi Dario R. , Campbell David G. , Shanahan Catherine , Cohen Philip , Cohen Patricia T.W.
Published: July 2002

The structures of the M110 and M21 regulatory subunits of protein phosphatase‐1M, the major enzyme which dephosphorylates myosin in smooth muscle, have been deduced from cloned cDNAs. The N‐terminus of the M110 subunit from rat aorta contains seven ankyrin repeats, while the C‐terminus of the M21 subunit from chicken gizzard contains a leucine zipper motif. The M110 subunit is expressed in two different forms which differ in their C‐terminal sequences. One of these is highly homologous to the whole of the M21 subunit.
Increase of c‐jun mRNA upon hypo‐osmotic cell swelling of rat hepatoma cells
Authors: Finkenzeller Günter , Newsome William , Lang Florian , Häussinger Dieter
Published: July 2002

c‐jun mRNA levels were increased in rat hepatoma cells (H4‐II‐E‐C3) when exposed to hypotonie medium (205 ) with a maximal induction observed after 1 h of hypotonie exposure. At this time point an approximate 5‐fold increase in c‐jun expression could be detected in relation to nonnotonic control incubations (305 ). Hypertonic exposure (405 mosmol/1) had only a slight effect on c‐jun expression. In contrast to the increased c‐jun mRNA levels under hypotonic conditions, expression of the c‐fos proto‐oncogene was unaffected by changes in the osmolarity. The hypotonicity‐induced increase in c‐jun expression was also detectable in the presence of a protein kinase C (PKC) inhibitor. This indicates that PKC is not involved in the signal transduction pathway leading to c‐jun expression upon hypotonic cell swelling in these cells.
Molecular mechanisms of tumor necrosis factor‐induced cytotoxicity
Authors: Beyaert Rudi , Fiers Walter
Published: July 2002

Although TNF plays an important role in several physiological and pathological conditions, the hallmark of this important cytokine has been its selective cytotoxic activity on tumor cells. Since its cloning in 1984, understanding of how TNF selectively kills tumor cells has been the subject of research in many laboratories. Here we review TNF‐induced post‐receptor signaling mechanisms which seem to be involved in the pathway to cytotoxicity.
An unusual metal‐binding cluster found exclusively in the avian breast muscle troponin T of Galliformes and Craciformes
Authors: Jin Jian-Ping , Smillie Lawrence B.
Published: July 2002

A repeating metal‐binding (Cu2+ > Ni2+ > Zn2+ ~ Co2+) sequence (HE/AEAH)4 has been identified in troponin T isoforms specifically expressed in the breast but not leg muscles of all Galliformes and Craciformes. It is absent in the skeletal and cardiac muscles of mammals and all other avian species investigated. Concentration of the metal‐binding sites is adequate to affect free metal levels in the muscle cell and we suggest a possible link between its presence in breast muscle of Galliformes and the high ratio of breast muscle to total body muscle mass and explosive but short‐lived flight pattern of these birds. This sequence can be used for a highly selective metal‐affinity chromatographic purification of muscle or engineered TnTs even in high salt and/or urea.
Synthesis and characterization of sapecin and sapecin B
Authors: Il Kim Jae , Iwai Hideo , Kurata Shoichiro , Takahashi Masami , Masuda Katuyoshi , Shimada Ichio , Natori Shunji , Arata Yoji , Sato Kazuki
Published: July 2002

Two insect defencins, sapecin and sapecin B, were chemically synthesized to confirm their structure and antibacterial activity and also to examine the possibility that these peptides bind to the same site on the large conductance calcium‐activated potassium channel as charybdotoxin. Both synthetic peptides showed the same antibacterial activity as native sapecins, indicating that the synthetic peptides folded correctly in the chemical synthesis. Synthetic sapecins did not show an inhibitory effect on [125I]charybdotoxin binding to rat brain synaptic membranes, suggesting that sapecin B recognizes a different binding site from that of charybdotoxin despite the similar structural motif.
GTP‐dependent binding of Gi, Go and Gs to the γ‐subunit of the effector of Gt
Authors: Otto-Bruc Annie , Vuong T.Minh , Antonny Bruno
Published: July 2002

The γ‐subunit of the cGMP‐phosphodiesterase (PDEγ) of retinal rods forms a tight complex with the activated α‐subunit of transducin (GtαGTPγS). We observe that while PDEγ is not the physiological effector of other Gα subtypes, it can still detectably interact with them. This interaction is strong with Gilα and Gi3α (K
d ≈ 10 nM) and weaker with Goα and Ggα (K
d ≈ 1 μM). For all these Gα subtypes, similar intrinsic fluorescence changes are observed upon PDEγ binding. Moreover, similar relative decreases in affinity are obtained when the GDP forms of Gilα, Gi3α or Gtα are used in lieu of the GTP forms. This points to a conserved GTP‐dependent effector‐interaction domain.
Expression of protein kinase C isoforms in smooth muscle cells in various states of differentiation
Authors: Assender Jean W. , Kontny Ewa , Fredholm Bertil B.
Published: July 2002

