bluuebunny/crossref_metadata_2025 huggingface dataset:
Sub‐compartmentation of the ‘cytosolic’ glucose 6‐phosphate pool in cultured rat hepatocytes
Authors: Christ Bruno , Jungermann Kurt
Published: July 2002

[14C]Glucose release either from endogenous 14C‐prelabelled glycogen or from added 14C‐labelled glucose 6‐phosphate was measured in filipin‐treated, permeabilized hepatocytes in 48 h culture. [14C]Glucose output from prelabelled glycogen was not altered by the addition of 5 mM glucose 6‐phospate to the incubation medium. Conversely, [14C]glucose release from 5 mM labelled glucose 6‐phosphate was not influenced by different glycogen concentrations in the cells. Moreover, in the permeabilized cells the anion transport inhibitor DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid) inhibited only the liberation of [14C]glucose from labelled glucose 6‐phosphate but not from glycogen. It is therefore concluded that there exist at least 2 separate, mutually non‐accessible glucose 6‐phosphate pools in cultured rat hepatocytes, one linked to glycogenolysis and the other to gluconeogenesis.
The processing and secretion of rat serum albumin by oocytes from Xenopus laevis
Authors: Foreman R.C. , Judah J.D.
Published: July 2002

Microinjection of rat liver mRNA into Xenopus oocytes led to the synthesis of intracellular proalbumin and the secretion of mature albumin into the incubation medium. The ionophore monensin abolished the secretion of albumin but not the processing of the precursor. A variety of protease inhibitors were added to the incubation medium but there was no detectable inhibition of proalbumin cleavage.
Sulfhydryl groups are involved in H+ translocation via the uncoupling protein of brown adipose tissue mitochondria
Authors: Ježek Petr
Published: July 2002

Mersalyl inhibits H+ transport via the uncoupling protein (UP) in brown adipose tissue (BAT) mitochondria estimated as swelling in potassium acetate (K
i 67 μM) or as valinomycin‐induced H+ extrusion in K
2SO4 (K
i 55 μM) and KCl. The swelling in KCl is depressed only slightly. Some other SH‐reagents (p‐hydroxymercuribenzoate, 5,5'‐dithiobis(2‐nitrobenzoate) and thiolyte DB), but not hydrophobic reagents (N‐ethylmaleimide and eosin‐5‐maleimide), exhibit analogous inhibition. Thus an essential SH‐group localized at the water‐accessible cytosolic surface of UP was found to be involved in H+ transport via UP but not in Cl− transport.
Identification of disulfide bonds in carboxy‐terminal propeptides of human type I procollagen
Authors: Koivu Juha
Published: July 2002

Intra‐chain and inter‐chain disulfide bonds within the carboxy‐terminal propeptides of human type I procollagen were studied using cyanogen bromide cleavage of the propeptides and electrophoresis on SDS‐polyacrylamide/glycerol gels. The results could provide a better understanding of the assembly of proα‐chains into procollagen.
Electron microscopy study of Qβ replicase
Authors: Berestowskaya N.H. , Vasiliev V.D. , Volkov A.A. , Chetverin A.B.
Published: July 2002

Purified preparations of Qβ replicase have been studied by electron microscopy using a negative staining technique, and a three‐dimensional model of the enzyme molecule has been constructed. The molecule of this four‐subunit protein appears to be a compact structure having a size of 100 ± 10 Å; it is subdivided into two unequal bipartite subparticles. The conclusion has been made that all the constituent subunits, including the ribosomal protein S1, acquire a globular conformation when associated in the replicase complex.
Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase
Authors: Vihko Pirkko , Virkkunen Pirjo , Henttu Pirkko , Roiko Katja , Solin Timo , Huhtala Marja-Liisa
Published: July 2002

λgt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354‐residue protein with a calculated molecular mass of 41 126 Da. In the 5′‐end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino‐terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu‐type repetitive sequence about 900 nucleotides downstream from the coding region in the 3′‐untranslated region. Two of our cDNA clones differed from others at the 5′‐ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family. as do alkaline phosphatases.
An increase in intracellular pH is a general response of promonocytic cells to differentiating agents
Authors: Ladoux Annie , Damais Chantal , Krawice Irène , Abita Jean Pierre , Frelin Christian
Published: July 2002

Intracellular pH (pHi) was measured in HL60 and U937 cells before and after differentiation into monocyte‐macrophage like cells. 12‐O‐Tetradecanoyl phorbol‐13‐acetate (PMA), butyrate, interferon, retinoic acid and 1,25‐dihydroxyvitamin D3 all increased pHi. The increases elicited were rapid with PMA, much slower with retinoic acid and interferon and still slower with 1,25‐dihydroxyvitamin D3. Increases in pHi are due to an activation of the Na+/H+ exchange system. High pHi values are unlikely to serve as an early intracellular signal for initiating monocytic differentiation.
Aorta caldesmon inhibits actin activation of thiophosphorylated heavy meromyosin Mg2+‐ATPase activity by slowing the rate of product release
Authors: Marston Steven
Published: July 2002

