bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of Factors That Have Affected the Outcomes of Road Traffic Accidents on Lithuanian Roads
Authors: Leonavičienė Teresė , Pukalskas Saugirdas , Pumputis Vidmantas , Kulešienė Erika , Žuraulis Vidas
Published: December 2020

The purpose of this paper is to analyse the possibility for predicting the outcome of a road traffic accident concerning the traffic environment, personal traits of the traffic participant and the vehicle, i.e. aiming to answer the question whether specific values of the factors analysed to increase the likelihood of a fatal accident. The logistic regression model that allows identifying the relationship between the dependent and independent variables were used in the research. Other methods for describing and analysing categorical variables were also used alongside the logistic regression. When analysing the results, it was recognised that the odds ratio above 1 shows a higher likelihood for a representative of the category in question to be involved in a fatal accident compared to a representative of the base category. Odds ratios of likelihoods for calculation of the road traffic accident types show that the likelihood of a fatal accident is statistically significant affected by rollovers or driving into obstacles, compared to vehicular collisions. When summarising the results, it was stated that most of the factors researched have an impact on the outcome of a road traffic accident. The influence of some factors has a higher probability of resulting in a fatal accident as compared to other factors.
Usi comunitari e conservazione della natura nell’area protetta di Ndoinet (foresta Mau, Kenya): elementi di conflitto
Authors: Albertazzi Stefania
Published: December 2020

The article illustrates two elements of conflict found in the Ndoinet Forest area (Mau complex, Kenya): the first concerns the contrasting vision of the local communities and Kenya Forest Service (KFS) of degraded forest areas. The second concerns the conservation practices promoted by the forest service, specifically reforestation initiatives and the proposal to erect an electric fence. After illustrating the most common uses of the forest by the local population, I reflect on the conflicting elements that are created between these and the conservation measures by referring to “classic” and new themes of political ecology.
Experimental practice in the ruins of the Green Revolution: commoning with/in a water-scarce field
Authors: Autorino Pietro
Published: December 2020

This article considers water scarcity as an expression of agrarian crisis in South India. It explores how the drought affects the everyday life of communities in the area of Kadavur, limiting their possibilities to choose what crops they can grow, what food they may eat, and what futures they will cultivate. Moreover, it draws from the framework of experimental practice proposed by Papadopoulos in order to understand how more-than-social movements engage in material organizing to transform their condition. In the first part, it constructs the biofinancialization of organic millet, in relation to the making of cheap rice. Hence, it presents an ethnography of alterontological politics through enhanced care and tinkering with food. Moreover, the article explores the role of water management technology in the troubled ecology of the drought. Finally, by discussing an ethnography of borewell repair, it sheds light on the odd entanglements between a global technology and this situated ecology. This brings the fieldworker into the picture and considers the emergent politics of communing with/in the field.
L’ecologia politica latinoamericana dei movimenti indigeni in Ecuador: il caso della CONAIE
Authors: Bronzi Matteo
Published: December 2020

This work is focused on the analysis of the interconnections between Latin American political ecology and socio-environmental movements in Ecuador. Specifically, through the practices of CONAIE (Confederación de las Nacionalidades Indígenas del Ecuador), it will analyzed the importance of indigenous movements in the political dynamics of the Country and the socio-ecological theories and practices that the indigenous confederation has introduced into the national public debate.
Gentrification e urban gardening a Berlino. Riflessioni da Tempelhofer Feld e Prinzessinnengärten
Authors: Giovansana Sara , Zanolin Giacomo
Published: December 2020

Il contributo è dedicato al tema della creatività urbana e si concentra su due esperienze particolari nella città di Berlino. A partire da un’indagine svolta sul campo presso gli urban garden di Tempelhofer Feld e Prinzessinnengärten, questo lavoro ha l’obiettivo di riflettere sulle potenzialità di esperienze messe in atto dalle classi creative in relazione al tema della gentrification. Sulla base di quanto rilevato in tali avamposti della sostenibilità urbana, si cercherà, pertanto, di delineare un quadro generale dell’esperienza green berlinese, come uno dei segni del mutamento dell’urbanità in questa città.
Para una ecología política del agua: análisis de la periferia metropolitana de Río de Janeiro (Brasil)
Authors: Rocha André , de Oliveira Leandro
Published: December 2020

This paper presents a debate on water policy and ecology focuses on access to water and sewage network in the outskirts of the Metropolitan Area of Rio de Janeiro (Brazil). A theoretical and critical discussion is held based on political ecology and an overview of access to water and the sewage network is presented, according to data from the Brazilian Institute of Geography and Statistics. Qualitative analysis of the data shows that access is inefficient for the majority of the population in the periphery and indicates the need for further observation on the quality of water and sewage services. Another highlight is the need for greater reflection on the sector’s privatization, since water cannot be treated as a commodity, but as an essential right for the populations.
Il metodo del vivente. L’ecologia politica e la rielaborazione del discorso geografico
Authors: Torre Salvo
Published: December 2020

Political ecology debate reshaped the relationship between the two subjects assumed to be fundamental in human geography, man and nature, and criticised their scientific status. Feminist political ecology and decolonial debate adopted a different perspective on the subjectivities that inhabit human communities. Political ecology recent debate suggest that the role of geographical knowledge in the construction of social hierarchies has to be reconsidered. In this framework, nature as a political project, could be analysed restructuring the methods of geographical research. In the recent re-elaboration of post-Marxist political ecology and world-ecology, for instance the new methods of investigation are related to the study of internal contradictions and social conflicts. The article suggests to consider the fundamental contradiction between capital and living being as a principle of general analysis of the relationship between human communities and biomes and as the core of the construction of a method of geographical analysis.
How well can we separate genetics from the environment?
Authors: Blanc Jennifer , Berg Jeremy J
Published: December 2020