We analysed the expression of protein kinase C (PKC) isoforms in smooth muscle cells (SMC) in various states of differentiation, using Western blots and thermocycle amplification of mRNA. SMC isolated from intact tissues express three isoforms of PKC, namely α, δ, and ζ‐ Following cell culture, SMC additionally express mRNA for PKC‐ϵ, but significant amounts of the corresponding protein were not detected. Transformed SMC, such as the cell line DDT, MF‐2, express both mRNA and protein for PKC‐ϵ, lack the δ isoenyzme, whilst maintaining the expression of the α and ζ isoforms. Thus PKC‐δ and ϵ isoenzyme expression appears to vary with the state of differentiation of these cells, with PKC‐ϵ expression increasing as the cells become proliferative.
Isolation and partial cloning of ryanodine‐sensitive Ca2+ release channel protein isoforms from human myometrial smooth muscle
Authors: Lynn Stephen , Morgan Joanna M. , Lamb Heather K. , Meissner Gerhard , Gillespie James I.
Published: July 2002

Partial cDNAs of the ryanodine receptor were cloned using PCR analysis from reverse transcribed total and mRNA, extracted from freshly isolated pregnant, non‐pregnant, and cultured human myometrial smooth muscle. The identity of these clones was confirmed by nucleotide sequencing of the fragments and indicate the expression of both the skeletal and brain ryanodine receptor isoforms in these preparations. In freshly isolated non‐pregnant myometrial tissue, membrane fractions displaying specific [3H]ryanodine binding activities were isolated using density gradient centrifugation. SDS‐PAGE of the sucrose gradient fractions indicated the specific comigration of a polypeptide with a molecular mass of ∼ 544 kDa with the ryanodine binding activity.
Slow Ca2+‐induced inactive/active transition of the energy‐dependent Ca2+ transporting system of rat liver mitochondria: clue for Ca2+ influx cooperativity
Authors: Kasparinsky F.O. , Vinogradov A.D.
Published: July 2002

Rat liver mitochondria essentially free of endogenous Ca2+ show low initial rate of energy‐dependent Ca2+ uptake. Preincubation of mitochondria under de‐energized conditions in the presence of small amounts of external Ca2+ results in a 8–10‐fold time‐dependent increase of energy‐dependent Ca2+ uptake. Ca2+‐dependent activation of the Ca2+‐transporting system follows first‐order kinetics ( in the presence of 5 μM Ca2+ at 20°C). Ca2+‐activated mitochondria demonstrate a simple hyperbolic initial rate‐Ca2+ concentration dependence, whereas strong apparent cooperativity is observed in the velocity‐substrate curves for Ca2+‐depleted mitochondria. It is concluded that apparent cooperativity of the energy‐dependent Ca2+ uptake is due to slow (as compared with the ‘turnover number’) activation of a Ca2+‐specific uniporter which is inactive in the absence of external Ca2+.
Thyroid hormone promotes BCL‐2 expression and prevents apoptosis of early differentiating cerebellar granule neurons
Authors: Muller Y. , Rocchi E. , Lazaro J.B. , Clos J.
Published: July 2002

Programmed cell death is a basic cellular process that has aroused much interest in recent years. Like immune cells, cultures of cerebellar granule neurons are very homogeneous and provide a unique opportunity for quantifying by flow cytometry one form of programmed cell death in the CNS, the apoptosis, and for studying its regulation by neurotrophic factors. We found that thyroid hormone promoted postmitotic survival by preventing the apoptosis of newly formed and early differentiated granule neurons in a dose‐dependent manner. This regulation could be through the protein bcl‐2, which is known to prevent cell death. This protein was present at all stages of granule neuron differentiation and appeared to be developmentally regulated. It was underexpressed in apoptotic granule neurons. The protein content of the cerebellum in hypothyroid rats was drastically reduced. In contrast, thyroid hormone caused a marked dose‐dependent increase in the amounts of this protein in granule neuron cultures. The possibility that thyroid hormone may be directly or indirectly required to promote cell survival is discussed, in terms of the hormone control of the local delivery of neurotrophins, such as NGF and NT‐3, as well as the expression of their low affinity receptors, gp75. We suggest that thyroid hormone has a permissive action on the developing CNS.
The α2 and α5 integrin genes: identification of transcription factors that regulate promoter activity in epidermal keratinocytes
Authors: Corbi Angel L , Jensen Uffe Birk , Watt Fiona M
Published: July 2002