Activation of aorta thiophosphorylated heavy meromyosin (HMM[SP]) Mg2+‐ATPase activity by aorta actin and the fraction of HMM[SP]‐substrate intermediate complexes bound to actin were measured simultaneously. At 25°C the K
m for ATPase activation and the dissociation constant for the binding reaction were similar, irrespective of the presence or absence of tropomyosin. Aorta caldesmon (0.1 mol/mol actin) inhibited ATPase activation by 80–90% but did not alter the binding of HMM[SP]‐product intermediates to actin. It is concluded that caldesmon inhibits by slowing the rate‐limiting release of products from the actin‐HMM[SP]·ADP·Pi complex.
β‐Adrenergic regulation of c‐fos gene expression in an epithelial cell line
Authors: Yeh Chih-Ko , Louis John M. , Kousvelari Eleni E.
Published: July 2002

Stimulation of β‐adrenoreceptors in the RSMT‐A5 epithelial cell line is accompanied by an early and transient increase in the expression of the proto‐oncogene c‐fos. Maximal induction was at 30 min, returning to basal levels after 2 h. Similar results were obtained when cells were incubated with 8‐bromo‐cAMP. The induction of c‐fos is specific since the expression of p53, a transformation‐related gene, is not modulated by isoproterenol or 8‐bromo‐cAMP. The increase in c‐fos gene expression is not associated with proliferative activity in these epithelial cells.
Ca2+‐dependent K+ channels in neuroblastoma hybrid cells activated by intracellular inositol trisphosphate and extracellular bradykinin
Authors: Higashida Haruhiro , Brown David A.
Published: July 2002

Bradykinin (BK) activation of phosphatidylinositide breakdown in NG108‐15 neuroblastoma x glioma hybrid cells in the generation of an outward K+ current through the release of Ca2+ by the intermediary messenger inositol 1,4,5‐trisphosphate (InsP3). Channels mediating this outward current were identified using cell‐attached patch electrodes. Intracellular iontophoretic injection of InsP3 or Ca2+, or extracellular application of BK, evoked bursts of K+ channel activity coincident with cell hyperpolarization measured with an intracellular recording micropipette. The most frequent channels had a mean single‐channel conductance of about 40 pS in symmetrical K+ solutions; additional openings of lower conductance (18 pS) channels were also detected. Bath application of phorbol dibutyrate (PDBu, 1 μM) increased the number and opening probability of the InsP3‐induced channels.
Functional expression of cloned cDNA encoding sodium channel III
Authors: Suzuki Harukazu , Beckh Synnöve , Kubo Hideo , Yahagi Naoki , Ishida Hiroyuki , Kayano Toshiaki , Noda Masaharu , Numa Shosaku
Published: July 2002

mRNA synthesized by transcription in vitro of the cloned cDNA encoding rat brain sodium channel III directs the formation of a functional sodium channel in Xenopus oocytes. The tissue distribution of the mRNAs encoding sodium channels I, II and III has been studied by blot hybridization analysis with specific probes.
The migratory behavior of avian embryonic cells does not require phosphorylation of the fibronectin‐receptor complex
Authors: Duband Jean-Loup , Dufour Sylvie , Yamada Kenneth M. , Thiery Jean Paul
Published: July 2002

When locomotory embryonic cells become stationary, they acquire new substratum‐adhesion properties. In particular, the distribution of fibronectin receptors shifts from diffuse and highly mobile on the cell membrane to immobilized in close association with fibronectin molecules and cytoskeletal elements in focal contacts. Receptor phosphorylation has been proposed as a possible regulator of the interaction between the receptor and its intracellular and extracellular ligands. In the present study, we have compared the phosphorylation state of the fibronectin receptor in motile neural crest and somitic cells, in stationary somitic cells, and in Rous‐sarcoma virus transformed‐chick embryo fibroblasts, using immunoprecipitation following metabolic labeling. While no receptor phosphorylation was detected in motile embryonic cells, the beta subunit of the receptor was phosphorylated in stationary cells. This subunit was also highly phosphorylated in Rous‐sarcoma virus‐transformed chicken cells. These results suggest that phosphorylation of the fibronectin receptor cannot account for its distribution in the cell membrane and for the nature of the interactions between this receptor and its ligands in embryonic cells.
The complete amino acid sequence of the ribosomal A protein (L12) from the archaebacterium Sulfolobus acidocaldarius
Authors: Matheson A.T. , Louie K.A. , Böck August
Published: July 2002

The ribosomal A protein (SacL12) from the archaebacterium Sulfolobus acidocaldarius has been sequenced. The protein contains 105 amio acids, has a composition of Asp2, Asn3, Thr4, Ser6, Glu17, Gln4, Pro3, Gly7, Ala18, Val7, Met2, Ile7, Leu9, Tyr2, Phe1, His1, Lys11, Arg1, and a molecular mass of 11 126 Da. The Sulfolobus protein shows many features in common with the equivalent proteins in the eukaryotes such as 35–40% sequence homology and similar hydrophilicity profiles, features much less evident when this protein is compared to eubacterial L12 proteins. SacL12 contains an unusual sequence of alternating clusters of lysine and glutamic acid (—EKKEEKKEEEKK—) in the C‐terminal region. Similar sequences are found in some eukaryotic L12 proteins.
A mutant LexA repressor harboring a cleavage motif cysteine‐glycine remains inducible
Authors: Granger-Schnarr Michèle , Oertel Pascale , Schnarr Manfred
Published: July 2002

