bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurophysiologic and clinical outcome following medial pectoral to long thoracic nerve transfer for scapular winging: A case report
Authors: Tomaino Matthew M.
Published: October 2002

The use of nerve transfers (neurotization) in the reconstruction of nerve palsy is not new, but its clinical efficacy is still largely based on reports of successful restoration of elbow flexion and shoulder abduction following brachial plexus avulsion. Although its potential application extends beyond the brachial plexus, little has been written about additional indications or associated postoperative outcomes. The case described in this report illustrates yet another indication for which neurotization may be a useful technique. Medial pectoral nerve transfer to the long thoracic nerve was performed via an 11‐cm sural nerve graft to treat scapular winging 4 months following nerve injury caused during axillary node dissection. Neurophysiologic and clinical outcome 18 months postoperatively revealed successful reinnervation of the serratus anterior muscle, decreased scapular winging, and symptomatic improvement from the patient's perspective. © 2002 Wiley‐Liss, Inc. MICROSURGERY 22:254–257 2002
Local heparin is superior to systemic heparin in preventing arterial thrombosis
Authors: Andresen Dorthe M. , Barker John H. , Hjortdal Vibeke E.
Published: October 2002

Thrombosis poses a significant problem in microvascular surgery, despite antithrombotic therapy. The purpose of the present study was to investigate whether a topical application of unfractionated heparin is equally efficient as a systemic bolus in avoiding thrombosis. A rat femoral artery model was used. Three different doses of systemic heparin (50, 100, and 200 U/kg) and one dose of locally administered heparin (100 U/ml) were evaluated and compared to a control group receiving isotonic saline. A thrombogenic injury, simulating poor microsurgical technique, was applied to the artery. The thrombus area was visualized by transillumination, and recorded for 60 min on video, for subsequent measurement. In addition, the level of activated partial thromboplastin time (APTT) and of anti‐activated clotting factor X (aXa) was determined. Local heparin significantly reduced thrombus size as compared to isotonic saline and systemic heparin (50 and 100 U/kg). High‐dose systemic heparin (200 U/kg) was equally potent, but local heparin had significantly less influence on the hemostatic parameters.© 2002 Wiley‐Liss, Inc. MICROSURGERY 22:265–272 2002
Effect of Timolol vs. a postural orthotic on hand tremor during microsurgery
Authors: Lubahn John D. , Dickson Brian G. , Cooney Timothy E.
Published: October 2002

The efficacy of beta blockers to control hand tremor remains equivocal. This study evaluated the effectiveness of Timolol®, as well as a postural orthotic, in reducing movement deviation during a laboratory exercise. Eleven volunteers completed three randomized trials involving administration of Timolol®, a placebo, or a postural orthotic. Drug administration was blinded. Each trial consisted of a simulated 2‐hr clinical procedure, punctuated by video recordings of the subject's hand motion during a standardized pattern of instrument movement. ECRL EMG activity and digital oxygen perfusion were also measured. Recordings were converted into estimates of linear variance at baseline and 1 and 2 hr. A repeated‐measures ANOVA, or the Freidman test, were used to assess significant differences. Movement deviation, EMG activity, and oxygen perfusion were unaffected by the duration of exercise or treatment (P ≥ 0.454). We conclude that none of the treatments accorded a significant benefit in allaying hand tremor. © 2002 Wiley‐Liss, Inc. MICROSURGERY 22: 273–276 2002
Mode I crack propagation in concrete under fatigue: microscopic observations and modelling
Authors: Toumi A. , Bascoul A.
Published: October 2002

In this study, three‐point bending tests were carried out on notched beams to investigate mode I crack propagation in plain concrete under fatigue. The first part of the study focused on microscopic observations of the crack growth features. Microscopic observations were made using the replica method associated with scanning electron microscopy (SEM). Observations of fatigue crack growth both on the surface and inside the specimens are presented as a comparison between the observed crack lengths and those estimated by the compliance calibration method. In the second part, a finite element model of mode I crack propagation under fatigue is presented. According to the cohesive crack concept, a cohesive force distribution on the crack at various loading stages is assumed, according to both the stress‐crack opening relation worked out by Hordijk (1991; Thesis, Technische Universiteit) and a new proposed relation with hysteresis loop. Finite element computation is used to evaluate the crack extension in the bending beams. Numerical predictions are discussed in comparison with experimental results. Copyright © 2002 John Wiley & Sons, Ltd.
A simplified analysis method for piled raft and pile group foundations with batter piles
Authors: Kitiyodom Pastsakorn , Matsumoto Tatsunori
Published: October 2002