We analysed the activity of the proximal promoters of the α2 and α5 integrin genes in human keratinocytes. An AP‐1 site, found in the α5 but not the α2 promoter, bound c‐Jun/c‐Fos dimers and contributed strongly to promoter activity. Both promoters had a CCAAT/enhancer binding protein (C/EBP) binding site: the α5 C/EBP element enhanced activity, while the α2 site was a negative regulatory element. C/EBP overexpression repressed the activity of both promoters, but the effect was independent of occupancy of the identified C/EBP binding sites, suggesting interactions with additional transcription factors. We propose that upregulation of C/EBPs contributes to the inhibition of integrin transcription during keratinocyte terminal differentiation, while AP‐1 factors play a role in the selective induction of the α5 gene during wound healing.
Myosin light chain kinase binding to actin filaments
Authors: Smith Lula , Stull James T
Published: July 2002

Smooth muscle myosin light chain kinase (MLCK) plays important roles in contractile‐motile processes of a variety of cells. Three DFRxxL motifs at the kinase N‐terminus (residues 2–63) are critical for high‐affinity binding to actin‐containing filaments [Smith et al. (1999) J. Biol. Chem. 274, 29433–29438]. A GST fusion protein containing residues 1–75 of MLCK (GST75‐MLCK) bound maximally to both smooth muscle myofilaments and F‐actin at 0.28 and 0.31 mol GST75‐MLCK/mol actin with respective K
D values of 0.1 μM and 0.8 μM. High‐affinity binding of MLCK to actin‐containing filaments may be due to each DFRxxL motif binding to one actin monomer in filaments.
Mechanism of nitric oxide‐induced apoptosis in human neuroblastoma SH‐SY5Y cells
Authors: Moriya Ryuichi , Uehara Takashi , Nomura Yasuyuki
Published: July 2002

We have attempted to elucidate the precise mechanism of nitric oxide (NO)‐induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD‐AFC, VDVAD‐AFC, and LEHD‐AFC (specific substrates for caspase‐3‐like protease (caspase‐3 and ‐7), caspase‐2, and caspase‐9, respectively) were observed by treatment with NO. Western blot analysis showed that pro‐forms of caspase‐2, ‐3, ‐6, and ‐7 are decreased during apoptosis. Interestingly, Ac‐DEVD‐CHO, a caspase‐3‐like protease inhibitor, blocked not only the decreases in caspase‐2 and ‐7, but also the formation of p17 from p20 in caspase‐3 induced by NO, suggesting that caspase‐3 exists upstream of caspase‐2 and ‐7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase‐activated DNase (CAD) (CAD activation).
Stimulation of p38 MAP kinase reduces acidosis and Na+ overload in preconditioned hepatocytes
Authors: Carini Rita , Grazia De Cesaris Maria , Splendore Roberta , Albano Emanuele
Published: July 2002

Ischemic preconditioning has been shown to improve liver resistance to hypoxia/reperfusion damage. A signal pathway involving A2A‐adenosine receptor, Gi‐proteins, protein kinase C and p38 MAP kinase is responsible for the development of hypoxic preconditioning in hepatocytes. However, the coupling of this signal pathway with the mechanisms responsible for cytoprotection is still unknown. We have observed that stimulation of A2A‐adenosine receptors or of p38 MAPK by CGS21680 or anisomycin, respectively, appreciably reduced intracellular acidosis and Na+ accumulation developing during hypoxia. These effects were reverted by p38 MAPK inhibitor SB203580 as well as by blocking vacuolar proton ATPase with bafilomycin A1. SB203580 and bafilomycin A1 also abolished the cytoprotective action exerted by both CGS21680 and anisomycin. We propose that the stimulation of p38 MAPK by preconditioning might increase hepatocyte resistance to hypoxia by activating proton extrusion through vacuolar proton ATPase, thus limiting Na+ overload promoted by Na+‐dependent acid buffering systems.
Malaria parasite Plasmodium gallinaceum up‐regulates host red blood cell channels
Authors: Thomas Serge L.Y. , Egée Stéphane , Lapaix Franck , Kaestner Lars , Staines Henry M. , Ellory J.Clive
Published: July 2002

The properties of the malaria parasite‐induced permeability pathways in the host red blood cell have been a major area of interest particularly in the context of whether the pathways are host‐ or parasite‐derived. In the present study, the whole‐cell configuration of the patch‐clamp technique has been used to show that, compared with normal cells, chicken red blood cells infected by Plasmodium gallinaceum exhibited a 5–40‐fold larger membrane conductance, which could be further increased up to 100‐fold by raising intracellular Ca2+ levels. The increased conductance was not due to pathways with novel electrophysiological properties. Rather, the parasite increased the activity of endogenous 24 pS stretch‐activated non‐selective cationic (NSC) and 62 pS calcium‐activated NSC channels, and, in some cases, of endogenous 255 pS anionic channels.
Association of phosphatidylinositol 3‐kinase with the photo‐oncogene product Cbl upon CD38 ligation by a specific monoclonal antibody in THP‐1 cells
Authors: Matsuo Tsuyoshi , Hazeki Kaoru , Tsujimoto Noriko , Inoue Shin-ichi , Kurosu Hiroshi , Kontani Kenji , Hazeki Osamu , Ui Michio , Katada Toshiaki
Published: July 2002