A simulation study demonstrates a better method for separating genetic effects from environmental effects in genome-wide association studies, but there is still some way to go before this becomes a "solved" problem.
Purification of a troponin I‐like factor from pig platelet
Authors: Terrossian E.der , Pruliere G. , d'Albis A.
Published: July 2002

A troponin I‐like factor has been purified from pig platelet by G150 Sephadex filtration of a low ionic strength extract, acidification at pH 4.2, ion exchange on DE‐52 cellulose, and affinity chromatography on calmodulin‐Sepharose. This protein (M
r 17000), together with pig brain calmodulin and platelet tropomyosin, is able to participate to the reconstitution in vitro of a thin filament‐like complex which modulates with 55% calcium sensitivity
1
the platelet actin‐activated Mg2+‐dependent ATPase activity of rabbit skeletal muscle myosin.
Effects of hypothyroidism on myosin isozyme transitions in developing rat muscle
Authors: Butler-Browne Gillian S. , Herlicoviez Danielle , Whalen Robert G.
Published: July 2002

Hypothyroidism was induced in young rats by methylthiouracil treatment of pregnant mothers from 18 days of gestation to 4 weeks after birth. Electrophoretic analysis of native myosin isozymes revealed a persistence of neonatal and embryonic myosin in developing fast and slow muscles up to at least 28 days after birth. The appearance of adult fast myosin was inhibited in 28‐day old animals, however adult slow myosin was found in the soleus muscle. Immunocytochemical results on the soleus demonstrate a cellular heterogeneity in the response to hypothyroidism. About half fibers have a normal complement of slow myosin and do not contain neonatal myosin. Only the remaining fibers contain the large amounts of neonatal myosin demonstrated by electrophoresis.
Atrial pronatriodilatin: a precursor for natriuretic factor and cardiodilatin
Authors: Lazure C. , Seidah N.G. , Chrétien M. , Thibault G. , Garcia R. , Cantin M. , Genest J.
Published: July 2002

Numerous peptides isolated from rat heart atria, including two containing 33 and 73 amino acids, were isolated and shown to exhibit natriuretic activities. Here, we describe the purification and partial amino acid sequence of a 106‐residue peptide containing the previously sequenced 33‐ and 73‐amino‐acid ANF peptides. The determined sequence is a novel one and is not significantly homologous to any known protein or segment thereof. In fact, this sequence shows significant homology only to another novel partial sequence obtained from sequence analysis of a porcine peptide, called cardiodilatin, also found in heart atria. This relationship is taken as evidence that ANF and cardiodilatin are part of the same precursor molecule which would contain at the very least 126 amino acids.
Decrease of platelet aggregation and spreading via inhibition of the cAMP phosphodiesterase by trapidil
Authors: Mazurov A.V. , Menshikov M.Yu. , Leytin V.L. , Tkachuk V.A. , Repin V.S.
Published: July 2002

Trapidil (N,N‐diethyl‐5‐methyl[l,2,4]triazolo[l,5‐α]pyrimidine‐7‐amine) inhibits platelet spreading and aggregation induced by arachidonic acid (AA), a stable analogue of prostaglandin (PG) endoperoxides (U46619), ADP, and low concentrations of thrombin, but not by A23187 and high concentrations of thrombin. Trapidil does not affect platelet adenylate cyclase but inhibits the cAMP PDE by approx. 50%. PDE inhibition proceeds via a competitive mechanism (K
i = 0.52 mM) and is not mediated by calmodulin inhibition. Trapidil does not change the platelet basal cAMP level but potentiates an increase of cAMP induced by the stable prostacyclin analogue (6β‐PGIi). These results suggest that trapidil antiplatelet effects may be due to the inhibition of platelet PDE.
(—)‐[3H]Desmethoxyverapamil, a novel Ca2+ channel probe
Authors: Goll A. , Ferry D.R. , Striessnig J. , Schober M. , Glossmann H.
Published: July 2002

(—)‐[3H]Desmethoxyverapamil (2,7‐dimethyl‐3‐(3,4‐dimethoxyphenyl)‐3‐cyan‐7‐aza‐9‐(3‐methoxyphenyl)‐nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (—)‐[3H]desmethoxyverapamil co‐purified with t‐tubule membrane markers in an established subcellular fractionation procedure. (—)‐[3H]Desmethoxyverapamil bound to partially purified t‐tubule membranes with a K
D of 2.2 ± 0.1 nM and a B
maxof 18 ± 4 membrane protein at 25°C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non‐competitive fashion by the benzoihiazepine Ca2+ antagonist d‐cis‐diltiazem and the 1,4‐dihydropyridine Ca2+ antagonist (+)‐PN 200‐110. Target size analysis of the (—)‐[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 ± 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d‐cis‐[3H]diltiazem, was 130.5 ± 4 kDa (p <0.01) and of the 1,4‐dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (—)‐[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine‐selective receptor site of the t‐tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites.
A novel radioiodinated high affinity ligand for the D2‐dopamine receptor
Authors: Amlaiky Nourdine , Kilpatrick Brian F. , Caron Marc G.
Published: July 2002

A novel high affinity dopaminergic ligand, N‐(p‐aminophenethyl)spiroperidol, has been synthesized and radioiodinated to a specific radioactivity of 2175 . Binding of this ligand to bovine anterior pituitary membranes is: (i) rapid (40–60 min to equilibrium at 25°C) and reversible t