A simplified method of numerical analysis has been developed to estimate the deformation and load distribution of piled raft foundations subjected to vertical, lateral, and moment loads, using a hybrid model in which the flexible raft is modelled as thin plates and the piles as elastic beams and the soil is treated as springs. Both the vertical and lateral resistances of the piles as well as the raft base are incorporated into the model. Pile–soil–pile, pile–soil–raft and raft–soil–raft interactions are taken into account based on Mindlin's solutions for both vertical and lateral forces. The validity of the proposed method is verified through comparisons with several existing methods for single piles, pile groups and piled rafts. Workable design charts are given for the estimation of the lateral displacement and the load distribution of piled rafts from the stiffnesses of the raft alone and the pile group alone. Additionally, parametric studies were carried out concerning batter pile foundations. It was found that the use of batter piles can efficiently improve the deformation characteristics of pile foundations subjected to lateral loads. Copyright © 2002 John Wiley & Sons, Ltd.
Implicit J2‐bounding surface plasticity using Prager's translation rule
Authors: Montáns Francisco J. , Borja Ronaldo I.
Published: October 2002

A bounding surface J2‐plasticity model that uses Prager's translation rule is presented. The model preserves Masing's rules and is developed from the same ideas as classical infinitesimal J2‐plasticity, resulting in the same formulation with the exception of the algorithm for the computation of the hardening function. Instead of utilizing a loading surface as in a previous formulation, hardening surfaces are introduced; the formulation is similar to that of multilayer plasticity using Prager's rule, presented in previous work. An implicit algorithm based on the radial return concept is used, and the consistent elastoplastic tangent is also developed in closed form. Examples illustrating anisotropic behaviour are presented and compared to that predicted by a multilayer J2‐plasticity model. The model is also applied to a soil dynamics problem to show the robustness of the algorithm and its applicability to complex loading. Copyright © 2002 John Wiley & Sons, Ltd.
Efficient mixed Timoshenko–Mindlin shell elements
Authors: Kulikov G. M. , Plotnikova S. V.
Published: October 2002

The precise representation of rigid body motions in the displacement patterns of curved Timoshenko–Mindlin (TM) shell elements is considered. This consideration requires the development of the strain–displacement relationships of the TM shell theory with regard to their consistency with the rigid body motions. For this purpose a refined TM theory of multilayered anisotropic shells is elaborated. The effects of transverse shear deformation and bending‐extension coupling are included. The fundamental unknowns consist of five displacements and eight strains of the face surfaces of the shell, and eight stress resultants. On the basis of this theory the simple and efficient mixed models are developed. The elemental arrays are derived using the Hu–Washizu mixed variational principle. Numerical results are presented to demonstrate the high accuracy and effectiveness of the developed 4‐node shell elements and to compare their performance with other finite elements reported in the literature. Copyright © 2002 John Wiley & Sons, Ltd.
Guaranteed‐quality triangular mesh generation for domains with curved boundaries
Authors: Boivin Charles , Ollivier‐Gooch Carl
Published: October 2002

Guaranteed‐quality unstructured meshing algorithms facilitate the development of automatic meshing tools. However, these algorithms require domains discretized using a set of linear segments, leading to numerical errors in domains with curved boundaries. We introduce an extension of Ruppert's Delaunay refinement algorithm to two‐dimensional domains with curved boundaries and prove that the same quality bounds apply with curved boundaries as with straight boundaries. We provide implementation details for two‐dimensional boundary patches such as lines, circular arcs, cubic parametric curves, and interpolated splines. We present guaranteed‐quality triangular meshes generated with curved boundaries, and propose solutions to some problems associated with the use of curved boundaries. Copyright © 2002 John Wiley & Sons, Ltd.
Transforming top‐down agricultural extension to a participatory system: a study of costs and prospective benefits in Egypt
Authors: Fleischer Gerd , Waibel Hermann , Walter‐Echols Gerd
Published: October 2002

