bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulfobacillus thermosulfidooxidans: a new lineage of bacterial evolution?
Authors: Karavajko G.I. , Bulygina E.S. , Tsaplina I.A. , Bogdanova T.I. , Chumakov K.M.
Published: July 2002

The nucleotide sequence of 5 S ribosomal RNA (rRNA) of type strain Sulfobacillus thermosulfidooxidans VKM B‐1269 was determined. This organism represents a group of moderately thermophilic acidophilic chemolithotrophic bacteria, able to use ferrous and sulfur compounds as the sole energy source. 5 S rRNA of this bacterium is drastically different from all other known bacterial 5 S rRNA sequences. It is suggested that S. thermosulfidooxidans represents a new lineage of bacterial evolution, that diverged from other bacteria at an early step of their evolution.
Transforming growth factor‐β regulates the splicing pattern of fibronectin messenger RNA precursor
Authors: Borsi Laura , Castellani Patrizia , Risso Anna Maria , Leprini Alessandra , Zardi Luciano
Published: July 2002

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions (ED‐A, ED‐B and IIICS) of the primary transcript of a single gene. Using monoclonal antibodies, we previously demonstrated that transforming growth factor‐β (TGF‐β) preferentially increases the accumulation of the FN isoforms containing the ED‐A sequence in cultured normal human fibroblasts [Balza et al., (1988) FEBS Lett. 228, 42‐44]. To determine the basis of this effect, we have examined through S1 nuclease analysis, the levels of ED‐A‐ and ED‐B‐containing mRNAs in cultured normal human skin flbroblasts before and after TGF‐β treatment. These experiments have shown that TGF‐β increases the relative amount of m‐RNA for ED‐A‐ and ED‐B‐containing FN isoforms. These data demonstrate that a growth factor may regulate the splicing pattern of a pre‐mRNA.
Interleukin 1β modulates hepatic synthesis of α1‐acid glycoprotein in the fetal rat
Authors: Durand Dominique , Bertaux Odile , Fournier Thierry , Porquet Dominique , Valencia Richard , Durand Geneviéve
Published: July 2002

The ability of the fetal rat to respond to interleukin 1β (IL1β) by expressing α1‐acid glycoprotein (AGP) was investigated. Eight and 20 h after injection of 7 ng IL1β into 19‐day fetuses, liver AGP mRNA increased by a factor of 66 and 82 respectively, while serum AGP levels increased by a factor of 3 and 5. Similar treatment of the mothers altered in the fetuses neither AGP serum levels nor the amount of liver AGP mRNA. The induction of AGP gene expression in the fetal liver in response to IL1β was similar to that observed in the adult liver. These results demonstrate that at day 19 the fetal rat liver has acquired a mature acute‐phase system.
Purification of a Ca2+/calmodulin‐dependent nitric oxide synthase from porcine cerebellum
Authors: Mayer Bernd , John Mathias , Böhme Eycke
Published: July 2002

L‐Arginine‐derived nitric oxide acts as an inter‐ and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine‐inducible enzyme in macrophages and a constitutive, Ca2+‐regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'‐ADP‐Sepharose. The enzyme was identified as an NO synthase with a specific NO‐chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO‐sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.
Synthesis and secretion of Alzheimer amyloid βA4 precursor protein by stimulated human peripheral blood leucocytes
Authors: Mönning Ursula , König Gerhard , Prior Reinhard , Mechler Hans , Schreiter-Gasser Ursula , Masters Colin L. , Beyreuther Konrad
Published: July 2002

Alzheimer amyloid precursor proteins (APP) are actively secreted by stimulated human peripheral mononuclear blood leucocytes (PMBLs). Induction of APP transcription, translation and secretion was observed with several T cell mitogens but was highest with phytohemagglutinin. The time course of induction is similar to that reported for IL‐2 and IL‐2 receptor. We suggest that APP may play an important role in the construction of the immunological network and the differentiation of T cells.
Do thin filaments of smooth muscle contain calponin?
Authors: Nishida Wataru , Abe Masahiro , Takahashi Katsuhito , Hiwada Kunio
Published: July 2002

A new method for the preparation of smooth muscle thin filaments which include calponin was established. We found that calponin readily separated from thin filaments in the presence of 10 mM ATP. By preventing thin filament extract from exposing to ATP, we obtained thin filaments which contained actin, tropomyosin, caldesmon and calponin in molar ratios of 7:0.9:0.6:0.7. We studied myosin Mg‐ATPase activity by using the thin filaments in comparison with classical thin filaments prepared by the method of Marston and Smith, which contained the same amounts of caldesmon and tropomyosin as our thin filaments but lost almost all calponin. The presence of calponin reduced the V
max value for thin filament‐activated myosin Mg‐ATPase activity by 33% without a significant change in K
m value. These findings suggest that calponin inhibits myosin Mg‐ATPase activity by modulation of a kinetic step as an integral component of smooth muscle thin filaments.
Degradation of type I and II collagen by human C1̄s
Authors: Yamaguchi Kiichiro , Sakiyama Hisako , Matsumoto Misako , Moriya Hideshige , Sakiyama Shigeru
Published: July 2002

The activated first component of human complement, C1̄s, was shown to cleave type I and II collagen and gelatin. The proteolytic activity was heat labile and was inhibited by a monoclonal antibody (M241) which recognized light chain of active human C1s or by a serine protease inhibitor, DFP, but not by a chelating agent.
Tyrosine‐specific phosphorylation of gpIIIa in platelet membranes
Authors: Elmore Moira A. , Anand Radhika , Horvath Andrea R. , Kellie Stuart
Published: July 2002