= 1 h at 25°C); (ii) saturable and of high affinity (K
D ~ 20 pM) and (iii) displays a typical D2‐dopaminergic specificity. The ligand, which identifies the same number of receptor sites as other tritiated antagonist ligands, can be used in different tissues and preparations to delineate the characteristics of the D2 receptor. Thus, this high affinity, high specific radioactivity ligand (N‐(p‐amino‐m‐[125I]iodophenethyl)spiroperidol) represents a tool which until now had not been available for the characterization of the D2‐dopamine receptor.
Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex
Authors: Racz K. , Kuchel O. , Cantin M. , De Léan A.
Published: July 2002

We have previously determined that atrial natriuretic factor (ANF) is a potent inhibitor of steroid secretion in cultured bovine zona glomerulosa and fasciculata cells. The present report describes a comparison of the effect produced by ANF on aldosterone, deoxycorticosterone and progesterone secretions by zona glomerulosa cells and on cortisol, corticosterone and progesterone secretions by zona fasciculata cells. The equipotent inhibitory action of ANF on the stimulated secretion of these steroids in both cell types indicates a common site of action prior to progesterone synthesis at which ANF inhibits the steroidogenic pathway.
Activation of phosphatidylinositol synthesis by different agonists in a primary culture of smooth muscle cells grown on collagen microcarriers
Authors: Berta Philippe , Sladeczek Fritz , Travo Pierre , Bockaert Joël , Haiech Jacques
Published: July 2002

Regulation of inositol phosphate synthesis was examined in a primary culture of vascular smooth muscle cells grown on collagen‐coated microcarriers. In the presence of Lid (10 mM), four agonists [serotonin, angiotensin, (arginine) vasopressin and noradrenaline] were found to stimulate the formation of inositol phosphates in a dose‐dependent manner. All agonists were found to have identical and additive effects on the time course of inositol phosphate formation. Therefore, our primary cell culture technique was proved to give smooth muscle cells suitable for the study of modulation of phosphoinositide metabolism in response to physiological effectors.
Calcium transients in single adrenal chromaffin cells detected with aequorin
Authors: Cobbold Peter H. , Cheek Timothy R. , Roy Cuthbertson K.S. , Burgoyne Robert D.
Published: July 2002

The effect of 55 mM K+ and nicotine on intracellular free calcium was monitored in bovine adrenal chromaffin cells microinjected with aequorin. In contrast to results with quin 2, which suggested that stimulation of chromaffin cells resulted in sustained rises in free calcium, aequorin measurements showed that 55 mM K+ and nicotine resulted in a transient (60–90 s) elevation of free calcium. The peak free calcium and duration of the transient elicited by nicotine were dose‐dependent. The concentration of nicotine (10 μM) giving a maximal secretory response gave a peak rise in free calcium of up to 1 μM. 55 mM K+ which only releases 30% of the catecholamine released by 10 μM nicotine generated a calcium transient indistinguishable from that due to 10 μM nicotine. These results support the idea that nicotinic agonists generate an alternative second messenger in addition to the rise in free calcium.
Pertussis toxin inhibits the angiotensin II and serotonin‐induced rise of free cytoplasmic calcium in cultured smooth muscle cells from rat aorta
Authors: Bruns Christian , Marmé Dieter
Published: July 2002

Angiotensin II, serotonin and K+‐depolarization cause an increase in free cytoplasmic Ca2+ in cultured smooth muscle cells. The involvement of a guanine nucleotide‐binding protein has been investigated by using pertussis toxin. When smooth muscle cells were pretreated with pertussis toxin angiotensin II and serotonin‐induced rise of cytosolic Ca2+ was found to be significantly reduced whereas the Ca2+ influx mediated by K+‐depolarization remained unchanged. These results suggest the participation of a guanine nucleotide‐binding protein in the receptor‐mediated rise of intracellular Ca2+.
Application of the small‐angle X‐ray scattering technique for the study of equilibrium enzyme‐substrate interactions of phenylalanyl‐tRNA synthetase from E. coli with tRNAPhe
Authors: Tuzikov F.V. , Zinoviev V.V. , Vavilin V.I. , Maligin E.G. , Ankilova V.N. , Moor N.A. , Lavrik O.I.
Published: July 2002

The small‐angle X‐ray scattering technique (SAXS) is proposed for the investigation of equilibrium macromolecular interactions of the enzyme‐substrate type in solution. Experimental procedures and methods of analysing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium and structural parameters of the enzyme‐substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of tRNAPhe with phenylalanyl‐tRNA synthetase have been determined and demonstrate negative cooperativity for binding of the two tRNAPhe molecules. The structural parameters (R
g, R
c, semi‐axes) have been determined for free phenylalanyl‐tRNA synthetase and tRNAPhe from E. coli MRE‐600 and of enzyme complexes possessing one and two tRNAPhe molecules, indicating structural rearrangements of the enzyme in the interaction with tRNAPhe.
Brain natriuretic factor Augmentation of cellular cyclic GMP, activation of particulate guanylate cyclase and receptor binding
Authors: Song Dong-Li , Kohse Klaus P. , Murad Ferid
Published: July 2002