We reported that ecto‐NAD+ glycohydrolase activity induced upon differentiation of HL‐60 cells with retinoic acid is localized on the extracellular domain of CD38 and that CD38 ligation by a specific monoclonal antibody, HB‐7, is followed by rapid tyrosine phosphorylation of cellular proteins including a proto‐oncogene product, Cbl. In the present study, we investigated intracellular signaling linked to the HB‐7‐induced Cbl phosphorylation in dibutyryl cAMP‐treated THP‐1 cells. The 85‐kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3‐kinase was immunoprecipitated with anti‐Cbl antibody in a manner dependent on the tyrosine phosphorylation of Cbl. PI 3‐kinase activity was also observed in the immunoprecipitated fractions containing tyrosine‐phosphorylated Cbl. The phosphorylated form of Cbl, which had been separated from the CD38‐stimulated cells, was capable of directly binding to a recombinant p85 fused to glutathione S‐transferase. Thus, the direct association of tyrosine‐phosphorylated Cbl with PI 3‐kinase, possibly leading to the kinase activation, appeared to be involved in intracellular signaling caused by the CD38 ligation.
Overexpression of Gsα in NG108‐15, neuroblastoma X glioma cells: effects on receptor regulation of the stimulatory adenylyl cyclase cascade
Authors: Mullaney Ian , Craig Carr I. , Milligan Graeme
Published: July 2002

Neuroblastoma X glioma hybrid, NG108‐15 cells were stably transfected with an epitope tagged variant of Gsα(HAGsα). The introduced HA‐Gsα was able to interact with the IP prostanoid receptor and was able to stimulate adenylyl cyclase activity as measured by an enhanced capacity of membrane extracts to reconstitute NaF‐dependent adenylyl cyclase activity to membranes of S49 lymphoma cyc− cells. Despite this, neither the maximal stimulation nor the potency of agonist ligands at the IP prostanoid, A2 adenosine or secretin receptors was altered substantially compared to the parental cells although the basal adenylyl cyclase activity was increased. These data indicate that cellular levels of Gsα do not limit signal transduction capacity in NG108‐15 cells, whereas enhanced expression of adenylyl cyclase allows greater maximal cAMP generation following receptor activation (MacEwan, D.J., Kim, G.D. and Milligan, G. (1996) Biochem. J. 318, 1033–1039).
Cloning and functional expression of a novel isoform of ROMK inwardly rectifying ATP‐dependent K+ channel, ROMK6 (Kir1.1 f)
Authors: Kondo Chikako , Isomoto Shojiro , Matsumoto Shigeto , Yamada Mitsuhiko , Horio Yoshiyuki , Yamashita Shizuya , Takemura-Kameda Kaoru , Matsuzawa Yuji , Kurachi Yoshihisa
Published: July 2002

We have identified from rat kidney a novel isoform of ROMK/Kir1.1, designated ROMK6/Kir1.1f. ROMK6 was nearly identical to ROMK1, but possessed an 122‐bp insertion in the 5′ region. Its deduced amino acid sequence was shorter by 19 amino acids than that of ROMK1 in the amino‐terminus. Unlike other previously reported ROMK isoforms, ROMK6 mRNA was ubiquitously expressed in various tissues, including kidney, brain, heart, liver, pancreas and skeletal muscle. Xenopus oocytes injected with ROMK6 cRNA expressed a Ba2+‐sensitive weakly inwardly rectifying K+ current. These results indicate that ROMK6 is a novel functional K+ channel and might be involved in K+ secretion in various tissues.
Cysteine‐rich regions of protein kinase Cδ are functionally non‐equivalent
Authors: Hunn Martin , Quest Andrew F.G
Published: July 2002

Regulatory domain elements of the non‐calcium‐dependent protein kinase Cδ (nPKCδ), including either or both of the cysteine‐rich regions Cys1(δ) and Cys2(δ), were expressed as fusion proteins with glutathione‐S‐transferase and characterized using liposomal or mixed micellar phorbol ester binding assays. Fusion proteins containing Cys2(δ) bound phorbol‐12,13‐dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(δ). Likewise, in mixed micellar assays, fusion proteins with Cys2(δ) bound PDBu with high affinity (K
d: 14–37 nM) and to significant stoichiometric levels (0.23–0.66 mol/mol), but no binding could be detected for proteins with Cys1(δ) only. The PS dependence of PDBu binding to Cys2(δ) was highly cooperative with Hill numbers lying in the range of 2.5–5.2. These results demonstrate the presence of striking functional differences between the cysteine‐rich regions of nPKCδ and the calcium‐dependent isoform, cPKCγ, where both cysteine‐rich regions represent functional PDBu binding elements.
Transforming growth factor‐β1 induces activation of Ras, Raf‐1, MEK and MAPK in rat hepatic stellate cells
Authors: Reimann Thomas , Hempel Ute , Krautwald Stefan , Axmann Andreas , Scheibe Roland , Seidel Dagmar , Wenzel Klaus-Wolfgang
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Structure–function relationships in human ribonucleases: main distinctive features of the major RNase types
Authors: Sorrentino Salvatore , Libonati Massimo
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Characterization of brefeldin A induced vesicular structures containing cycling proteins of the intermediate compartment/cis‐Golgi network
Authors: Füllekrug Joachim , Sönnichsen Birte , Schäfer Ulrike , Nguyen Van Phuc , Söling Hans-Dieter , Mieskes Gottfried
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Novel gene delivery to liver cells using engineered virosomes
Authors: Ramani Komal , Bora Roop S , Kumar Mukesh , Tyagi Sandeep K , Sarkar Debi P
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Immune responses, not promoter inactivation, are responsible for decreased long‐term expression following plasmid gene transfer into skeletal muscle
Authors: Wells K.E , Maule J , Kingston R , Foster K , McMahon J , Damien E , Poole A , Wells D.J
Published: July 2002