In vitro phosphorylation of platelet subcellular fractions revealed that most of the alkali‐resistant phosphoproteins and the majority of pp60c‐src were in the surface membrane fraction. An alkali‐resistant phosphoprotein of about 100 kDa was also immune precipitated by an anti‐phosphotyrosine antibody and comigrated with gpIIIa. The phosphorylation of gpIIIa, but not gpIIb, was confirmed by the comparison of reduced and non‐reduced gels, and this protein was phosphorylated exclusively on tyrosine. In contrast, both gpIIb and gpIIIa were phosphorylated when the purified complex was added to immunopurified, immobilised pp60c‐src. A synthetic peptide with partial homology to a putative tyrosine phosphorylation site in the cytoplasmic domain of gpIIIa was phosphorylated by antibody‐purified pp60c‐src. Our results indicate that tyrosine‐specific phosphorylation of gpIIIa by pp60c‐src may play a role in the regulation of platelet function.
Resonance Raman studies of the [4Fe‐4S] to [2Fe‐2S] cluster conversion in the iron protein of nitrogenase
Authors: Fu Weiguang , Morgan T.Vance , Mortenson Leonard E. , Johnson Michael K.
Published: July 2002

Resonance Raman spectroscopy has been used to investigate the Fe‐S stretching modes of the [4Fe‐4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe‐4S] cluster having effective Td
symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118–2122], treatment with the iron chelator α,α′‐dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe‐2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe‐2S]2+ cluster is induced by thionine‐oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP‐binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine‐oxidized Clostridium pasteurianum bidirectional hydrogenase.
The specific production of the third component of complement by osteoblastic cells treated with 1α,25‐dihydroxyvitamin D3
Authors: Sato Toshiyuki , Hong Mei Hua , Jin Cheng He , Ishimi Yoshiko , Udagawa Nobuyuki , Shinki Toshimasa , Abe Etsuko , Suda Tatsuo
Published: July 2002

A 190 kDa protein was purified from conditioned media of mouse marrow‐derived stromal cell (ST2) cultures treated with 1α 25‐dihydroxyvitamin D3 (1α,25(OH)2D3) and identified as the third component of mouse complement (C3). Northern and Western blot analysis revealed that the production of C3 by ST2 and primary osteoblastic cells was strictly dependent on 1α,25(OH)2D3, but the production by hepatocytes was not. Adding 1α,25(OH)2D3 together with mouse C3 antibody to bone marrow cultures greatly inhibited the formation of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like multinucleated cells. Adding C3 alone induced no TRAP‐positive cell formation. These results suggest that, in bone tissues, C3 is specifically produced by osteoblasts in response to 1α,25(OH)2D3 and somehow involved in inducing differentiation of bone marrow cells into osteoclasts in concert with other factors produced by osteoblasts in response to 1α,25(OH)2D3.
Oxidative damage to plasma constituents by ozone
Authors: Cross Carroll E. , Motchnik Paul A. , Bruener Bernd A. , Jones Daniel A. , Kaur Harparkash , Ames Bruce N. , Halliwell Barry
Published: July 2002

The reaction of ozone (O3) with human blood plasma was studied to help understand possible events that could occur in the respiratory tract. Uric acid (quantitatively the most important scavenger) and ascorbic acid were oxidized quickly, protein‐SH groups were lost more slowly, and there was no loss of bilirubin or α‐tocopherol. There was little formation of lipid hydroperoxides and no detectable formation of 4‐hydroxynonenal, hexanal or nonanal, or changes in lipoprotein electrophoretic mobility. Uric acid in human upper airway secretions may play a significant role in removing inhaled O3. Oxidative damage to lipids must not be assumed to be the key mechanism of respiratory tract O3 toxicity.
Aziridine‐2‐carboxylic acid A reactive amino acid unit for a new class of cysteine proteinase inhibitors
Authors: Morodor L. , Musiol H.-J. , Scharf R.
Published: July 2002

The three‐membered ring of aziridine‐2‐carboxylic acid, which is susceptible to opening by nucleophiles, has been analyzed as a potential useful handle for the design of specific irreversible inhibitors of cysteine proteinases. For this thiol‐reactive amino acid, an imino analogue of proline, a second‐order rate constant of 17.07 M−1 s−1 for inactivation of papain was determined. Thus, the aziridine moiety proved to be remarkably more reactive than activated double bonds, e.g. N‐ethylmaleimide, or halides such as α‐iodopropionic acid or chloroacetic acid. Since it does not alkylate histidine under conditions in which quantitative alkylation occurs with N‐ethyl‐maleimide, it could represent an interesting reactive amino acid unit for the synthesis of a new class of irreversible inhibitors, at least in terms of specificity of the chemical reaction involved in the inactivation process.
A novel coumarin‐labelled peptide for sensitive continuous assays of the matrix metalloproteinases
Authors: Knight C.Graham , Willenbrock Frances , Murphy Gillian
Published: July 2002

(7‐methoxycoumarin‐4‐yl)Acetyl‐Pro‐Leu‐Gly‐Leu‐(3‐[2,4‐dinitrophenyl]‐l‐2,3‐diaminopropionyl)‐Ala‐Arg‐NH2 (Mca‐Pro‐Leu‐Gly‐Leu‐Dpa‐Ala‐Arg‐NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly flourescent 7‐methoxycoumarin group is efficiently quenched by energy transfer to the 2,4‐dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3,4,24,23) cleaves the substrate at the Gly‐Leu bond with a 190‐fold increase in fluorescence (λcm 328 nm, λcm 393 nm). In assays of the human matrix metalloproteinases, Mca‐Pro‐Leu‐Gly‐Leu‐Dpa‐Ala‐Arg‐NH2 is about 50 to 100 times more sensitive than dinitrophenyl‐Pro‐Leu‐Gly‐Leu‐Trp‐Ala‐d‐Arg‐NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (k
cal/K
m) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.
Determination of proteinase 3—α1‐antitrypsin complexes in inflammatory fluids
Authors: Dolman Koert M. , van de Wiel Bart A. , Kam Chih-Min , Abbink Jannie J. , Hack C.Erik , Sonnenberg Arnoud , Powers James C. , von dem Borne Albert E.G.Kr. , Goldschmeding Roel
Published: July 2002

Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is α1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3—α1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3—α1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)—α1AT complexes. Thus, in vivo α1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.
Class II tubulin, the major brain β tubulin isotype is polyglutamylated on glutamic acid residue 435
Authors: Rüdiger Manfred , Plessman Uwe , Klöppel Klaus-Dieter , Wehland Jürgen , Weber Klaus
Published: July 2002

Protein sequencing shows that porcine brain tubulin retains the N‐terminal sequences of α and β tubulin after a mild treatment with subtilisin, C‐terminal peptides released by subtilisin were purified and characterized by automated Edman degradation and mass spectrometry. We confirm the polyglutamylation of α tubulin on glutamic acid residue 445 reported by others and show in addition that class 11 β tubulin, the major β tubulin isotype of adult brain, is also polyglutamylated. The substitution is restricted to glutamic acid residue 435. Thus all major tubulin isotypes of adult brain are subjected to polyglutamylation.
Effect of RU 38486 on TNF production and toxicity
Authors: Lázár George , Duda Ernö , Lázár George
Published: July 2002

Gluracarticoid steroids provide considerable protection against the systemic toxicity or tumor necrosis factor‐α (TNF‐α, cachexin). In animal experiments RU 38486 (mircpristone), a steroid antagonist, increased the synthesis of TNF and sensitized the animals to the cytotoxic action of TNF. As compared to the control and methylprednisolone‐treated groups, mifepristone significantly increased the level of TNF in the serum, liver and spleen or lipopolysaccharide (LPS)‐treated animals. In tissue cultures F.U. 38486 induced the TNF synthesis of mycloid cells and increase the TNF production or genetically modified HeLa cells, which synthesize TNF constitutively. Normal and tumor cell cultures exhibited increased sensitivity toward TNF in the presence of mifepristone.
Interactions of calreticulin with proteins of the endoplasmic and sarcoplasmic reticulum membranes
Authors: Burns Kimberly , Michalak Marek
Published: July 2002

The ability of [125I]calreticulin to bind to membrane fractions isolated from different muscle and non‐muscle tissues was examined by a protein overlay technique. Specific [125AI]calreticulin binding proteins were detected in rat liver smooth and rough endoplasmic reticulum and Golgi, in canine pancreatic microsomes, and in rabbit skeletal muscle sarcoplasmic reticulum. These proteins were confined only to membranes that contain calreticulin; they were not found in rat liver mitochondria or cytosol. [125I]Calreticulin binds to a 50‐kDa protein and a number of lower Mr
(20,000–38,000) endoplasmic reticulum membrane proteins and to 30‐kDa protein in skeletal muscle sarcoplasmic reticulum. Full‐length calreticulin and the carboxyl‐terminal region (C‐domain) of the protein both competed with [125I]calreticulin for binding to the membrane proteins. Binding of [125I]calreticulin to pancreatic microsomes was also partially inhibited by the N‐domain and to a lesser extent by the P‐domain of the protein. We conclude that calreticulin interacts with the endoplasmic reticulum membrane proteins mainly through its carboxyl‐terminal domain and that the endoplasmic and sarcoplasmic reticulum membranes may contain different calreticulin binding proteins.
Control of myogenic factor genes by the membrane depolarization/protein kinase C cascade in chick skeletal muscle
Authors: Huang Chang-Fen , Neville Craig M. , Schmidt Jakob
Published: July 2002

Myogenic factor genes were found to respond differentially to electrical stimulation of denervated chick skeletal muscle. Myogenin gene activity declined rapidly (t
½:~2 min), comparable to the rate of acetylcholine receptor (AChR) gene inactivation, while other myogenic bHLH genes either lost activity more slowly (MyoD) or not at all (myf5, herculin). Protein kinase C (PKC) is known to couple membrane activity to AChR gene inactivation; myogenin gene transcription was also rapidly blocked by the PKC activator PMA, whereas electrostimulation remained without effect on myogenin gene activity in muscle that was either exposed to the kinase inhibitor staurosporine or chronically treated with PMA to deplete PKC. These results attest to a special role for myogenin in the activation of AChR genes in denervation supersensitivity.
Sulfhydryl oxidation induces rapid and reversible closure of the ATP‐regulated K+ channel in the pancreatic β‐cell
Authors: Islam Md.Shahidul , Berggren Per-Olof , Larsson Olof
Published: July 2002

Effects of sulfhydryl modification on the ATP regulated K1 channel (KATP channel) in the pancreatic β‐cell were studied, using the patch clamp technique. Application of the sulfhydryl oxidizing agents thimerosal and 2,2'‐dithio‐bis(5‐nitropyridine) (DTBNP), in micromolar concentrations, caused complete inhibition of the kATP channel, in inside‐out patches. The inhibition was rapid and was reversed by the disulfide reducing agents dithiothreitol and cysteine. Thimerosal, which is poorly membrane permeable, inhibited channel activity, only when applied to the intracellular face of the plasma membrane. In contrast, DTBNP, which is highly lipophilic, caused closure of the kATP channel and consequent depolarization of the membrane potential, also when applied extracellularly. Our results indicate the presence of accessible free SH groups on the cytoplasmic side of the kATP channel in the pancreatic β‐cell. These SH groups are essential for channel function and it is possible that thiol‐dependent redox mechanisms can modulate KATP channel activity.
Regulation of spermidine/spermine N1‐acetyltransferase by intracellular polyamine pools
Authors: Shappell N.W. , Fogel-Petrovic M.F. , Porter C.W.
Published: July 2002