This article challenges some previous assessments of agricultural extension relying on simple measures of costs per farmer trained. Taking the case of Egypt, five pilot projects that aim to transform the existing agricultural extension system to a participatory system are analysed as regards their cost‐effectiveness and prospective cost–benefits. It is shown that the intensity and likely impact of participatory approaches among the different projects vary. Hence, it is insufficient to judge extension programmes by their cost‐effectiveness alone. In the case of cotton, for example, the costs per farmer trained are considerably lower than in horticultural crops but there are large differences in prospective benefits which would make investment in participatory extension in the latter more promising. The article calls for a more careful analysis of the costs of extension programmes in agricultural development and identifies four major cost categories, namely base costs, start‐up costs, recurrent and farmers' costs. The article also submits that in the context of the debate on privatization of agricultural extension there is a role to be played for the public sector in agriculture. The success of participatory approaches to extension will depend on the quality of services provided in connection with farmer training programmes. Copyright © 2002 John Wiley & Sons, Ltd.
The random development of LOH on chromosome 9q in superficial bladder cancers
Authors: van Tilborg Angela AG , de Vries Annie , de Bont Maarten , Groenfeld Lilian E , Zwarthoff Ellen C
Published: October 2002

Allelic loss on chromosome 9q is a very frequent event in bladder carcinogenesis. In recent years, efforts have been directed towards identifying the postulated tumour suppressor genes on this chromosome arm by deletion mapping and mutation analysis. However, no convincing candidate genes have been identified. This paper describes the development of chromosome 9q alterations in multiple recurrent superficial bladder cancers of ten patients and shows that loss of heterozygosity (LOH) on this chromosome is almost never the characteristic first step. The regions of loss are multiple and variable in different tumours from the same patient and expand in subsequent tumours. Moreover, the regions of loss vary from patient to patient. It is concluded that even if 9q harbours a bladder cancer gatekeeper gene, it is unlikely that the gene will be identified through LOH analysis alone. Copyright © 2002 John Wiley & Sons, Ltd.
A Markov model for sample size calculation and inference in vaccine cost‐effectiveness studies
Authors: Byrnes G. B.
Published: October 2002

Sample size and power formulae are derived for designing trials of vaccine cost‐effectiveness in healthy working adults. When existing trials of influenza vaccine have presented sample size calculations, these have been based on vaccine effectiveness. A Markov model is introduced to account for non‐independence of working days lost. Two effect sizes need to be specified, one for a reduction in the number of episodes of absence and the second for a reduction in the mean duration of episodes. The relative cost of vaccine provision is also incorporated. Applying the resulting formulae to published studies indicates that most were underpowered to detect any financial benefit. Moreover, biased estimates of variance have led to led to incorrect inference. Copyright © 2002 John Wiley & Sons, Ltd.
Multilevel modelling of medical data
Authors: Goldstein Harvey , Browne William , Rasbash Jon
Published: October 2002

This tutorial presents an overview of multilevel or hierarchical data modelling and its applications in medicine. A description of the basic model for nested data is given and it is shown how this can be extended to fit flexible models for repeated measures data and more complex structures involving cross‐classifications and multiple membership patterns within the software package MLwiN. A variety of response types are covered and both frequentist and Bayesian estimation methods are described. Copyright © 2002 John Wiley & Sons, Ltd.
Inhibition of the post‐translational processing of microvillar hydrolases is associated with a specific decreased expression of sucrase‐isomaltase and an increased turnover of glucose in Caco‐2 cells treated with monensin
Authors: Rousset Monique , Trugnan Germain , Brun Jean-Louis , Zweibaum Alain
Published: October 2002

The biosynthesis and post‐translational processing of sucrase‐isomaltase and dipeptidylpeptidase IV were studied by L‐[35S]methionine labeling, immunoisolation with monoclonal antibodies and SDS‐PAGE in post‐confluent Caco‐2 cells treated with monensin (10 μM, 48 h). In addition to its classical effect on the post‐translational processing of both hydrolases, i.e. an inhibition of the conversion of the high‐mannose to the complex glycosylated form of the enzymes, monensin was found to have two other effects: a marked decrease of sucrase‐isomaltase expression, but not of dipeptidylpeptidase IV; an increased turnover of glucose, as substantiated by increased rates of glucose consumption and lactic acid production and a decreased glycogen content. Whether these two effects are related to the particular differentiation and metabolic status of Caco‐2 cells is discussed, as well as a possible role for the drug‐induced modifications of glucose turnover on the decreased expression of sucrase‐isomaltase.
Effects of magnesium and sodium ions on the outer membrane permeability of cephalosporins in Escherichia coli
Authors: Yamaguchi Akihito , Yanai Misa , Tomiyama Noribumi , Sawai Tetsuo
Published: October 2002