The newly discovered peptide, brain natriuretic factor (BNF), caused a concentration‐dependent increase (up to 400‐fold) in intracellular cyclic GMP levels in cultured endothelial, smooth muscle and fibroblast cells. The extent of cGMP augmentation was comparable to that produced by atrial natriuretic factor (ANF). The activity of the membrane‐bound guanylate cyclase of different rat tissues and cultured cells was markedly stimulated by the peptide and the addition of ATP potentiated the stimulation. As opposed to tissue particulate guanylate cyclase, the enzyme in cell membranes was slightly more sensitive to activation by BNF than to stimulation by ANF. On bovine aortic smooth muscle (BASM) cells, specific high‐affinity binding sites (B
max=398 fmol/106 cells, K
d=0.52 nM) for BNF were observed for which ANF could compete with apparently equal affinity. These results suggest that activation of the cGMP pathways constitutes a common mechanism of action for both BNF and ANF.
Synthesis of the vasoconstrictor peptide endothelin in kidney cells
Authors: Kosaka Takuo , Suzuki Nobuhiro , Matsumoto Hirokazu , Itoh Yasuaki , Yasuhara Tadashi , Onda Haruo , Fujino Masahiko
Published: July 2002

The expression plasmid containing human prepro‐endothelin cDNA was constructed and introduced into COS‐7 cells. Mature endothelin, consisting of 21 amino acid residues, was secreted into the culture medium of the transfected cells and was also synthesized by non‐transfected COS‐7 cells. Normal kidney cells derived from other species also synthesized and secreted endothelins. Partial characterization of endothelins produced by kidney cells suggested the existence of new types of endothelin. This is the first report of the vasoconstrictor peptide endothelin being synthesized in kidney cells.
Y1 and Y2 receptors for neuropeptide Y
Authors: Sheikh Søren P. , Håkanson Rolf , Schwartz Thue W.
Published: July 2002

By using monoiodinated radioligands of both intact neuropeptide Y (NPY) and of a long C‐terminal fragment, NPY13–36, two subtypes of binding sites, which differ in affinity and specificity, have been characterized. The Y1 type of binding site, characterized on a human neuroblastoma cell line, MC‐IXC, and a rat pheochromocytoma cell line, PC‐12, binds NPY with a dissociation constant (K
d) of a few nanomolar but does not bind NPY13–36. The Y2 type of binding site, characterized on porcine hippocampal membranes and on another human neuroblastoma cell line, SMS‐MSN, is of higher affinity and binds both NPY and NPY13–36. None of the binding sites distinguish between NPY and the homologous peptide YY (PYY). It is concluded that NPY/PYY‐binding sites occur in two subtypes which may represent two types of physiological receptors.
GTP interacts through its ribose and phosphate moieties with different subunits of the eukaryotic initiation factor eIF‐2
Authors: Bommer Ulrich-Axel , Kurzchalia Teymuras V.
Published: July 2002

We have previously shown that a GTP derivative bearing p‐azidoaniline at the γ‐phosphate group specifically labels the γ‐subunit of eukaryotic initiation factor eIF‐2. In the present study a new GTP derivative carrying the photoreactive group at the ribose moiety of GTP was applied for affinity labeling of eIF‐2 in different initiation complexes. Using this GTP analogue the β‐subunit of eIF‐2 was found to be specifically labeled in all complexes investigated. It is concluded that GTP interacts with both the β‐ and γ‐subunit of eIF‐2: the guanosine moiety is in contact with the β‐subunit and the γ‐phosphate group with the γ‐subunit.
Evidence for the existence of a cardiac specific isoform of the α1 subunit of the voltage dependent calcium channel
Authors: Slish Donald F. , Engle Dorothy B. , Varadi Gyula , Lotan Ilana , Singer Datna , Dascal Nathan , Schwartz Arnold
Published: July 2002

Biochemical, pharmacological and electrophysiological evidence implies the existence of tissue specific isoforms of the L‐type VDCC. The α1 and α2 subunits of the skeletal muscle calcium channel have been previously cloned and their amino acid sequence deduced. Here we report the isolation and sequencing of a partial cDNA that encodes a heart specific isoform of the α1 subunit. The amino acid sequence deduced from this part cDNA clone shows 64.7% similarity with the skeletal muscle α1 subunit. Northern analysis reveals 2 hybridizing bands, 8.5 and 13 kb, in contrast to one 6.5 kb band in the skeletal muscle. Selective inhibition of mRNA expression in Xenopus oocytes by complementary oligodeoxynucleotides derived from the heart clone provides further evidence that the cDNA corresponds to an essential component of the VDCC. These data further support the existence of tissue‐specific isoforms of the L‐type VDCC.
The activity of the mouse renin promoter in cells that do not normally produce renin is dependent upon the presence of a functional enhancer
Authors: Ekker Marc , Sola Christophe , Rougeon François
Published: July 2002

The expression of a hybrid gene containing the promoter region of the mouse Ren‐1 or Ren‐2 genes and the chloramphenicol acetyl transferase (CAT) gene coding region was analysed in five cell lines that do not normally express the renin gene. The renin promoter is inactive in each of these cell lines unless the SV40 enhancer is also present in the construct. In the latter case, transcription initiates at the normal renin start site. This is in contrast to the situation observed in lymphoid cells where the renin promoter is inactive even when coupled to a functional enhancer [(1987) EMBO J. 6, 1685–1690]. Furthermore, 5′‐flanking sequences of Ren‐2, placed upstream of the thymidine kinase or SV40 early region promoters do not alter the activity of these promoters in three different cell lines. The results suggest that, except for B cells, the renin promoter is not tissue‐specific but that its lack of activity in cells that do not express the gene is not the result of repression but may be due to the absence in these cells of one or several trans‐acting positive factors.
Potassium channels from NG108‐15 neuroblastoma‐glioma hybrid cells
Authors: Yokoyama Shigeru , Imoto Keiji , Kawamura Tetsuro , Higashida Haruhiro , Iwabe Naoyuki , Miyata Takashi , Numa Shosaku
Published: July 2002