Through its role in polyamine acetylation and the back‐conversion pathway, spermidine/spermine N
1‐acetyltransferase (SSAT) has the potential to control intracellular polyamine pools by facilitating their catabolism and/or excretion. The possibility that the enzyme is subject to regulation by intracellular polyamine pools was investigated in MALME‐3 human melanoma cells. Increases in intracellular polyamine pools by treatment with 3 μM exogenous spermidine or spermine for 48 h caused SSAT activity to increase 111% and 226%, respectively, and SSAT‐specific mRNA to rise 19% and 66%, respectively. Decreases in polyamine pools by treatment with inhibitors of polyamine biosynthesis caused SSAT activity to decrease by 46% and mRNA to fall by 89%. Both SSAT activity and mRNA were more sensitive to changes in spermine than spermidine. The identification of a positive regulatory relationship between SSAT and intracellular polyamine pools further implicates this enzyme in a proposed model for polyamine pool homeostasis.
Inhibitory effect of neopterin on NADPH‐dependent superoxide‐generating oxidase of rat peritoneal macrophages
Authors: Kojima Shuji , Nomura Takafumi , Icho Takeshi , Kajiwara Yoshio , Kitabatake Katsuaki , Kubota Kazuhiko
Published: July 2002

The effect of the oxidized form of neopterin (NP) on the NADPH‐dependent superoxide‐generating oxidase (NADPH‐oxidase) was investigated in both whole‐cell and cell‐free activation systems by using peritoneal macrophages of rats which had received an intraperitoneal injection of mineral oil. In the whole‐cell system, NP remarkably inhibited the generation of Superoxides in macrophages stimulated with phorbol myristate acetate (PMA). NP also showed an significant suppression of the activation of superoxide‐generating NADPH‐oxidase in the cell‐free system using solubilized membranes and sodium dodecyl sulfate (SDS) as a stimulant. The 50%‐inhibitory concentration (IC50) of NP was about 1 μM in both assay systems. In a kinetic study, competitive inhibition of the NADPH‐oxidase by NP was observed in the cell‐free system with a calculated inhibition constant (K
i) of 6.50 μM. These findings suggest that NP may play an important role in the suppression of Superoxide generation via the inhibition of the NADPH‐oxidase in phagocytes.
Expression of mRNA for cyclooxygenase‐1 and cyclooxygenase‐2 in human tissues
Authors: O'Neill Gary P. , Ford-Hutchinson Anthony W.
Published: July 2002

The rate‐limiting step in the formation of prostanoids is the conversion of arachidonic acid to prostaglandin H2 by cyclooxygenase, also known as prostaglandin G/H synthase/cyclooxygenase. Two forms of cyclooxygenase have been characterized: a ubiquitously expressed form (COX‐1) and a recently described second form (COX‐2) inducible by various factors including mitogens, hormones, serum and cytokines. Here we quantitate by the reverse transcriptase‐polymerase chain reaction (RT‐PCR) the expression of COX‐1 and COX‐2 mRNA in human tissues including lung, uterus, testis, brain, pancreas, kidney, liver, thymus, prostate, mammary gland, stomach and small intestine. All tissues examined contained both COX‐1 and COX‐2 mRNA and could be grouped according to the level of COX mRNA expression. The highest levels of COX mRNAs were detected in the prostate where approximately equal levels of COX‐1 and COX‐2 transcripts were present. In the lung high levels of COX‐2 were observed whereas COX‐1 mRNA levels were about 2‐fold lower. An intermediate level of expression of both COX‐1 and COX‐2 mRNA was observed in the mammary gland, stomach, small intestine, and uterus. The lowest levels of COX‐1 and COX‐2 mRNA were observed in the testis, pancreas, kidney, liver, thymus, and brain.
Interaction between calponin and smooth muscle myosin
Authors: Szymanski Pawel T. , Tao Terence
Published: July 2002

Calponin is a thin filament‐associated protein in smooth muscle that has been shown to bind actin, tropomyosin and calmodulin, and has been implicated to play a role in regulation of smooth muscle contractility. Using a centrifugation assay we found that calponin interacts with unphosphorylated filamentous smooth muscle myosin. We found that this calponin‐myosin interaction is reversed by Ca2+‐CaM, and depends on ionic strength. At 50 mM NaCI the binding constant and the stoichiometry of this interaction were estimated to be 2 × 106 M−1, and 1.2–2.4 calponin per myosin, respectively. We suggest that the calponin‐myosin interaction could be involved in regulation of smooth muscle contractility by anchoring myosin to actin.
Improved gene transfer by direct plasmid injection associated with regeneration in mouse skeletal muscle
Authors: Wells Dominic J.
Published: July 2002

Gene transfer into skeletal muscle via simple plasmid injection in vivo has many potential uses but these are severely constrained by the low efficiency of this technique. Muscle regeneration, induced by the myotoxic local anaesthetic bupivacaine, significantly increased gene expression following plasmid injection 3–7 days after bupivacaine treatment. Much of this effect can be attributed to uptake and expression of the plasmid by a greater number of muscle fibres, up to 9% of the mouse tibialis anterior muscle. A similar significant increase in expression was observed in the naturally regenerating muscle of the dystrophic mdx mouse when compared to the control C57B1/10 strain.
Intron and intronless transcription of the chicken polyubiquitin gene UbII
Authors: Mezquita Jovita , López-Ibor Blanca , Pau Montserrat , Mezquita Cristóbal
Published: July 2002