Using site‐directed mutagenesis of the lexA gene we have changed the amino acid Ala‐84 of the LexA repressor for a cysteine. The reason for this change was the striking homology between LexA and UmuD and the comparable size of the two amino acid side chains. Using an in vivo repression/induction assay it is shown that the LexA‐Cys‐84 mutant remains inducible by mitomycin C and UV irradiation essentially in the same way as the wild‐type repressor.
Stimulation of hepatic T‐kininogen production by interferon
Authors: Itoh Norio , Yayama Katsutoshi , Okamoto Hiroshi
Published: July 2002

T‐kininogen is known to be an acute‐phase reactant as well as a kininogen in rat plasma. Three kinds of cytokines, interleukin‐1, tumor necrosis factor and interferon, were assayed for their abilities to stimulate hepatic production of T‐kininogen. Of these cytokines, interferon was able to stimulate hepatic production of T‐kininogen, but few effects were observed for interleukin‐1 and tumor necrosis factor. In addition, the stimulatory effect of interferon was inhibited by tumor necrosis factor. Our data suggest that interferon is a candidate for the leukocyte‐derived factor mediating the acute‐phase response of T‐kininogen.
ω‐Conotoxin binding and effects on calcium channel function in human neuroblastoma and rat pheochromocytoma cell lines
Authors: Sher E. , Pandiella A. , Clementi F.
Published: July 2002

Binding of ω‐conotoxin, a peptide toxin specific for some subtypes of voltage‐operated calcium channels (VOCCs), was investigated in IMR32 neuroblastoma and PC12 pheochromocytoma cell lines. In both cell types, binding was specific, saturable and of high affinity. Association was rapid and dissociation almost non‐existent. Dihydropyridines and verapamil failed to affect toxin binding, while high concentrations of CaCl2 completely antagonized it. Depolarization with high K+ induced a [Ca2+]i rise (revealed by the fura‐2 fluorimetric technique) that consisted of an initial (0.5–1 min) peak followed by a prolonged (several minutes) plateau phase. ω‐Conotoxin blocked mainly the first phase, while the dihydropyridine Ca2+ channel blocker, nitrendipine, primarily affected the plateau. This result suggests that in the two cell lines investigated, ω‐conotoxin acts mainly on a subgroup of VOCCs that is resistant to dihydropyridines.
In situ binding of a photo‐affinity GTP analog to synaptic membrane G‐proteins Distribution of bound GTP analog reflects the status of adenylate cyclase
Authors: Gordon John H. , Rasenick Mark M.
Published: July 2002

Regulation of synaptic membrane adenylate cyclase is likely to involve interaction between neurotransmitter receptors, G‐proteins and the adenylate cyclase catalytic unit as well as several other membrane proteins and lipids. Despite intensive study of this system, regulation of guanine nucleotide binding by the G‐proteins which stimulate [Gs] or inhibit [Gi] adenylate cyclase has been examined only when those proteins have been purified and removed from the influence of the membrane environment. The hydrolysis‐resistant photoaffinity GTP‐analog, P3‐(4‐azidoanilido)‐P1 5′‐GTP (AAGTP) is able to bind specifically to the G‐proteins in rat cerebral cortex synaptic membranes and, in this study, we have used this probe to examine the specificity and selectivity of guanine nucleotide binding to each G‐protein without removing those proteins from the synaptic membrane. Marked differences were noted between guanine nucleotide binding data obtained with detergent‐soluble G‐proteins and data from this in situ approach. In these studies it was found that the affinity of the G‐proteins binding AAGTP correlated well with the expression of adenylate cyclase activity, the affinity of both forms of Gs, increasing under conditions favoring the stimulation of that enzyme.
Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles
Authors: Schilling K. , Gratzl M.
Published: July 2002

Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.
Family of human Na+,K+‐ATPase genes Structure of the gene for the catalytic subunit (αIII‐form) and its relationship with structural features of the protein
Authors: Ovchinnikov Yu.A. , Monastyrskaya G.S. , Broude N.E. , Ushkaryov Yu.A. , Melkov A.M. , Smirnov Yu.V. , Malyshev I.V. , Allikmets R.L. , Kostina M.B. , Dulubova I.E. , Kiyatkin N.I. , Grishin A.V. , Modyanov N.N. , Sverdlov E.D.
Published: July 2002

The primary structure of a gene of the Na+,K+‐ATPase multigenic family in the human genome has been determined. The gene corresponds to a hypothetical αIII‐form of the enzyme catalytic subunit. The gene comprises over 25000 bp, and its protein coding region includes 23 exons and 22 introns. Possible correlation between structural features of the protein and location of introns in the gene are discussed.
Electrostimulation‐induced fast‐to‐slow transitions of myosin light and heavy chains in rabbit fast‐twitch muscle at the mRNA level
Authors: Kirschbaum Bernhard J. , Heilig Annegard , Härtner Karl-Thomas , Pette Dirk
Published: July 2002