Long‐term high‐level in vivo gene expression appears to depend on the promoter chosen to drive the gene of choice. In many cases the promoter appears to ‘switch off’ some time after in vivo gene transfer. We demonstrate that, following intramuscular injection of β‐galactosidase reporter plasmids, promoter ‘switch off’ is due to elimination of fibres expressing the transferred reporter gene by activation of a Th1 (cytotoxic) immune response. This finding, in the absence of stimulation of the immune system by viral vector proteins, has implications not only for gene transfer experiments but for the future of muscle‐directed gene therapy.
DNA fragmentation is a feature of cystic fibrosis epithelial cells: a disease with inappropriate apoptosis?
Authors: Maiuri Luigi , Raia Valeria , De Marco Giulio , Coletta Salvatore , de Ritis Giorgio , Londei Marco , Auricchio Salvatore
Published: July 2002

Cystic fibrosis (CF) is a single‐gene disease caused by mutations in the CFTR gene, which result in disrupted chloride secretions with inspissated mucous secretions by exocrine glands. Nick‐end labelling was used to assess DNA fragmentation in 14 CF and 24 control duodenal samples, and in two CF and two control lung tissues. In CF small intestine median 46% (range: 30–82) villus enterocytes show DNA fragmentation (vs. 3% (range: 1–7) in controls P<0.001) and median 37.5% (range: 23–79) crypt enterocytes show Ki67 antigen (P<0.001). In CF airways 57% (range: 54–70) of epithelial cells show DNA fragmentation. Inappropriate high DNA fragmentation is a feature of various CF epithelia. This could have great impact in understanding the mechanisms leading to disease.
Essential role of the beta subunit in modulation of C‐class L‐type Ca2+ channels by intracellular pH
Authors: Schuhmann Klaus , Voelker Christof , Höfer Gerhard F , Pflügelmeier Helmut , Klugbauer Norbert , Hofmann Franz , Romanin Christoph , Groschner Klaus
Published: July 2002

Elevation of intracellular pH (pHi) enhances the activity of native L‐type Ca2+ channels in cardiac and smooth muscle. We studied the modulation by pHi of expressed L‐type Ca2+ channels comprised of either the α1C subunits alone or of α1C plus β2a subunits. Ca2+ channels were expressed in human embryonic kidney cells (HEK 293) and pHi was increased from a basal level of 7.3 to 8.3 by exposure of cells to NH4Cl (20 mM) or by elevation of extracellular pH to 8.5. Elevation of pHi enhanced the activity of Ca2+ channels derived by coexpression of α1C and β2a subunits. This alkalosis‐induced stimulation of channel activity was mainly due to an increase in channel availability. Channels derived by expression of α1C alone were not affected by intracellular alkalosis. Our results demonstrate that the pHi sensitivity of L‐type Ca2+ channels is conferred by the β subunit of the channel complex.
High expression of the yeast syntaxin‐related Vam3 protein suppresses the protein transport defects of a pep12 null mutant
Authors: Götte M. , Gallwitz D.
Published: July 2002

The Pep12 protein of Saccharomyces cerevisiae is a member of the syntaxin family thought to function as target membrane receptor (t‐SNARE) for vesicular intermediates travelling between the Golgi apparatus and the vacuole. Exploiting the temperature‐sensitive growth phenotype of pep12 deletion strains, we identified VAM3 as a multicopy suppressor. Vam3p is another syntaxin‐related protein which on high expression restored vacuole acidification of pep12 null mutants and effectively suppressed their sorting and maturation defects of vacuolar hydrolases. We conclude that Vam3p acts either as a bypass suppressor or by functionally replacing Pep12p at an endosomal, prevacuolar compartment.
Sequence and expression of a novel member (LTBP‐4) of the family of latent transforming growth factor‐β binding proteins
Authors: Giltay Richard , Kostka Günter , Timpl Rupert
Published: July 2002