The complete amino acid sequences of two potassium channel proteins from NG108‐15 neuroblastoma‐glioma hybrid cells have been deduced by cloning and sequencing the cDNAs. One of these proteins (NGK2) is structurally more closely related to the Drosophila Shaw gene product than to the Shaker and Shab gene products, whereas the other (NGK1) is identical with a rat brain potassium channel protein (BK2) which is more closely related to the Drosophila Shaker gene product. mRNAs derived from both the cloned cDNAs, when injected into Xenopus oocytes, direct the formation of functional potassium channels with properties of delayed rectifiers.
Ca2+‐dependent formation of an L‐arginine‐derived activator of soluble guanylyl cyclase in bovine lung
Authors: Mayer Bernd , Böhme Eycke
Published: July 2002

Arginine; Ca2+; Guanylyl cyclase; NADPH
Diphosphorylation of myosin light chain in smooth muscle cells in culture
Authors: Sasaki Yasuharu , Seto Minoni , Komatsu Ken-Ichi
Published: July 2002

Prostaglandin (PG) F2α (30 μM) stimulated both monophosphorylation and diphosphorylation of myosin light chain (MLC) in a smooth muscle cell line (SM‐3). The diphosphorylation was significantly decreased by treatment with the protein kinase C inhibitor staurosporine (30 nM, 30 min) from 20.1% of total MLC to 4.5%. The protein kinase C down‐regulation treatment of SM‐3 cells with phorbol dibutyrate suppressed to 8.7% the MLC diphosphorylation activity in the SM‐3 cells. This down‐regulation treatment had little effect on the monophosphorylation. We propose that the MLC diphosphorylation in PGF2α‐stimulated SM‐3 cells in culture may be regulated through mechanisms sensitive to protein kinase C.
Long term culture of rat soleus muscle in vitro
Authors: Stace Pamela B. , Marchington David R. , Kerbey Alan L. , Randle Philip J.
Published: July 2002

Rat soleus muscle strips cultured for 24 h in medium 199 were well preserved in terms of electron microscopy; ATP and creatine phosphate concentrations; rates of glucose utilization, glycogen and protein synthesis, and effects of insulin thereon. Culture led to modest changes in fluid spaces and intracellular (K+); increased basal glucose utilization up to two‐fold; had no effect on the maximum response to insulin; and had no effect on sensitivity to insulin except in the presence of adenosine deaminase. Thus in vitro neither denervation nor absence of insulin had any marked effects in 24 h to decrease responses to insulin.
Alteration of the enzymatic properties of smooth muscle myosin by a monoclonal antibody against subfragment 2
Authors: Higashihara Masaaki , Ikebe Mitsuo
Published: July 2002

A monoclonal antibody against subfragment 2 (S‐2) of smooth muscle myosin, designated MM‐9, was generated and characterized. MM‐9 potently inhibited subfragment 1 (S‐1) release by papain proteolysis of myosin, suggesting that the epitope of MM‐9 is at or very close to the S‐1/S‐2 junction. The depression of Ca2+ ‐ and Mg2+‐ATPase activities of myosin at low ionic strength was significantly reduced by MM‐9. MM‐9 increased the acto dephosphorylated HMM ATPase activity about 3‐fold. On the other hand, the antibody had no effect on the KCl‐dependence of viscosity of monomeric myosin. These results suggest that the folding of the myosin rod is not the direct determinant of enzymatic activity, and that the subtle conformational change at the S‐1/S‐2 junction (head‐neck region) plays a critical role in determining enzymatic activities.
Endothelin‐induced intracellular Ca2+ mobilization through its specific receptors in murine peritoneal macrophages
Authors: Kishino Junji , Hanasaki Kohji , Kato Toshiyuki , Arita Hitoshi
Published: July 2002

We studied the presence of specific binding sites for endothelin (ET) and the effect of ET on cytosolic free Ca2+ concentration ([Ca2+]i) in murine thiogylcolate‐activated peritoneal macrophages. Scatchard analysis for binding experiments using [125I]ET‐1 or [125I]ET‐3 revealed the existence of a single class of binding sites. The binding parameters (K??? and B???) for [125I]ET‐1 were almost identical to those for [125I]ET‐3. In addition, unlabeled 3 ET isopeptides (ET‐1, ET‐2 and ET‐3) inhibited the specific binding of both ET‐1 and ET‐3 with similar inhibitory potencies. All 3 ET isopeptides caused an increase in [Ca2+]i in the same dose‐dependent manner (0.01–100nM). These results demonstrate the existence of an ET receptor with the same affinity for all isoforms that mediates the ET‐induced intracellular Ca2+ mobilization in murine peritoneal macrophages.
Translation enhancing properties of the 5′‐leader of potato virus X genomic RNA
Authors: Zelenina D.A. , Kulaeva O.I. , Smirnyagina E.V. , Solovyev A.G. , Miroshnichenko N.A. , Fedorkin O.N. , Rodionova N.P. , Morozov S.Yu. , Atabekov J.O.
Published: July 2002