Chronic low‐frequency stimulation of rabbit fast‐twitch muscle induces progressive increases in slow myosin light chain mRNAs followed by an increase in the slow myosin heavy chain HCI mRNA. Therefore, the effects of chronic stimulation are more pronounced in rabbit than in rat fast‐twitch muscle. The latter responds mainly with a rearrangement of its fast isomyosin pattern.
Differences in 23 S rRNA‐protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex
Authors: Chistyakov P.G. , Venjaminova A.G , Vladimirov S.N. , Graifer D.M. , Kasakov S.A. , Karpova C.G
Published: July 2002

rRNA‐protein cross‐links in free E. Coli
35S‐labeled 70 S ribosomes and in the initiation complex 35‐labeled 70 S ribosome · AUGU6 · Met
f‐tRNAMet
f were studied with the aid of a new type of binuclear (PtII) compound — dichlorotetra‐ammine (1,6‐hexamethylenediaminediplatinum) dichloride. The use of this reagent allowed us to reveal differences in the rRNA‐protein neighbourhood in free 70 S ribosomes and in the initiation complex. Proteins L3, L6, L23 and L25 were shown to cross‐link to 23 S rRNA only in the initiation complex, whereas proteins L1, L13, L14, L16. L17, L18, L22, L28 and S1 did so in both free ribosomes and the complex. 16 S rRNA was found to be cross‐linked preferentially to a single protein, S1, in both states of the ribosomes.
The refined 2.0 Å X‐ray crystal structure of the complex formed between bovine β‐trypsin and CMTI‐I, a trypsin inhibitor from squash seeds (Cucurbita maxima) Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes
Authors: Bode Wolfram , Greyling H.Johann , Huber Robert , Otlewski Jacek , Wilusz Tadeusz
Published: July 2002

The stoichiometric complex formed between bovine β‐trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI‐I) was crystallized and its X‐ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0 — 2.0 Å). CMTI‐I is of ellipsoidal shape; it lacks helices or β‐sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS‐3I‐20I, Cys‐10I‐22I and Cys‐16I‐28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val‐2I (P4) — Glu‐9I (P4′) which contains the reactive site bond Arg‐5I — Ile‐6I and is in a conformation observed also for other serine proteinase inhibitors.
Receptor‐independent metabolism of platelet‐activating factor by myelogenous cells
Authors: O'Flaherty Joseph T. , Chabot Marie C. , Redman Jimmy , Jacobson David , Wykle Robert L.
Published: July 2002

Human neutrophils incorporate and metabolize platelet‐activating factor (PAF). We dissociated these events from PAF binding to its receptors. Cells were pretreated with either pronase, a PAF antagonist (L652731), or excess PAF. This reduced PAF receptor numbers by 70 to almost 100% but had no comparable effect upon the neutrophil's ability to metabolize PAF. Furthermore, HL‐60 cells efficiently metabolized, but did not specifically bind, PAF. Thus, PAF receptor availability did not correlate with PAF metabolic capacity and we conclude that myelogenous tissues can process this bioactive ligand by a receptor‐independent pathway.
Developmental regulation of expression of the α1 and α2 subunits mRNAs of the voltage‐dependent calcium channel in a differentiating myogenic cell line
Authors: Varadi Gyula , Orlowski John , Schwartz Arnold
Published: July 2002

The voltage‐dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC α1 and α2 mRNAs is developmentally regulated in differentiating C2Cl2 myogenic cells. The α1 mRNA is not detectable in the myoblast form of C2Cl2 cells while its expression is induced 20‐fold in differentiated myotubes. In contrast, the α2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.
Bradykinin inhibits a potassium M‐like current in rat pheochromocytoma PC12 cells
Authors: Villarroel Alvaro , Marrion Neil V. , Lopez Hector , Adams Paul R.
Published: July 2002

We studied the action of bradykinin (BK) on ionic currents in fused pheochromocytoma PC12 cells under voltage‐clamp in whole‐cell mode, and on intracellular calcium using fura‐2. BK induced the development of an outward current associated with an increase in intracellular calcium, followed by inhibition of an M‐like current. The outward current was blocked by (+)‐tubocurarine, and prevented when the calcium chelator BAPTA or high concentrations of inositol 1,4,5‐triphosphate were introduced into the cell, whereas the M‐like current and its inhibition by BK remained unaffected. The protein kinase activator phorbol 12,13 dibutyrate partially reduced the M‐current. M‐current density did not substantially change after prolonged treatment with nerve growth factor.
Pertussis toxin activates protein kinase C and a tyrosine protein kinase in the human T cell line Jurkat
Authors: Thom Rebecca E. , Casnellie John E.
Published: July 2002

Pertussis toxin activates T lymphocytes by a mechanism that is independent of its ADP‐ribosylation activity. The toxin stimulates increases in diacylglycerol and intracellular calcium apparently by interacting with a cell surface receptor. Consistent with the production of these second messengers we have found that pertussis toxin activates protein kinase C in the Jurkat cell line. The toxin was also found to activate a tyrosine protein kinase in these cells in a manner similar to that observed with phytohemagluttinin. These results provide evidence that the mechanism of activation of T cells by pertussis toxin involves stimulating the activity of protein kinase C and a tyrosine protein kinase.
Purification of a p47 phosphoprotein from Xenopus laevis oocytes and identification as an in vivo and in vitro p34cdc2 substrate
Authors: Mulner-Lorillon Odile , Poulhe Robert , Cormier Patrick , Labbe Jean-Claude , Doree Marcel , Belle Robert
Published: July 2002

This paper describes the purification of a 47 kDa protein from Xenopus laevis oocytes that becomes phosphorylated when the oocytes undergo meiotic maturation. This protein (p47) is part of a high molecular mass complex containing at least two other proteins of molecular mass 30 and 36 kDa. This complex can be isolated from stage VI oocytes before maturation. We obtained a pattern for phosphopeptides in p47 phosphorylated in vivo very similar to that of the purified protein phosphorylated in vitro by p34cdc2 (a H1 kinase which is a component of the M‐phase promoting factor) and [γ‐32P]ATP. Therefore, the purified p47, already described as a marker of MPF activity, is the first reported in vivo substrate for the cell division control kinase.
Bradykinin blocks the action of EGF, but not PDGF, on fibroblast division
Authors: Newman E.Luke , Hyldahl Louise , Larsson Olle , Engström Wilhelm , Rees Anthony R.
Published: July 2002