Overlapping cDNA clones from human heart and melanoma libraries were used to establish the 1587‐residue sequence of a novel protein (LTBP‐4) belonging to the family of extracellular microfibrillar proteins which also bind transforming growth factor‐β. LTBP‐4 consists of 20 EG modules, 17 of them with a consensus sequence for calcium binding, 4 TB modules with 8 cysteines and several proline‐rich regions. Northern blots demonstrated a single 5 kb mRNA which is highly expressed in heart but also present in skeletal muscle, pancreas, placenta and lung. The modular structure predicts that LTBP‐4 should be a microfibrillar protein which probably also binds TGF‐β.
Role of glutathione and nitric oxide in the energy metabolism of rat liver mitochondria
Authors: Nishikawa Manabu , Sato Eisuke F. , Kuroki Tetsuo , Inoue Masayasu
Published: July 2002

Previous studies have suggested that nitric oxide (NO) inhibited mitochondrial respiration. NO and/or its intermediate(s) react with various molecules, such as hemeproteins and free SH groups. The inhibitory effect of NO on mitochondrial respiration was decreased by exogenously added glutathione (GSH). However, a decrease of intramitochondrial GSH by pretreating animals with l‐buthionine sulfoximine had no appreciable effect on the inhibitory effect of isolated mitochondria. Furthermore, the effect of NO was not affected by depleting free SH residues in mitochondria by N‐ethylmaleimide. These results suggest that cytosolic but not intramitochondrial GSH might be an important factor that determines the NO‐dependent regulation of mitochondrial energy metabolism.
Modulation of the DNA binding activity of transcription factors CREP, NFκB and HSF by H2O2 and TNFα. Differences between in vivo and in vitro effects
Authors: Jornot Lan , Petersen Hilke , Junod Alain F
Published: July 2002

Human endothelial cells exposed to H2O2 showed reduced CREP DNA binding activity, enhanced HSF activation, and no induction of NFκB binding activity. Interestingly, H2O2 was able to induce NFκB subunit p65 translocation in the nucleus. In contrast, cells exposed to TNFα showed enhanced CREP binding activity, activation of NFκB and no induction of HSE‐HSF complex. Addition of H2O2, diamide and iodoacetic acid to the binding reaction mixture markedly reduced the DNA binding ability of the three transcription factors. Thus free sulfhydryls were important in DNA binding activity of CREP, NFκB and HSF, and the lack of induction of NFκB by H2O2 in intact cells was likely caused by oxidation on a thiol, and not by a deficiency in the activation pathway.
Expression cloning and characterization of a renal organic anion transporter from winter flounder
Authors: Wolff N.A , Werner A , Burkhardt S , Burckhardt G
Published: July 2002

The cDNA coding for a renal p‐aminohippurate (PAH) transporter from winter flounder (Pseudopleuronectes americanus), designated fROAT, was cloned by functional expression in Xenopus laevis oocytes. fROAT is approximately 2.8 kbp in length and encodes a protein of 562 amino acids, related to the rat renal organic anion transporter ROAT1/OAT1 and the organic cation transporters OCT1 and OCT2. In oocytes, fROAT mediated probenecid‐sensitive PAH uptake, with a K
m for PAH of about 20 μM, and inhibited by external glutarate (GA) (1 mM). The functional characteristics suggest that fROAT is the basolateral PAH/dicarboxylate exchanger of the flounder kidney.
Cyclosporin A‐sensitive release of Ca2+from mitochondria in intact thymocytes
Authors: Chernyak Boris V.
Published: July 2002

Release of Ca2+from mitochondria into cytosol in intact thymocytes was studied using the fluorescent dye Fluo‐3. It was shown that the release of Ca2+induced by the dithiol cross‐linking agent phenylarsine oxide or by uncoupler was strongly inhibited by cyclosporin A, a specific inhibitor of the permeability transition pore (PTP) in mitochondria. Oxidative stress sensitized the pore so even partial uncoupling caused rapid cyclosporin A‐sensitive release of Ca2+. The experiments on digitonin‐permeabilized cells confirmed that uncoupling induced opening of the PTP, which forms the major pathway for rapid release of Ca2+from thymocyte mitochondria.
Contractile impairment and structural alterations of skeletal muscles from knockout mice lacking type 1 and type 3 ryanodine receptors
Authors: Barone Virginia , Bertocchini Federica , Bottinelli Roberto , Protasi Feliciano , Allen Paul D. , Franzini Armstrong Clara , Reggiani Carlo , Sorrentino Vincenzo
Published: July 2002