The double‐stranded DNA copy corresponding to the 5′‐nontranslated αβ‐leader of potato virus X (PVX) genomic RNA (positions −3 to −85 according to AUG initiator) was chemically synthesized and fused to the transcription plasmids containing three different reporter genes: neomycinphosphotransferase type II (NPT II) gene, Bacillus thuringiensis coleopteran‐specific toxic protein gene and β‐glucuronidase (GUS) gene. Expression of the reporter genes in vitro and in plant protoplasts (in the case of GUS gene) reveals that the αβ‐leader of PVX RNA acts as a translation enhancer despite the presence of the upstream vector‐derived sequence and irrespective of the length of the spacer sequence preceding the reporter genes.
Physical and chemical characterization of the oligomerization state of the Aeromonas hydrophila lipase/acyltransferase
Authors: Ausio Juan , van der Goot F.Gisou , Buckley J.Thomas
Published: July 2002

Aeromonas glycerophospholipid:cholesterol acyl transferase undergoes a conformational transition upon activation by treatment with trypsin. Chemical cross‐linking and sedimentation velocity analysis showed that the lipase dimerizes due to removal of a region near its C‐terminus. The lipase monomer has a sedimentation coefficient s
20,w = 2.83 S, whereas the dimer has s
20,w = 3.65 ± 0.22 S. Hydrodynamic analysis using these sedimentation values and the masses determined by mass spectrometry indicated that the monomers are aligned side‐by‐side in the dimer. An important change occurs in the apparent partial specific volume of the molecule upon activation.
Chloride‐depletion of photosynthetic water oxidase
Authors: Lübbers Karin , Drevenstedt Wolfgang , Junge Wolfgang
Published: July 2002

Chloride depletion blocks the normal four‐step progress of photosynthetic water oxidation. We studied proton release in chloride‐depleted thylakoids which were dark‐adapted and excited by flashing light. Proton release was blocked from the second flash on, possibly leaving an uncompensated positive charge in the catalytic centre. The reduction of P+
680 by Tyrz was still very rapid (⪡ 10 μs). From the third flash on, P+
680 was reduced more slowly (), and by an electrogenic back‐reaction. The uncompensated positive charge may be the reason why the rapid reduction of P+
680 by Tyrz is prevented and the transmembrane charge‐pair recombination is facilitated.
GTP analogs suppress uptake but not transport of d‐glucose analogs in Glut1 glucose transporter‐expressing Xenopus oocytes
Authors: Wellner Maren , Mueckler Mike M. , Keller Konrad
Published: July 2002

A Xenopus oocyte expression‐co‐injection system was used to study the influence of guanine nucleotides on D‐glucose uptake. GTP analogs like GTPγS and GppNHp had no effect on 3‐O‐methylglucose transport determined by zero‐trans uptake or equilibrium exchange, but suppressed 2‐deoxyglucose uptake into Glutl glucose transporter‐expressing oocytes by up to 86%. Both GTP analogs showed concentration dependence of their effectiveness, with GTPγS being more potent than GppNHp. No statistically significant differences were observed between groups of oocytes co‐injected with water or GDPβS (250 and 500 μM intracellular concentration). Glut1 transporter expression in plasma membrane was not different between water or GTPγS‐co‐injected oocytes. Thus, inhibition of hexokinase catalytic activity is the most likely causative factor for down‐regulation of 2‐deoxyglucose uptake.
Primary structure of crayfish visual pigment deduced from cDNA
Authors: Hariyama Takahiko , Ozaki Koichi , Tokunaga Fumio , Tsukahara Yasuo
Published: July 2002

The primary structure of opsin of the crayfish Procambarus clarkii has been deduced from the cDNA sequence. The opsin is composed of 376 amino acid residues including all the conservative residues characteristic of other members of the rhodopsin family. Comparison of sequences of all known opsins reveals that the major Drosophila rhodopsin is more similar to the crayfish rhodopsin than to the Drosophila UV‐sensitive pigments. The phylogenetic trees of invertebrate opsins are constructed.
A molecular basis for gating mode transitions in human skeletal muscle Na+ channels
Authors: Bennett Paul B. , Makita Naomasa , George Alfred L.
Published: July 2002

Recombinant sodium channel α subunits expressed in Xenopus oocytes display an anomalously slow rate of inactivation that arises from channels that predominantly exist in a slow gating mode [1,2]. Co‐expression of Na− channel β1 subunit with the human skeletal muscle Na+ channel α subunit increases the Na+ current and induces normal gating behavior in Xenopus laevis oocytes. The effects of the β1 subunit can be explained by an allosterically induced conformational switch of the α subunit protein that occurs upon binding the β1 subunit. This binding alters the free energy barriers separating distinct conformational states of the channel. The results illustrate a fundamental modulation of ion channel gating at the molecular level, and specifically demonstrate the importance of the β1 subunit for gating mode changes of Na+ channels.
GRP78 induction by cyclosporin A in human HeLa cells
Authors: Paslaru Liliana , Pinto Moïse , Morange Michel
Published: July 2002

Immunosuppressive drugs such as cyclosporin A (CsA) and FK506 are known to have pleiotropic effects on cells. Here we demonstrate that treatment of HeLa cells with low concentrations of CsA (but not of FK506) induces the synthesis of a stress protein, GRP78, located inside the endoplasmic reticulum. High concentrations of CsA lead to a general decrease in protein synthesis. When cells are stressed (heat‐shocked) during the CsA treatment, the synthesis of heat shock proteins is reinforced. FK506 has no detectable effects at any concentration. The mechanism of induction of GRP78 by CsA remains presently unknown. Whatever the mechanism involved, GRP78 overexpression might be responsible for some of the physiological effects of CsA.
Molecular cloning and evolutional analysis of a mammalian homologue of the Distal‐less 3 (Dlx‐3) homeobox gene
Authors: Shirasawa Takuji , Sakamoto Kyoichi , Tkahashi Hiroshi
Published: July 2002