Both Mg2+ and Na+ stimulated the outer membrane permeation of negatively charged cephalosporins in Escherichia coli without any significant alteration of the permeation of a zwitterionic cephalosporin. Such stimulation was not observed in an E. coli mutant lacking porins. The stimulation was caused by the direct interaction between the cations and the porin pores, which resulted in a decrease in cation selectivity of both the Omp F and Omp C porin pores.
Different interferon‐producing capacities of L929 cell sublines and the enhancement of interferon production by priming are controlled pretranslationally
Authors: Rosztóczy I. , Papós M. , Megyeri K.
Published: October 2002

Two sublines of mouse L929 cells designated L929B and L929M were studied. The L929B cells, which displayed a 2–3‐fold higher IFN production in response to Sendai virus than that of the L929M cells, had a higher sensitivity to the antiviral and priming effects of IFN and were more resistant to VSV. In good accord with the amount of IFN produced, more translatable IFN mRNA was isolated from the L929B cells. IFN production and IFN mRNA activities were proportionally increased in the IFN‐primed cultures of both sublines. Results indicate that both inherent and priming‐induced increased‐IFN production are based on pretranslational control mechanisms.
Regulation of protein synthesis in rabbit reticulocyte lysates
Authors: Singh Ranu Rajinder
Published: October 2002

The heme‐regulated protein kinase, which specifically phosphorylates the 38‐kDa subunit of initiation factor eiF‐2, can utilize adenosine 5′‐O‐(3‐thiotriphosphate) (ATP[γS]) as a substrate. The rate of thiophosphorylation is 5‐6‐times slower than that observed with ATP. It is of special interest that thiophosphorylated derivatives of eIF‐2 are resistant to dephosphorylation catalyzed by eIF‐2 phosphoprotein phosphatase. The thiophosphorylated eIF‐2 is less effective in promoting protein synthesis in hemin‐deficient lysates under physiological conditions. In addition, ATP[γS] could also be utilized by the self‐phosphorylation activity intrinsically associated with HRI.
Differential effects of noradrenaline and glucagon on lipolysis and fatty‐acid utilization in brown adipose tissue
Authors: Howland Roger J. , Benning Andrew D.
Published: October 2002

The relative effects of noradrenaline (300 pM‐3 μM) and glucagon (30 pM–300 nM) upon lipolysis and fatty acid utilization rates in brown adipose tissue from warm‐ (WA) and cold‐acclimated (CA) rats, were: (i) lipolytic sensitivity and responsiveness to the agonists were reduced in CA tissue; (ii) in CA tissue, at 300 pM, glucagon promoted fatty acid utilization more than noradrenaline; (iii) glucagon at 300 pM increased fatty acid utilization in WA tissue. The data suggest that glucagon has a physiological role in brown adipose tissue, modulating events subsequent to NA‐ and glucagon‐induced lipolysis, promoting fatty acid utilization.
Evidence for a high proton translocation stoichiometry of the H+‐ATPase complex in well coupled proteoliposomes reconstituted from a thermophilic cyanobacterium
Authors: van Walraven H.S. , Haak N.P. , Krab K. , Kraayenhof R.
Published: October 2002

Evidence is presented for a high proton translocation stoichiometry (H+/ATP) of approx. 9 in ATPase proteoliposomes with extremely low permeability for ions, reconstituted from a thermophilic cyanobacterium. A proportional relation between the phosphate potential (ΔG
fp) and the proton‐motive force (Δp) was observed in thermodynamic equilibrium. A bulk‐to‐bulk Δp was imposed by valinomycin‐induced K− diffusion potentials of different size while the initial ΔG
fp was varied. In all cases equilibrium was reached in about 1.5 h. A high H−/ATP ratio was also deduced from the relation between the initial rates of ATP synthesis or hydrolysis at varying ΔG
fp and Δp. The implications of these results for the mechanism of energy transduction in energy‐conserving membranes are discussed.
A temperature‐sensitive mutant of the yeast plasma membrane ATPase obtained by in vitro mutagenesis
Authors: Serrano Ramón , Montesinos Consuelo , Cid Angel
Published: October 2002

The yeast plasma membrane ATPase is a proton pump essential for cell growth. We have further analyzed the physiological role of the enzyme by constructing a temperature‐sensitive mutant. The cloned gene was treated with hydroxylamine and introduced into a haploid strain in which the constitutive promoter of the ATPase gene had been replaced by a galactose‐dependent promoter. One transformant exhibited thermosensitive growth on glucose but not on galactose. Under non‐permissive conditions the mutant is defective in proton efflux and amino acid uptake and it stops growing either unbudded or with an elongated bud. These results constitute the first genetic evidence for the chemiosmotic role of the enzyme suggested by biochemical and physiological studies.
Evidence that a nicked C4b, C4b′, is a functionally active C4b derivative
Authors: Yamazaki Mina , Ichihara Chikako , Nagasawa Shigeharu
Published: October 2002