Skeletal muscle contraction is triggered by the release of Ca2+ from the sarcoplasmic reticulum through the type 1 ryanodine receptor (RyR1). Recently it has been shown that also the type 3 isoform of ryanodine receptor (RyR3), which is expressed in some mammalian skeletal muscles, may participate in the regulation of skeletal muscle contraction. Here we report the generation and the characterization of double mutant mice carrying a targeted disruption of both the RyR1 and the RyR3 genes (RyR1−/−;RyR3−/−). Skeletal muscles from mice homozygous for both mutations are unable to contract in response to caffeine and to ryanodine. In addition, they show a very poor capability to develop tension when directly activated with micromolar [Ca2+]i after membrane permeabilization which indicates either poor development or degeneration of the myofibrils. This was confirmed by biochemical analysis of contractile proteins. Electron microscopy confirms small size of myofibrils and shows complete absence of feet (RyRs) in the junctional SR.
Inhibition of sperm glutathione S‐transferase leads to functional impairment due to membrane damage
Authors: Gopalakrishnan B , Shaha Chandrima
Published: July 2002

The role of glutathione S‐transferase (GST) in the defense mechanisms of sperm is not known. We report here interference with normal motility, acrosome reaction and fertilizing ability of the goat sperm as a consequence of inhibition of GST activity. That these functional impairments were due to membrane changes was evident from the alteration in the lipid peroxidation status of these cells after GST inhibitor treatment. Increased reactive oxygen species production by the cell which occurred when GST activity was suppressed may be the mediator for membrane damage. The data argue for a role of GST in maintaining sperm membrane status.
Estradiol binding to cell surface raises cytosolic free calcium in T cells
Authors: Benten W.Peter M , Lieberherr Michèle , Giese Günter , Wunderlich Frank
Published: July 2002

The Fura‐2 method is used to examine a possible action of 17β‐estradiol (E2) on [Ca2+]i of splenic T cells isolated from female C57BL/10 mice. E2 concentrations between 10 fM and 10 nM induce a rapid and dose‐dependent increase in [Ca2+]i due to Ca2+ influx and release of Ca2+ from intracellular stores. Ca2+ influx is mediated by Ca2+ channels which are completely blockable by Ni2+ and partly by nifedipine. The antiestrogen tamoxifen does not inhibit the E2‐induced rise in [Ca2+]i. Ca2+ influx and Ca2+ release from intracellular stores is also inducible by plasma membrane impermeable E2 conjugated to BSA. E2‐BSA‐FITC binds to the surface of T cells of both the CD4+ and CD8+ subset. Our data suggest a novel E2‐signalling pathway in T cells which is not mediated through the classical nuclear estrogen receptor response but rather through putative plasma membrane receptors for E2.
Heparin inhibits phorbol ester‐induced ornithine decarboxylase gene expression in endothelial cells
Authors: Pintus Gianfranco , Tadolini Bruna , Maioli Margherita , Posadino Anna M. , Bennardini Federico , Bettuzzi Saverio , Ventura Carlo
Published: July 2002

Glycosaminoglycans regulate angiogenesis by affecting the availability of different growth factors for the endothelial cell (EC). However, little is known about the molecular and functional consequences resulting from direct interaction of these polyelectrolytes with the EC. Here we show that heparin markedly inhibited serum‐stimulated DNA synthesis and ornithine decarboxylase (ODC) mRNA expression in human endothelial cells (HEC). About 50% of the serum effect on DNA synthesis and ODC gene expression was prevented by the selective protein kinase C (PKC) inhibitor chelerythrine or by PKC down‐regulation. Heparin was ineffective in counteracting that part of the effect of serum that was resistant to PKC inhibition or down‐regulation. In serum‐free cultured HEC, heparin completely abolished the increase in DNA synthesis and ODC mRNA expression elicited by a number of PKC activators. Cell exposure to difluoromethylornithine, an irreversible inhibitor of ODC enzyme, dramatically antagonised both serum‐ and phorbol 12‐myristate 13‐acetate (PMA)‐stimulated DNA synthesis. These results suggest that inhibition of PKC‐mediated ODC gene expression by glycosaminoglycans may represent an important mechanism in the regulation of HEC proliferation.
The TRP Ca2+ channel assembled in a signaling complex by the PDZ domain protein INAD is phosphorylated through the interaction with protein kinase C (ePKC)
Authors: Huber Armin , Sander Philipp , Bähner Monika , Paulsen Reinhard
Published: July 2002

Photoreceptors which use a phospholipase C‐mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cβ (PLCβ), the light‐activated Ca2+ channel TRP, and an eye‐specific protein kinase C (ePKC) are clustered by the PDZ domain protein INAD. Here we investigated the function of ePKC by cloning the Calliphora homolog of Drosophila ePKC, by precipitating the TRP signaling complex with anti‐ePKC antibodies, and by performing phosphorylation assays in isolated signaling complexes and in intact photoreceptor cells. The deduced amino acid sequence of Calliphora ePKC comprises 685 amino acids (MW=78 036) and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti‐ePKC antibodies led to the co‐precipitation of PLCβ, TRP, INAD and ePKC but not of rhodopsin. Phorbolester‐ and Ca2+‐dependent protein phosphorylation revealed that, apart from the PDZ domain protein INAD, the Ca2+ channel TRP is a substrate of ePKC. TRP becomes phosphorylated in isolated signaling complexes. TRP phosphorylation in intact photoreceptor cells requires the presence of extracellular Ca2+ in micromolar concentrations. It is proposed that ePKC‐mediated phosphorylation of TRP is part of a negative feedback loop which regulates Ca2+ influx through the TRP channel.
Specific reduction of insulin disulfides by macrophage migration inhibitory factor (MIF) with glutathione and dihydrolipoamide: potential role in cellular redox processes
Authors: Kleemann Robert , Mischke Ralf , Kapurniotu Aphrodite , Brunner Herwig , Bernhagen Jürgen
Published: July 2002