Quiescent fibroblasts derived from human fetal lung can be stimulated to reinitiate DNA synthesis by sequential addition of 3 nM IGF‐1 and a low concentration (8 pM) of EGF or by continuous exposure to 10% fetal calf serum or 10 ng/ml PDGF. Bradykinin blocks the IGF‐1 and EGF‐dependent signals without affecting the response to serum or PDGF. it activates protein kinase C and its anti‐mitogenic effect is abolished after this kinase has been down‐regulated. Bradykinin has no effect on the binding affinity of the EGF receptor whereas phorbol ester induces its ‘transmodulation’ to low affinity.
Identification of induced protein kinase activities specific for the ribosomal proteins uniquely phosphorylated during infection of HeLa cells with vaccinia virus
Authors: Beaud Georges , Masse Thierry , Madjar Jean-Jacques , Leader David P.
Published: July 2002

We have examined the ribosomal protein kinase activities in partially purified cytoplasmic extracts from HeLa cells infected with vaccinia virus. We found an activity or activities, absent from mock‐infected cells, that was capable of phosphorylating the proteins S2 and S13 in vitro. The ribosomes phosphorylated in vitro exhibited the same multiple phosphorylation of S2 found in vivo, at least 3 phosphoryl residues being seen, and the samemono‐phosphorylation of S13. Also as in vivo, ribosomal protein S2 contained phosphothreonine as well as phosphoserine, whereas S13 contained only phosphoserine. This strongly suggests that these new protein kinase activities are responsible for the ribosomal protein phosphorylations that occur during infection with vaccinia virus.
Histamine and bradykinin stimulate the phosphoinositide turnover in human umbilical vein endothelial cells via different G‐proteins
Authors: Voyno-Yasenetskaya T.A. , Panchenko M.P. , Nupenko E.V. , Rybin V.O. , Tkachuk V.A.
Published: July 2002

The G‐proteins which regulate hormonal turnover of phosphoinositide (PI) in human umbilical vein endothelial cells have been investigated. A 40–41 kDa doublet present in the membranes of these cells was selectively ADP ribosylated by pertussis toxin (PTx), and this doublet was Gα2
and Gα3 according to immunoblotting with specific antisera. By contrast, a doublet of 24–26 kDa proteins in the same membrane preparations was ADP ribosylated by the C3 component of botulinum toxin (BoTx). PTx‐dependent ADP ribosylation blocked stimulation of PI turnover by histamine, but did not affect stimulation by bradykinin, whereas BoTx (C2 + C3 components) had the opposite effect. Thus two different groups of G‐proteins may be involved in hormone‐dependent stimulation of PI turnover in human umbilical vein endothelial cells.
Glucose and nitrogen rapidly activate a Ca2+‐inhibitable, serine/threonine kinase activity toward microtubule‐associated protein 2 in Saccharomyces cerevisiae
Authors: Hoshi Minako , Nishida Eisuke , Matsumoto Seiji , Akiyama Tetsu , Kawakami Minoru , Yahara Ichiro , Sakai Hikoichi
Published: July 2002

Treatment of the glucose‐starved yeast cells (Saccharomyces cerevisiae) with 1% glucose or 2‐deoxyglucose induced a rapid increase in a protein kinase activity in cell extracts that phosphorylated microtubule‐associated protein 2 (MAP2) in vitro. Addition of 0.5% ammonium sulfate to the nitrogen‐starved yeast cells also stimulated the kinase activity toward MAP2. The stimulated MAP2 kinase activities had the following common properties: (i) Activation was rapid and transient in response to stimuli; (ii) The kinase activity was serine/threonine‐specific; and (iii) The kinase activity was inhibited by micromolar concentrations of free Ca2+. These properties are very similar to those of the mitogen‐activated, Ca2+‐sensitive MAP2 kinase we have recently found in mammalian fibroblastic cells. The MAP2 kinase activation may be involved in initiation of proliferation of yeast cells.
Thin laminar microcrystals of 70 S ribosomes from Thermus thermophilus
Authors: Shirokov V.A. , Ryazantsev S.N. , Sergeev Yu.V. , Garber M.B. , Vasiliev V.D.
Published: July 2002

A new crystalline form, thin laminar microcrystals of 70 S ribosomes from Thermus thermophilus, have been obtained. Negatively stained ordered sheets derived from these crystals were studied by electron microscopy with subsequent computer image processing. The sheets have two‐dimensional unit cell parameters a = b = 480 Å, γ = 90° and a resolution limit of about 30 Å. The microcrystals are suitable for three‐dimensional image reconstruction.
Small conductance chloride channels in the apical membrane of thyroid cells
Authors: Champigny G. , Verrier B. , Gérard C. , Mauchamp J. , Lazdunski M.
Published: July 2002