We have cloned and sequenced a cDNA encoding the mammalian homologue of Distal‐less 3 (Dlx‐3) from a rat embryonic brain cDNA library. The primary structure of rat Dlx‐3 showed a cDNA with an open reading frame of 290 amino acids with a molecular weight of 31 kDa harboring a homeodomain sequence characteristic of the Distal‐less homoeobox gene. In addition to the highly conserved homeodomain sequence, we newly found the consensus motifs in the N‐terminal and C‐terminal region, which were specifically conserved among Distal‐less members of different species. Phylogenetic analysis of the Distal‐less homeodomain sequence also showed a possible association between the sequence similarities in the homeodomain and the spatial specifications of homeobox genes expressed in the developing central nervous system.
A delayed rectifier potassium channel cloned from bovine adrenal medulla Functional analysis after expression in Xenopus oocytes and in a neuroblastoma cell line
Authors: García-Guzmán Miguel , Sala Francisco , Criado Manuel , Sala Salvador
Published: July 2002

Using a cDNA library from bovine adrenal medulla, and, subsequently, a bovine genomic library, we have isolated the gene coding for a non inactivating potassium channel. This gene encodes a 597‐amino acid protein which we have called BAK5 as its sequence is very similar to members of Kv1.5 potassium channel family. Neuroblastoma cells (Neuro‐2a cell line) were stably transfected with BAK5 DNA. Protein expression was under the control of a heat‐shock promoter. Transfected cells showed a current highly selective for potassium, insensitive to tetraethylammonium but reversibly blocked by 4‐aminopyridine. Oocytes injected with BAK5 mRNA also expressed a potassium current with the same characteristics.
Modification of isolated subunit c of the F1F0‐ATPase from Propionigenium modestum by dicyclohexylcarbodiimide
Authors: Kluge Claudia , Dimroth Peter
Published: July 2002

Subunit c of the F1F0‐ATPase from Propionigenium modestum was extracted from the particulate cell fraction with chloroform/methanol. The protein was further purified by carboxymethyl cellulose chromatography and anion exchange HPLC in the organic solvent. SDS‐PAGE of the purified protein indicated a single stained protein band migrating as expected for the c‐subunit. Incubation of isolated subunit c in chlorform/methanol or aqueous buffer containing dodecyl‐β‐d‐maltoside with [14C]dicyclohexylcarbodiimide (DCCD) resulted in the incorporation of radioactivity into the protein. The rate of this reaction depended on the external pH; it was significantly faster in the more acidic than in the alkaline pH range. In the presence of Na+ subunit c was partially protected from labeling with [14C]DCCD at pH 6.1 and at pH 7.5, whereas no protection was evident at pH 5.5. At pH 7.5, the rate of subunit c labeling by [14C]DCCD in the presence of 20 mM NaCl was about 50% lower than in the absence of Na+ ions. The isolated c‐subunit therefore apparently retains in part the Na+ binding site which, when occupied, diminishes the reactivity of the protein towards DCCD.
Molecular determinants of external barium block in Shaker potassium channels
Authors: Hurst Raymond S. , Toro Ligia , Stefani Enrico
Published: July 2002

Mutations in the outer pore region of Shaker K+ channels (T449 and D447) can influence external Ba2+ block. Substitution of T449 by A, V or Y differentially reduced Ba2+ block primarily by decreasing the blocking rate. Substitution of D447 by N resulted in a non‐conducting channel with apparently normal gating currents. External Ba2+ can speed the OFF gating current of a different non‐conducting mutant, W434F; this effect was markedly attenuated by the D447N substitution. These results suggest that D447 contributes to an external Ba2+ binding site while T449 imposes a barrier to the access of that site.
Fatty acids bind to the fungal elicitor cryptogein and compete with sterols
Authors: Osman H. , Mikes V. , Milat M.-L. , Ponchet M. , Marion D. , Prangé T. , Maume B.F. , Vauthrin S. , Blein J.-P.
Published: July 2002

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid–cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.
S100A1 modulates skeletal muscle contraction by desensitizing calcium activation of isometric tension, stiffness and ATPase
Authors: Adhikari Bishow B , Wang Kuan
Published: July 2002

S100, a subfamily of the EF‐hand type calcium sensing proteins, is implicated in many cellular functions including muscle contractility. Two isoforms, S100A1 and S100B, at 2–10 μM significantly inhibit active tension, stiffness and ATPase of skinned single rabbit psoas muscle fibers at sub‐maximal (pCa ∼6.1‐5.6), but not at maximal levels of activation (pCa 4.0). S100A1 is a more potent inhibitor than S100B. Hill analysis of the ATPase–pCa and tension–pCa curves indicates that these proteins reduce calcium sensitivity and enhance the cooperativity toward calcium. We propose S100A1, and perhaps S100B, are viable candidates as physiological modulators of muscle contraction.
Cloning and functional expression of the murine homologue of proteinase 3: implications for the design of murine models of vasculitis
Authors: Jenne Dieter E. , Fröhlich Leopold , Hummel Amber M. , Specks Ulrich
Published: July 2002