The molecular mechanism of action of MIF, a cytokine that plays a critical role in the host immune and inflammatory response, has not yet been identified. We recently demonstrated that MIF is an enzyme that exhibits oxidoreductase activity by a cysteine thiol‐mediated mechanism. Here we further investigated this function by examining the reduction of insulin disulfides by wild‐type human MIF (wtMIF) using various substrates, namely glutathione (GSH), dihydrolipoamide, l‐cysteine, β‐mercaptoethanol and dithiothreitol. The activity of wtMIF was compared to that of the relevant cysteine mutants of MIF and to two carboxy‐truncated mutants. Only GSH and dihydrolipoamide were found to serve as reductants, whereas the other substrates were not utilized by MIF. Reduction of insulin disulfides by MIF was closely dependent on the presence of the Cys57‐Ala‐Leu‐Cys60 (CALC) motif‐forming cysteines C57 and C60, whereas C81 was not involved (activities: 51±13%, 14±5%, and 70±12% of wtMIF, respectively, and 20±3% for the double mutant C57S/C60S). Confirming the notion that the activity of MIF was dependent on the CALC motif in the central region of the MIF sequence, the C‐terminal deletion mutants MIF(1–105) and MIF(1–110) were found to be fully active. The favored use of GSH and dihydrolipoamide indicated that MIF may be involved in the regulation of cellular redox processes and was supported further by the finding that MIF expression by the cell lines COS‐1 and RAW 264.7 was significantly induced upon treatment with the oxidant hydrogen peroxide.
Detection of pro‐caspase‐3 in cytosol and mitochondria of various tissues
Authors: Samali Afshin , Zhivotovsky Boris , Jones Dean P , Orrenius Sten
Published: July 2002

Caspases are a family of cysteine proteases of critical importance in the apoptotic cell death process. They are normally present as zymogens (pro‐caspases) in the cytoplasm of vertebrate and other organisms. In this study we have shown that pro‐caspase‐3 is localized to cytosol and mitochondria of various rat tissues (brain, heart, kidney, liver, spleen and thymus). Although the majority of pro‐caspase‐3 was localized in the cytosol, the amount of mitochondrial pro‐caspase‐3 was significant. The ratio of cytosolic and mitochondrial pools of pro‐caspase‐3 appeared to vary between different tissues. The higher amount of mitochondrial pro‐caspase‐3 was found in thymus and spleen, i.e. tissues in which spontaneous apoptosis plays an important role. Our findings provide further support for mitochondrial localization of pro‐caspase‐3 and the critical role of this organelle in apoptosis.
The expression of epidermal growth factor and transforming growth factor‐α mRNA in the small intestine of suckling rats: organ culture study
Authors: Dvořák Bohuslav , Kolı́nská Jiřina , McWilliam Debra L , Williams Catherine S , Higdon Travis , Zákostelecká Marie , Koldovský Otakar
Published: July 2002

Epidermal growth factor (EGF) and transforming growth factor‐α (TGF‐α) are associated with regulation of various gastrointestinal functions. In order to better understand their role in developing small intestine EGF, TGF‐α and EGF‐R steady‐state mRNA levels and transcript stability were determined. Reverse transcription (RT) competitive‐polymerase chain reaction (PCR) revealed that intestinal TGF‐α mRNA levels were 10‐fold higher in comparison with EGF mRNA. The primary intestinal culture technique was used to evaluate mRNA stability. The stability of TGF‐α mRNA was remarkably lower than the stability of EGF mRNA. High levels of TGF‐α mRNA accompanied by high degradation rate of this mRNA suggested a rapid turnover of intestinal TGF‐α mRNA.
Endomorphins fully activate a cloned human mu opioid receptor
Authors: Gong Jianhua , Strong Judith A , Zhang Shengwen , Yue Xia , DeHaven Robert N , Daubert Jeffrey D , Cassel Joel A , Yu Guangling , Mansson Erik , Yu Lei
Published: July 2002

Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin‐1 and endomorphin‐2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin‐stimulated increase of cAMP in a dose‐dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein‐activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose‐dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [d‐Ala2,N‐Me‐Phe4,Gly‐ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G‐protein‐activated inwardly rectifying potassium (GIRK) channels.