A small conductance chloride channel has been identified on the apical membrane of porcine thyroid cells using the patch‐clamp technique. In cell attached membrane patches with NaCl in the pipette, the single channel conductance is 5.5 pS. The channel is highly selective for chloride over gluconate and iodide, and is impermeable to Na+, K+ and tetraethylammonium ions. The open state probability of the channel is not affected by voltage. The channel activity disappears after excision of the patch. The Cl− channel Mocker 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) did not affect the activity of the thyroid Cl− channels. Treatment of thyroid cells with 8‐(4‐chlorophenylthio)adenosine‐3',5'‐cyclic monophosphate (8‐chloro‐cAMP) (0.5 mM) prior to giga‐seal formation increased Cl− channel activity in the apical membrane of thyroid cells.
Heterogeneity of protein kinase C expression and regulation in T lymphocytes
Authors: Lucas S. , Marais R. , Graves J.D. , Alexandere D. , Parker P. , Cantrell D.A.
Published: July 2002

The purpose of the present study was to examine protein kinase C (PKC) isotype expression in T lymphoblasts derived from peripheral blood and the T leukaemic cell Jurkat. Using antisera reactive with PKC α,β1, and β2 and γ, it was observed that T cells expressed two PKC isotypes, PKC α and β. No PKC γ was detected in T lymphocytes. In lymphoblasts, high levels of PKC β compared to PKC α were found whereas Jurkat cells expressed high levels of α compared to PKC β. Differences in the calcium sensitivity of phorbol ester‐induced phosphorylation were observed in Jurkat and T lymphoblasts which correlated with the relative levels of PKC α and β isotypes expressed by the cells.
Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes
Authors: Lupu-Meiri Monica , Shapira Hagit , Oron Yoram
Published: July 2002

We tested the contribution of extracellular calcium (Cao
2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Cao
2+ removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage‐sensitive calcium channels. When oocytes were challenged with ACh in Ca2+‐free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Cao
2+‐dependent component of membrane electrical responses, the Ca2+ ‐evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage‐sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2+‐evoked current, implying negative feedback of calcium channels by calcium. Injection of 10–100 fmol ofinositol 1,4,5‐trisphosphate (IP3) resulted in a two‐component depolarizing current. IP3 injection promoted the appearance of Cao
2+‐evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell‐membrane muscarinic receptors causes opening of apparently voltage‐insensitive and verapamil or diltiazem‐resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor‐operated channels described in other model systems.
Selective inhibitors of KCl cotransport in human red cells
Authors: Ellory J.C. , Hall A.C. , Ody S.O. , Englert H.C. , Mania D. , Lang H.-J.
Published: July 2002

Two analogues of the loop diuretics furosemide and bumetanide have been identified as differential inhibitors of KCl and NaKCl cotransport systems, assayed by measuring K+ influx in ‘young’ human red cells. H25 inhibited both NaKCl and KCl cotransport, with I
50% values of 0.03 and 30 μM respectively; H74 had no effect on NaKCl cotransport, even at 0.3 mM, but inhibited KCl cotransport with an I
50% of 75 μM. These compounds are therefore useful for resolving the two transport systems.
Effects of tissue absorbance on NAD(P)H and Indo‐1 fluorescence from perfused rabbit hearts
Authors: Fralix Teresa A. , Heineman Frederick W. , Balaban Robert S.
Published: July 2002

The effects of tissue optical absorbance on intracellular NAD(P)H and Indo‐1 fluorescence emission have been evaluated in the perfused rabbit heart. These results demonstrate that the tissue optical absorbance significantly modifies the emission characteristics of these fluorophores. This tissue ‘inner filter’ effect, observed with both probes, changed as a function of tissue oxygenation and redox state in a wavelength‐dependent manner. Pathlength calculations from these results indicate that this inner filter effect could occur with a mean pathlength of 310 μm due to the extremely high extinction coefficient of heart tissue. It is concluded that tissue optical absorbance significantly affects the fluorescent emission characteristics of both intrinsic and extrinsic probes in the intact heart, under a variety of conditions. Several potential methods of correcting for these tissue inner filter effects are discussed.
Staurosporine enhances gonadotrophin‐releasing hormone‐stimulated luteinizing hormone secretion
Authors: van der Merwe P.Anton , Millar Robert P. , Wakeneld Ian K. , Macrae M.Bruce , Davidson James S.
Published: July 2002

In intact sheep gonadotropes, the protein kinase inhibitor, staurosporine, inhibited the stimulatory effect of phorbol 12‐myristate 13‐acetate (PMA) on luteinizing hormone (LH) secretion. Under the same conditions staurosporine enhanced gonadotrophin‐releasing hormone (GnRH)‐stimulated LH exocytosis without altering the EC50 of GnRH and without affecting basal LH exocytosis. These results suggest that PKC does not play a major role in mediating acute GnRH‐stimulated LH exocytosis. Furthermore, they demonstrate that staurosporine enhances GnRH stimulus‐secretion coupling. Both extracellular Ca2+ ‐dependent and Ca2+ ‐independent components of GnRH‐stimulated LH secretion were enhanced by the drug. Staurosporine had no effect on GnRH stimulation of cAMP and inositol phosphate synthesis. In permeabilized cells staurosporine did not enhance Ca2+ and cAMP‐stimulated LH exocytosis. Based on these results we hypothesize that staurosporine inhibits a protein kinase which is activated by GnRH and which negatively modulates GnRH stimulus‐secretion coupling.
An element of symmetry in yeast TATA‐box binding protein transcription factor IID
Authors: Stucka Rolf , Feldmann Horst
Published: July 2002