We have previously reported that the chicken polyubiquitin gene UbII is preferentially expressed during spermatogenesis and we show here that UbII is the predominant polyubiquitin gene expressed in early embryogenesis. Two main initiation sites were detected. Transcription from the initiation site used in early embryos results in the presence of an intron in the 5'‐untranslated region of the transcripts as has been reported for other polyubiquitin messages. In mature testis, however, the use of a different initiation site, located within the intron, produces intronless transcripts. Distinct promoter sequences, present in each initiation site, may regulate the differential expression observed in this gene.
Topology of Na,K‐ATPase α subunit epitopes analyzed with oligopeptide‐specific antibodies and double‐labeling immunoelectron microscopy
Authors: Ning Gang , Maunsbach Arvid B. , Lee Yang-Jae , Møller Jesper Vuust
Published: July 2002

Using four oligopeptide‐specific polyclonal antibodies, we mapped the α subunit ofNa,K‐ATPase by double‐labeling immunoelectron microscopy combined with negative staining. The results show that the epitopes of the N‐tenninus (Gly1‐His13), C‐terminus (Ile1002‐Tyr1016) and Leu815‐Gln828 are located on the same face of crystallized Na,K‐ATPase membranes from pig kidney, whereas the epitope Asn889‐Gln903 is present on the opposite side. The present study demonstrates the cytoplasmic location of C‐terminus and that Leu828‐Gln828 is exposed on the eytoplasmic and Asn889‐Gln903 on the extracellular side. The results are consistent with an eight‐ or ten‐segment model, and support the existence of an M5/M6 loop and the presence of one transmembrane segment between Leu815‐Gln828 and Asn889‐Gln903.
Activation of phosphatidylinositol lipid‐specific phospholipase C‐β3 by G‐protein βγ subunits
Authors: Carozzi Amanda , Camps Montserrat , Gierschik Peter , Parker Peter J.
Published: July 2002

A novel member of the inositol lipid‐specific phospholipase C family, PtdIns‐PLCβ3, is shown to be activated by βγ subunits of the heterotrimeric GTP‐binding protein, transducin. The activation is a direct effect since it is observed with the purified proteins. Furthermore, the activation is blocked by the GDP‐liganded α subunit of transducin, confirming that the effect is due to free βγ subunits. The implications with respect to receptor‐PtdIns‐PLC coupling are discussed.
Oxidative stress during recovery from muscle atrophy
Authors: Kondo Hisao , Kodama Junko , Kishibe Takako , Itokawa Yoshinori
Published: July 2002

Single ankle joints of male Wistar rats (15‐week‐old) were immobilized in the extended position for 7 days and remobilized for 5 days after the immobilization period. Atrophic and contralateral soleus, typical slow red muscles, were collected and their levels of thiobarbituric acid‐reactive substance (TBARS) and glutathione were measured. Five‐day remobilization did not increase muscle weight significantly. However, there were significant increases in TBARS and oxidized glutathione in the recovering muscle, which strongly suggested that enhanced oxidative stress occurred during the recovery from disuse muscle atrophy. Vitamin E injection accelerated the recovery from atrophy, thus showing that oxidative stress slowed it down.
The protein coded by the X‐adrenoleukodystrophy gene is a peroxisomal integral membrane protein
Authors: Contreras M. , Mosser J. , Mandel J.L. , Aubourg P. , Singh I.
Published: July 2002

The gene for adrenoleukodystrophy (X‐ALD), a peroxisomal disease characterized by excessive accumulation of very long‐chain (VLC) fatty acids (> C22;0), has recently been identified by positional cloning, and it is predicted to encode a protein (ALD‐P) of 745 amino acids [(1993) Nature 361, 726]. Using Western blot analysis of subcellular organelles purified by isopycnic density gradient centrifugation from X‐ALD and control fibroblasts, we show that the monoclonal antibodies directed against ALD‐P cross‐react with a 75 kDa protein in intact peroxisomes and that ALD‐P is an integral component of the peroxisomal membrane. Moreover, no signal for ALD‐P was detected in peroxisomes from X‐ALD patients with deletion of the ALD gene.
No requirement of reactive oxygen intermediates in Fas‐mediated apoptosis
Authors: Hug Hubert , Enari Masato , Nagata Shigekazu
Published: July 2002

Fas is a cell surface molecule that mediates apoptosis, but the intracellular mechanisms leading to apoptosis are not well understood. It is known that diethylmaleate (DEM)‐induced cell death can be blocked by substances with antioxidant activity. Here we have studied whether antioxidants have any effect on Fas‐mediated apoptosis and show that they are not able to block Fas‐mediated apoptosis. Therefore, it seems that reactive oxygen intermediate (ROI)‐dependent and ‐independent mechanisms which lead to apoptosis do exist. Fas‐mediated apoptosis probably proceeds via a ROI‐independent pathway.
Sensory rhodopsin I photocycle intermediate SRI380 contains 13‐cis retinal bound via an unprotonated Schiff base
Authors: Haupts Ulrich , Eisfeld Wolf , Stockburger Manfred , Oesterhelt Dieter
Published: July 2002

Sensory rhodopsin I (SRI), the mutated derivative SRI‐D76N and the complex of SRI with its transducer HtrI were overexpressed in Halobacterium salinarium and analyzed by resonance Raman spectroscopy. In the initial state SRI contains all‐trans retinal bound via a protonated Schiff base as confirmed by retinal extraction which yields 95 ± 3% all‐trans retinal. The photocycle intermediate absorbing maximally at 380 nm (SRI380) contains a Schiff base linkage between the protein and 13‐cis retinal. Extraction of illuminated SRI yields up to 93% 13‐cis retinal. Neither the mutation D76N nor HtrI changed the vibrational pattern of the chromophore.
Induction of the small stress protein, hsp25, in Ehrlich ascites carcinoma cells by anticancer drugs
Authors: Bielka Heinz , Hoinkis Günter , Oesterreich Steffi , Stahl Joachim , Benndorf Rainer
Published: July 2002