Anti‐neutrophil cytoplasmic autoantibodies recognizing conformational epitopes (c‐ANCA) of proteinase 3 (PR3) from azurophil granules are a diagnostic hallmark in Wegener's granulomatosis (WG). Because a functional PR3 homologue has not been identified in rodents, it is difficult to assess immunopathological responses in rats or mice immunized with patients' derived c‐ANCA or human PR3. Here we report the full length cDNA cloning and functional expression of murine PR3 in HMC‐1 cells. Recombinant murine PR3 shows highly similar substrate specificities towards synthetic peptides and is inhibited by human α1‐proteinase inhibitor like human PR3. However, neither human c‐ANCA, rabbit sera nor mouse monoclonal antibodies to human PR3 recognize the murine homologue. Consequently, it is unlikely that disease observed in mice after immunization with c‐ANCA or human PR3 is caused by pathogenic antibodies directed against mouse PR3. Recombinant human‐mouse chimaeric variants will be a valuable new tool to localize the disease‐specific immunodominant epitopes in human PR3.
Functional expression of the murine D2, D3 and D4 dopamine receptors in Xenopus laevis oocytes
Authors: Skaaning Jensen Bo , Levavi-Sivan Berta , Fishburn C.Simone , Fuchs Sara
Published: July 2002

The different murine D2‐type dopamine receptors (D2L, D2S, D3L, D3S, and D4) were expressed in Xenopus laevis oocytes. The D2‐type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [125I]sulpride. They were all shown to activate Cl− influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na+/K+/2Cl− cotransporter component of the Cl− influx from the total unidirectional Cl− influx. The D3L subtype was found to operate exclusively through the bumetanide‐insensitive Cl− influx whereas the other D2‐type receptors acted on the Na+/K+/2Cl− cotransporter as well. The pertussis toxin sensitivity of the receptor‐activated chloride influx via the Na+/K+/2Cl− cotransporter varied between the various D2‐type receptors showing that they may couple to different G proteins, and activate different second messenger systems.
Overexpression of Bcl‐XL prevents caspase‐3‐mediated activation of DNA fragmentation factor (DFF) produced by treatment with the photochemotherapeutic agent BPD‐MA
Authors: Granville David J , Jiang Huijun , An Mary T , Levy Julia G , McManus Bruce M , Hunt David W.C
Published: July 2002

Photodynamic therapy (PDT) is a clinically effective cancer treatment. For human promyelocytic leukemia HL‐60 cells, cleavage of pro‐caspase‐3 (CPP32/Yama/apopain) into its proteolytically active subunits rapidly follows the photodynamic treatment of these cells with cytotoxic levels of the photosensitizer benzoporphyrin derivative monoacid ring A and visible light. Cleavage of a recently identified cytosolic 45 kDa protein, DNA fragmentation factor (DFF), is required for endonuclease activation leading to DNA fragmentation. In the present study, DFF was rapidly processed following PDT. Overexpression of the anti‐apoptotic Bcl‐XL gene in HL‐60 cells prevented PDT‐induced caspase activation, DFF cleavage and DNA fragmentation. These results demonstrate for the first time an example of chemotherapeutic drug‐induced activation of DFF and its regulation by Bcl‐XL.
Two genes, COL4A3 and COL4A4 coding for the human α3(IV) and α4(IV) collagen chains are arranged head‐to‐head on chromosome 2q361
Authors: Momota Ryusuke , Sugimoto Manabu , Oohashi Toshitaka , Kigasawa Kazuteru , Yoshioka Hidekatsu , Ninomiya Yoshifumi
Published: July 2002

We first isolated and characterized genomic DNA fragments that cover the 5′ flanking sequences of COL4A3 and COL4A4 encoding the human basement membrane α3(IV) and α4(IV) collagen chains, respectively. Nucleotide sequence analysis indicated that the two genes are arranged head‐to‐head. To determine transcription start site for COL4A4 gene, we performed RACE and RNase protection assays, indicating that there are two alternative transcripts presumably derived from two different promoters. Interestingly, one transcription start site (from exon 1′) of COL4A4 is only 5 bp away from the reported transcription start site of COL4A3, whereas the other transcript (from exon 1) starts 373 nucleotides downstream from the first one, generating the two kinds of transcripts that differ in the 5′ UTR regions. Expression of these two transcripts appears tissue‐specific; exon 1 transcript was expressed predominantly in epithelial cells, while exon 1′ transcript showed rather ubiquitous and low expression. The nucleotide sequence of the promoter region is composed of dense CpG dinucleotides, GC boxes, CTC boxes and a CCAAT box but no TATA box. These results provide information to delineate the promoter activity for the tissue‐specific expression of the six type IV collagen genes and basement membrane assembly in different tissues and organs.
Folding and cell surface expression of the vasopressin V2 receptor: requirement of the intracellular C‐terminus
Authors: Oksche Alexander , Dehe Marcel , Schülein Ralf , Wiesner Burkhard , Rosenthal Walter
Published: July 2002

We characterized truncations of the human vasopressin V2 receptor to determine the role of the intracellular C‐terminus (comprising about 44 amino acids) in receptor function and cell surface expression. In contrast to the wild‐type receptor, the naturally occurring mutant R337X failed to confer specific [3H]AVP binding to transfected cells. In addition, no vasopressin‐sensitive adenylyl cyclase was detectable in membrane preparations of these cells. Laser scanning microscopy revealed that c‐myc epitope‐ or green fluorescent protein‐tagged R337X mutant receptors were retained within the endoplasmic reticulum. Increasing the number of C‐terminal residues (truncations after codons 348, 354 and 356) restored G protein coupling, but revealed a length‐dependent reduction of cell surface expression. Replacement of positively charged residues within the C‐terminus by glutamine residues also decreased cell surface expression. A chimeric V2 receptor with the C‐terminus replaced by that of the β2‐adrenergic receptor did not bind [3H]AVP and was retained within the cell. These data suggest that residues in the N‐terminal part of the C‐terminus are necessary for correct folding and that C‐terminal residues are important for efficient cell surface expression.