TATA‐box binding factor TFIID is one of the key factors in transcriptional activation. Surprisingly, the yeast TFDII protein [(1989) Nature 341, 299‐303; (1989) Cell 56, 1173‐1181; (1989) Proc. Natl. Acad. Sci USA 86, 7785‐7789] reveals only limited homology with other DNA‐binding proteins. From computer‐assisted searches we infer that yeast TFIID possesses a domain structure in which homologous segments are repeated. The greatest similarity is found between two segments, each 33 amino acids in length, in which the positions of four basic residues are strictly conserved. The high homology is also reflected at the gene level. Implications of this novel type of domain structure for possible interactions in transcriptional activation are discussed.
Insulin‐responsive glucose transporters are concentrated in a cell surfacederived membrane fraction of 3T3‐L1 adipocytes
Authors: Lange Klaus , Brandt Ursula
Published: July 2002

The recently proposed mechanistic concept of a receptor‐regulated entrance compartment for hexose transport formed by microvilli on 3T3‐L1 adipocytes predicted a preferential localization of glucose transporters in these structures. The cytochalasin B‐binding technique was used to determine in basal and insulin‐stimulated cells the distribution of glucose transporters between plasma membranes, low density microsomes (LDM) and two cell surface‐derived membrane fractions prepared by a hydrodynamic shearing technique. The shearing procedure applied prior to homogenization yielded a low density surface‐derived vesicle (LDSV) fraction which contained nearly 60% of the cellular glucose transporters and the total insulin‐sensitive transporter pool. The rest of the glucose transporter population was localized within the plasma membrane (5%) and the LDM fraction (37%). Pretreatment of the cells with insulin (20 for 10 min) reduced the transporter content of the LDSV fraction by 40% and increased that of the plasma membrane fraction 4‐fold. The transporter containing LDSV fraction was clearly differentiated from the LDM fraction by its low specific galactosyltransferase activity and its insulin‐sensitivity. Scanning electron microscopy revealed that the LDSV fraction contained a rather uniform population of spherical vesicles of 100–200 nm in diameter.
Molecular cloning and functional expression of a potassium channel cDNA isolated from a rat cardiac library
Authors: Tseng-Crank Julie C.L. , Tseng Gea-Ny , Schwartz Arnold , Tanouye Mark A.
Published: July 2002

A full‐length K+ channel cDNA (RHK1) was isolated from a rat cardiac library using the polymerase chain reaction (PCR) method and degenerate oligonucleotide primers derived from K+ channel sequences conserved between Drosophila Shaker H4 and mouse brain MBK1. Although RHK1 was isolated from heart, its expression was found in both heart and brain. The RHK1‐encoded protein, when expressed in Xenopus oocytes, gated a 4‐aminopyridine (4‐AP)‐sensitive transient outward current. This current is similar to the transient outward current measured in rat ventricular myocytes with respect to voltage‐dependence of activation and inactivation, time course of activation and inactivation, and pharmacology.
Coexistence of different forms ofNa,K‐ATPase in two‐dimensional membrane crystals
Authors: Hebert Hans , Skriver Elisabeth , Kavéus Urban , Maunsbach Arvid B.
Published: July 2002

Two‐dimensional membrane crystals of renal Na,K‐ATPase were analyzed by electron microscopy and image processing. The particular property of the crystals in this work was that they showed unit cell parameters similar to the previously studied p21 crystals but lacked the dyad axis as observed in nominal 0°‐projections. A three‐dimensional reconstruction revealed that structural differences between αβ‐units of the enzyme gave rise to the asymmetry. A high degree of two‐fold rotational symmetry was observed in the middle of the structure while the protein units had different three‐dimensional shapes at levels above and below the central sections. The simultaneous coexistence of different forms of Na,K‐ATPase suggests that the conformational flexibility of the enzyme plays an important role in the pumping process.
Low‐conductance chloride channel activated by cAMP in the epithelial cell line T84
Authors: Tabcharani J.A. , Low W. , Elie D. , Hanrahan J.W.
Published: July 2002

We have studied the modulation and pharmacological properties of two anion channels in T84 cells by recording single channel and transepithelial currents. One channel had an outwardly rectifying current‐voltage I/V curve, was rarely active in cell‐attached patches, and was unaffected by cAMP. The other channel had lower conductance (8.7 pS at 37°C) and a more ohmic I/V relationship. Exposure to cAMP increased the probability of observing low‐conductance channel activity in cell‐attached patches > 6‐fold. Extracellular DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulfonic acid) or IAA‐94 (an indanyloxyacetic acid) inhibited the outward rectifier but did not affect the low‐conductance channel or cAMP‐stimulated transepithelial current. These results suggest the low‐conductance Cl channel may contribute to apical membrane conductance during cAMP‐stimulated secretion.
Regulation of plant gene expression by antisense RNA
Authors: Mol J.N.M. , van der Krol A.R. , van Tunen A.J. , van Blokland R. , de Lange P. , Stuitje A.R.
Published: July 2002