Treatment of in vitro cultured Ehrlich ascites carcinoma cells with cisplatin, daunomycin, doxorubicin, cytosine arabinoside, 3'‐fluorodeoxythymidine, colchicine and vincristine in cytostatically effective concentrations results in significantly increased levels of the small stress protein, hsp25, as analyzed by immunoblotting. However, no induction of hsp25 could be detected after treatment of the tumour cells with 5‐fluorouracil, aminopterin, amethopterin, mithramycine and cyclophosphamide. None of these cytostatic drugs induces hsp70.
Inhibition of the Ca2v+‐activated K+‐channel by sapecin B, an insect antibacterial protein
Authors: Shimoda Masafumi , Takagi Hiroshi , Kurata Shoichiro , Yoshioka Tohru , Natori Shunji
Published: July 2002

Sapecin is an antibacterial protein of the flesh fly and sapecin B is its homologue structurally similar to charybdotoxin of scorpion venom, which is known to be a K+ channel inhibitor. We found that, like charybdotoxin, sapecin B inhibits part of the voltage pulse‐induced K+ currents of rat cerebellar Purkinje cells. We suggest that this effect is due to inhibition of the Ca+‐activated K+ channel. Probably, sapecin B is a naturally occurring K+ channel inhibitor as well as an antibacterial protein.
The effect of haloperidol on D2 dopamine receptor subtype mRNA levels in the brain
Authors: Fishburn C.Simone , David Carol , Carmon Sara , Fuchs Sara
Published: July 2002

Chronic neuroleptic treatment induces an increase in the density of D2 dopamine receptors in the striatum. The effect of prolonged administration of haloperidol on mRNA levels of the short (D2s) and long (D2L) isoforms of the D2 dopamine receptor and the D3 dopamine receptor in different brain regions was examined. Haloperidol caused a significant rise in both D2L and D2s mRNA levels in the striatum, but had no effect on D3 mRNA levels. This rise was restricted to the striatum, showing that the effect of haloperidol on dopamine receptor mRNA is both subtype‐ and tissue‐specific.
Molecular cloning, functional expression and localization of a novel inward rectifier potassium channel in the rat brain
Authors: Koyama Hidekazu , Morishige Ken-Ichirou , Takahashi Naohiko , Zanelli Jill S. , Fass David N. , Kurachi Yoshihisa
Published: July 2002

We have cloned a novel inward rectifier potassium channel from a rat brain cDNA library and designated it RB‐IRK2. The rat brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. The amino acid sequence of RB‐IRK2 shares 70%, 40% and 45% identity to mouse IRK1, rat ROMK1 and rat GIRK1, respectively. Xenopus oocytes injected with cRNA derived from RB‐IRK2 expressed a potassium current which showed inward‐rectifying channel characteristics similar to the IRK1 current, but distinct from the ROMK1 or the GIRK1 currents. However, the localization of RB‐IRK2 mRNA in rat tissues, assessed by the Northern blot analysis, differed from that of mouse IRK1. These results indicate that the IRK family is composed of multiple genes, which express in different tissues and therefore may play heterogenous functional roles in various organs, including rat central nervous system.
Cloning and expression of the vesamicol binding protein from the marine ray Torpedo
Authors: Varoqui Hélène , Diebler Marie-Françoise , Meunier François-Marie , Rand James B. , Usdin Ted B. , Bonner Tom I. , Eiden Lee E. , Erickson Jeffrey D.
Published: July 2002

Complementary DNA clones corresponding to a messenger RNA encoding a 56 kDa polypeptide have been obtained from Torpedo marmorata and Torpedo ocellata electric lobe libraries, by homology screening with a probe obtained from the putative acetylcholine transporter from the nematode Caenorhabditis elegans. The Torpedo proteins display approximately 50% overall identity to the C. elegans unc‐17 protein and 43% identity to the two vesicle monoamine transporters (VMAT1 and VMAT2). This family of proteins is highly conserved within 12 domains which potentially span the vesicle membrane, with little similarity within the putative intraluminal glycosylated loop and at the N‐ and C‐termini. The ~ 3.0 kb mRNA species is specifically expressed in the brain and highly enriched in the electric lobe of Torpedo. The Torpedo protein, expressed in CV‐1 fibroblast cells, possesses a high‐affinity binding site for vesamicol (K
d = 6 nM), a drug which blocks in vitro and in vivo acetylcholine accumulation in cholinergic vesicles.
Mitochondrial free calcium transients during excitation‐contraction coupling in rabbit cardiac myocytes
Authors: Chacon Enrique , Lemasters John J , Ohata Hisayuki , Harper Ian S , Trollinger Donna R , Herman Brian
Published: July 2002

Mitochondrial free Ca2+ may regulate mitochondrial ATP production during cardiac exercise. Here, using laser scanning confocal microscopy of adult rabbit cardiac myocytes co‐loaded with Fluo‐3 to measure free Ca2+ and tetramethylrhodamine methylester to identify mitochondria, we measured cytosolic and mitochondrial Ca2+ transients during the contractile cycle. In resting cells, cytosolic and mitochondrial Fluo‐3 signals were similar. During electrical pacing, transients of Fluo‐3 fluorescence occurred in both the cytosolic and mitochondrial compartments. Both the mitochondrial and the cytosolic transients were potentiated by isoproterenol. These experiments show directly that mitochondrial free Ca2+ rises and falls during excitation‐contraction coupling in cardiac myocytes and that changes of mitochondrial Ca2+ are kinetically component to regulate mitochondrial metabolism on a beat‐to‐beat basis.
Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy
Authors: Hellwig Petra , Rost Borries , Kaiser Ulrike , Ostermeier Christian , Michel Hartmut , Mäntele Werner
Published: July 2002