Regulation of gene expression by antisense RNA was first discovered as a naturally‐occurring phenomenon in bacteria. Recently natural antisense RNAs have been found in a variety of eukaryotic organisms; their in vivo function is, however, obscure. Deliberate expression of antisense RNA in animal and plant systems has lead to successful down‐regulation of specific genes. We will review the current status of antisense gene action in plant systems. The recent discovery that ‘sense’ genes are able to mimic the action of antisense genes indicates that (anti)sense genes must operate by mechanisms other than RNA‐RNA interaction.
Activation of phospholipase D in rabbit neutrophils by fMet‐Leu‐Phe is mediated by a pertussis toxin‐sensitive GTP‐binding protein that may be distinct from a phospholipase C‐regulating protein
Authors: Kanaho Yasunori , Kanoh Hiroyuki , Nozawa Yoshinori
Published: July 2002

Stimulation by N‐formyl‐Met‐Leu‐Phe (fMLP) of rabbit peritoneal neutrophils, in which phosphatidylcholine was preferentially labeled with l‐O‐[3H]octadecyl lyso platelet‐activating factor, activated phospholipase D, resulting in the formation of [3H]PA from [3H]PC. A direct activator of GTP‐binding proteins (G‐proteins), NaF, also stimulated [3H]PA formation. fMLP‐stimulated [3H]PA formation was inhibited by pertussis toxin (IAP) in a time‐ and dose‐dependent manner. IAP also inhibited fMLP‐stimulated IP3 formation, but the inhibition of IP3 formation was significantly greater than that of [3H]PA formation. These results indicate that activation of phospholipase D by fMLP in rabbit neutrophils is mediated by an IAP‐sensitive G‐protein that may be distinct from a phospholipase C‐regulating protein.
The repression of the reverse‐oriented transcription from the adenovirus terminus by NFI in competition with TFIID
Authors: Matsumoto Ken , Nagata Kyosuke , Hanaoka Fumio , Ui Michio
Published: July 2002

Nuclear factor 1 (NFI) represses the transcription which is promoted by the cloned adenovirus (Ad) type 5 DNA replication origin and is reverse‐oriented with respect to the direction of the replication. The mechanism of this repression by NFI was investigated. In the cell‐free transcription system, the repression was observed only when NFI was present during the formation of the transcription initiation complex. From the results of DNase I protection experiments, it was indicated that NFI bound to its binding site in the Ad replication origin prevents TFIID from proper binding to the adjacent AT‐rich region and consequently represses the transcription.
Identification of the C2‐1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C‐1H heteronuclear multiple quantum coherence method
Authors: Derrick J.P. , Lian L.-Y. , Roberts G.C.K. , Shaw W.V.
Published: July 2002

Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75000. [ring2‐13C]Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CATIII) using a histidine auxotroph of E. coli. Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring. One‐ and two‐dimensional spectra revealed six signals out of a total of seven histidine residues in CATIII. pH titration, chemical modification and ligand binding were used to demonstrate that the signal from H195, the histidine at the active site, is not among those observed. Nevertheless, this work demonstrates that selective isotopic enrichment and multiple quantum coherence techniques can be used to distinguish proton resonances in a protein of high molecular mass.
The effects of glucocorticoid hormone on the expression of c‐jun
Authors: Lee Heng , Shaw Yueh-Tsern , Chiou Shean-Tai , Chang Wen-Chang , Lai Ming-Derg
Published: July 2002

The effects of glucocorticoid hormone on the expression of c‐jun in the fibroblasts were studied. The expression of c‐jun was repressed by dexamethasone in the NIH3T3 cells, but not in the transformed BI04‐1 or EJ‐Ras cells. The repression was not relieved by the addition of cycloheximide.
ATP‐sensitive K channels in heart muscle Spare channels
Authors: Findlay Ian , Faivre Jean-François
Published: July 2002

We show that ATP‐sensitive K+ channels of excised inside‐out membrane patches of rat ventricular myocytes show considerable variation in their sensitivity to ATP. In 102 different membrane patches IC50 values ranged from 9 to 580 μM ATP and Hill coefficient from 1% to 6.41% of patches showed openings of ATP‐sensitive K+ channels in the presence of 1 mM ATP. These results considerably widen the range of internal ATP concentrations over which one might expect activation of the ATP‐sensitive K+ current in cardiac myocytes.
Different cellular receptors for human placental laminin and murine EHS laminin
Authors: Brown Judith C. , Goodman Simon L.
Published: July 2002

Human placental laminin purified without the use of proteases promotes similar levels of cell attachment ? murine EHS laminin. The major HT1080 cell binding site of human placental laminin is thermally less labile than that of EHS laminin. Monoclonal antibody GOH3, which recognises the integrin α6 subunit, inhibits HT1080 and B16 cell attachment to EHS laminin but not to human placental laminin, indicating that the cells use different receptors to bind to these two types of laminin. Antibodies P1E6 and P1B5, which recognise the integrin α2 and α3 subunits respectively, do not affect HT1080 cell binding to either type of laminin.
Developmental expression of cloned cardiac potassium channels
Authors: Roberds Steven L. , Tamkun Michael M.
Published: July 2002

Cardiac K+ channels are responsible for repolarization of the action potential and are the targets of several antiarrhythmic drugs. This study examines the differential expression of six K+ channel mRNAs during rat heart development. RK1 and RK2 K+ channel transcripts were undetectable prior to 10 days after birth. In contrast, RK4 mRNA was present at equivalent levels from day 14 in utero to 20 days after birth. RK3 and RK5 were detected as early as 14 days in utero. These data indicate that K+ channel expression in the heart is closely regulated and further argue for physiologically distinct roles for K+ channel isoforms.