The redox reactions of the cytochrome c oxidase from Paracoccus denitrificans were investigated in a thin‐layer cell designed for the combination of electrochemistry under anaerobic conditions with UVIVIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surface‐modified electrode for all cofactors as indicated by the optical signals in the 400–700 nm range. Fourier transform infrared (FTIR) difference spectra of reduction and oxidation (reduced‐minus‐oxidized and oxidized‐minus‐reduced, respectively) obtained in the 1800‐1000 cm−1 range reveal highly structured band features with major contributions in the amide I (1620–1680 cm−1) and amide II (1580‐1520 cm−1) range which indicate structural rearrangements in the cofactor vicinity. However, the small amplitude of the IR difference signals indicates that these conformational changes are small and affect only individual peptide groups. In the spectral region above 1700 cm−1, a positive peak in the reduced state (1733 cm−1) and negative peak in the oxidized state (1745 cm−1) are characteristic for the formation and decay of a COOH mode upon reduction. The most obvious interpretation of this difference signal is proton uptake by one Asp or Gin side chain carboxyl group in the reduced state and deprotonation of another Asp or Glu residue. Moreover, both residues could well be coupled as a donor‐acceptor pair in the proton transfer chain. An alternative interpretation is in terms of a protonated carboxyl group which shifts to a different environment in the reduced state. The relevance of this first direct observation of protein protonation changes in the cytochrome c oxidase for vectorial proton transfer and the catalytic reaction is discussed.
Immunolocalization of an inwardly rectifying K+ channel, KAB‐2 (Kir4.1), in the basolateral membrane of renal distal tubular epithelia
Authors: Ito Minoru , Inanobe Atsushi , Horio Yoshiyuki , Hibino Hiroshi , Isomoto Shojiro , Ito Hiroyuki , Mori Keiji , Tonosaki Akira , Tomoike Hitonobu , Kurachi Yoshihisa
Published: July 2002

Immunolocalization of KAB−2 (Kir4.1), an inwardly ectifying K+ channel with a putative ATP‐binding domain, was examined in rat kidney where expression of KAB−2 mRNA was previously shown. Anti‐KAB−2 antibody was raised in rabbit and then affinity‐purified. An immunohistochemical study revealed that KAB‐2 immunoreactivity was detected specifically in the basolateral membrane of distal tubular epithelia. Therefore, KAB‐2 is the first K+ channel shown to be localized in the basolateral membrane of renal epithelia. The finding suggests that KAB‐2 may contribute to supplying K+ to the Na+‐K+ pump, which is abundant in the basolateral membrane of distal tubular epithelia, as well as to maintenance of the deep negative membrane potential of these cells.
A study of approximating the moments of the job completion time in PERT networks
Authors: Mehrotra Kishan , Chai John , Pillutla Sharma
Published: July 2002

The importance of proper management of projects has not gone unrecognized in industry and academia. Consequently, tools like the Critical Path Method (CPM) and the Program Evaluation Review Technique (PERT) for project planning have been the focus of attention of both practitioners and researchers. Determination of the Time to Complete the Job (TCJ) in PERT networks is important for planning and bidding purposes. The complexity involved in accurately determining the TCJ has led to the development of many approximating procedures. Most of them ignore the dependence between paths in the network. We propose an approximation to determine the TCJ which explicitly recognizes this dependency. Dependency in networks arises due to commonality of activities among various paths in the network. We develop an approximation which is simple to use and makes use of readily available tables. Also, the approximation employs the traditional concept of the critical path which is easy to understand and to operationalize. The activities on the critical paths are divided into an independent portion and a dependent portion. The dependent portion comprises activities common to various critical paths. Order statistics are used in computing the time for the dependent portion of the critical path. We present the theoretical underpinnings of our approach and illustrate its application via an example. In the absence of other measures, we use simulation results as a proxy for the TCJ and as a benchmark to measure the accuracy of our approximation. Empirical results are obtained for a variety of networks in the literature. We show that the distribution of the TCJ is better approximated by a mixture of distributions. Comparison with other approaches from the literature indicates that our approximation yields estimates for the TCJ which are closer to the simulation results.
Comparative study of the effects of zopiclone (7.5 mg), zolpidem, flunitrazepam and a placebo on nocturnal cognitive performance in healthy subjects, in relation to pharmacokinetics
Authors: Allain H , Patat A , Lieury A , Le Coz F , Janus C , Menard G , Gandon JM
Published: July 2002

The effect of zopiclone (7.5 mg) on attention, vigilance and memory components was evaluated during a nocturnal period in comparison to a placebo, to zolpidem (10 mg) and to flunitrazepam (1 mg) in a double blind, randomized study, after administration of a single dose in 16 young healthy volunteers. It appears that there is a clear effect on attention and vigilance; this effect is apparent during the kinetic phase of the absorption of the medication. The effect on memory is transient and is absent four hours after the ingestion of the drug. The objective results are not strictly consistent with the chronology of the subjective parameters (Leeds scale — Visual Analogue Scale). The three hypnotics under comparison do not fundamentally differ except in their kinetic/pharmacodynamic effect relationship. One important fact, taking the parameters as a whole, is that there is no objective “residual” effect.
EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two‐photon and fluorescence lifetime microscopy
Authors: Jakobs Stefan , Subramaniam Vinod , Schönle Andreas , Jovin Thomas M. , Hell Stefan W.
Published: July 2002

The green fluorescent protein (GFP) has become an invaluable marker for monitoring protein localization and gene expression in vivo. Recently a new red fluorescent protein (drFP583 or DsRed), isolated from tropical corals, has been described [Matz, M.V. et al. (1999) Nature Biotech. 17, 969–973]. With emission maxima at 509 and 583 nm respectively, EGFP and DsRed are suited for almost crossover free dual color labeling upon simultaneous excitation. We imaged mixed populations of Escherichia coli expressing either EGFP or DsRed by one‐photon confocal and by two‐photon microscopy. Both excitation modes proved to be suitable for imaging cells expressing either of the fluorescent proteins. DsRed had an extended maturation time and E. coli expressing this fluorescent protein were significantly smaller than those expressing EGFP. In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy.
Nuclear factor Y activates the human xanthine oxidoreductase gene promoter
Authors: Martelin Eeva , Palvimo Jorma J. , Lapatto Risto , Raivio Kari O.
Published: July 2002

To study the regulation of the human xanthine oxidoreductase (XOR) gene, we cloned 1.9 kb of the promoter region. In reporter gene assays, a construct encompassing nucleotides between −142 to +42 conferred maximal basal activity of the XOR promoter in 293T cells, in comparison with shorter (−92 to +42) or longer (up to −1937 to +42) constructs. The promoter activity was low in NIH‐3T3 cells. The most active construct contained a putative CCAAT motif at −119 to −123. Electrophoretic mobility shift assays showed that this sequence binds the ubiquitous nuclear factor Y (NF‐Y). Mutation of the CCAAT motif (CTGAT) abolished the NF‐Y binding and considerably reduced the promoter activity. Our data suggest an important functional role for NF‐Y in the transcriptional activation of the human XOR gene.
Crystal structure of S‐glutathiolated carbonic anhydrase III
Authors: Mallis Robert J. , Poland Bradley W. , Chatterjee Tapan K. , Fisher Rory A. , Darmawan Steven , Honzatko Richard B. , Thomas James A.
Published: July 2002

S‐Glutathiolation of carbonic anhydrase III (CAIII) occurs rapidly in hepatocytes under oxidative stress. The crystal structure of the S‐glutathiolated CAIII from rat liver reveals covalent adducts on cysteines 183 and 188. Electrostatic charge and steric contacts at each modification site inversely correlate with the relative rates of reactivity of these cysteines toward glutathione (GSH). Diffuse electron density associated with the GSH adducts suggests a lack of preferred bonding interactions between CAIII and the glutathionyl moieties. Hence, the GSH adducts are available for binding by a protein capable of reducing this mixed disulfide. These properties are consistent with the participation of CAIII in the protection/recovery from the damaging effects of oxidative agents.
Phosphatidylinositol 4,5‐bisphosphate specifically stimulates PP60c‐src catalyzed phosphorylation of gelsolin and related actin‐binding proteins
Authors: De Corte Veerle , Gettemans Jan , Vandekerckhove Joël
Published: July 2002

Gelsolin is a widely distributed Ca2+‐dependent regulator of the cortical actin network. We demonstrate that gelsolin is phosphorylated by pp60c‐src and that this phosphorylation is dramatically enhanced by phosphatidylinositol 4,5‐bisphosphate (PIP2), known to specifically interact with gelsolin. Other phospholipids display only a marginal effect. pp56lck, a tyrosine kinase of the same family, does not phosphorylate gelsolin. Other mammalian actin‐binding proteins such as profilin and CapG but also fragmin from Physarum polycephalum are similar targets for PIP2‐stimulated pp60c‐src phosphorylation.
Rab proteins of Drosophila melanogaster: Novel members of the Rab‐protein family
Authors: Satoh Akiko K. , Tokunaga Fumio , Ozaki Koichi
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Interaction of human retinal RGS with G‐protein α‐subunits
Authors: Natochin Michael , Lipkin Valery M , Artemyev Nikolai O
Published: July 2002

A novel family of RGS proteins negatively regulates signaling via heterotrimeric G‐proteins by accelerating the GTPase activity of G‐protein α subunits. We have investigated interaction of human retinal RGS protein (hRGSr) with in vitro translated G
α
subunits: Gtα
, Giα1, Goα
and Gsα
. hRGSr binds well to Gtα
, Giα1 and Goα
in the presence of AlF4
−, but does not interact with Gsα
. The N‐ and C‐terminally truncated G
α
subunits interact with hRGSr similarly to the intact G
α
polypeptides. Analysis of interaction between hRGSr and Goα
/Gsα
chimeras suggests that a region of Goα
, Goα
22–212, contains major structural determinants for binding to RGS proteins.
TNF‐α downregulates the peroxisome proliferator activated receptor‐α and the mRNAs encoding peroxisomal proteins in rat liver
Authors: Beier Konstantin , Völkl Alfred , Fahimi H.Dariush
Published: July 2002

We have studied the effects of TNF‐α on the mRNAs coding for the peroxisome proliferator activated receptor α (PPAR‐α), and for catalase (Cat), acyl‐CoA oxidase (AOX), multifunctional enzyme (PH), and β‐actin in rat liver. Total RNA was isolated from livers of male SD‐rats 16 h after administration of a single dose of 25 μg TNF‐α and mRNAs were analyzed by a novel dot blot RNase protection assay. The mRNAs for PPAR‐α and for Cat, AOX and PH were significantly reduced by TNF‐treatment. In addition, the level of PPAR‐α protein was also decreased after TNF. In contrast, the mRNA for β‐actin was markedly increased implying that the effect of TNF on PPAR‐α and the peroxisomal mRNAs is highly selective. This effect may have important implications in perturbation of the lipid metabolism induced by TNF‐α.