Factor I‐catalyzed C4b cleavage is a regulatory reaction for the classical pathway of the complement system. Although the reaction was shown to be a two‐step reaction, (i) production of a nicked form of C4b, C4b′, as an intermediate cleavage product and (ii) subsequent splitting of C4b′ into C4c and C4d, it is not known which of the two steps represents the inactivation of the C4b function in the assembly of C3 convertase, C4b,2a. We have purified C4b′ and assessed the ability of C4b′ to assemble C3 convertase with C2 by utilizing size exclusion high performance liquid chromatography. Evidence was obtained demonstrating that C4b′ still retains the function of C4b to assemble C3 convertase. Thus, the substantial step for the inactivation of the C4b function appears to be the second cleavage reaction, that is, the cleavage of C4b′ into C4c and C4d.
(HX)n repeats: a pH‐controlled protein—protein interaction motif of eukaryotic transcription factors?
Authors: Janknecht Ralf , Sander Chris , Pongs Olaf
Published: October 2002

A characteristic sequence repeat of type His‐X, repeated several times in a row, is present in several eukaryotic transcription factors, e.g. HPHAHPHP in paired protein. Detailed molecular modelling and database searches lead to the suggestion that (HX)
n
repeats can mediate interaction between transcription factors in a pH‐controlled fashion.
A novel strategy for production of a highly expressed recombinant protein in an active form
Authors: Blackwell John R. , Horgan Roger
Published: October 2002

Under standard growth conditions, E. coli transformed with the high‐level expression vector pMON5525 produces recombinant DMAPP/AMP transferase in inactive, insoluble complexes. We have produced large amounts of active, soluble protein by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine. This caused an increase of up to 427‐fold in the active yield, and the disappearance of the protein from the pelletable fraction of cell extracts. This treatment may have wide applicability.
Relationship between DNA methylation and cell proliferation in Trypanosoma cruzi
Authors: Rojas M.Verónica , Galanti Norbel
Published: October 2002

5‐Azacytidine treatment of T. cruzi epimastigotes in culture induces active cell proliferation. This effect was detected as an increase in the cell number and [3H‐methyl]thymidine incorporation into DNA. 5‐Azacytidine does not alter other metabolic parameters. We have previously demonstrated that 5‐azacytidine induces DNA hypomethylation in T. cruzi. Accordingly, we suggest that this chemical modification may be related to the control of T. cruzi cell division.
Muramyl peptide‐binding sites are located inside target cells
Authors: Sumaroka M.V. , Litvinov I.S. , Khaidukov S.V. , Golovina T.N. , Kamraz M.V. , Komal'eva R.L. , Andronova T.M. , Makarov E.A. , Nesmeyanov V.A. , Ivanov V.T.
Published: October 2002

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide‐binding sites were shown to be located inside T‐lymphocytes, macrophages and neuroblastoma cells, but not inside B‐cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEH1‐3 cells with K
fd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D‐configuration of isoglutamine residue are important.
Uncoupling effect of fatty acids on heart muscle mitochondria and submitochondrial particles
Authors: Dedukhova V.I. , Mokhova E.N. , Skulachev V.P. , Starkov A.A. , Arrigoni-Martelli E. , Bobyleva V.A.
Published: October 2002

The effect of ATP/ADP‐antiporter inhibitors on palmitate‐induced uncoupling was studied in heart muscle mitochondria and inside‐out submitochondrial particles. In both systems palmitate is found to decrease the respiration‐generated membrane potential. In mitochondria, this effect is specifically abolished by carboxyatractylate (CAtr) a non‐penetrating inhibitor of antiporter. In submitochondrial particles, CAtr does not abolish the palmitate‐induced potential decrease. At the same time, bongkrekic acid, a penetrating inhibitor of the antiporter, suppresses the palmitate effect on the potential both in mitochondria and particles. Palmitoyl‐CoA which is known to inhibit the antiporter in mitochondria as well as in particles decreases the palmitate uncoupling efficiency in both these systems. These data are in agreement with the hypothesis that the ATP/ADP‐antiporter is involved in the action of free fatty acids as natural uncouplers of oxidative phosphorylation.
A C‐terminal proline is required for bioluminescence of the Ca2+‐binding photoprotein, aequorin
Authors: Nomura Midori , Inouye Satoshi , Ohmiya Yoshihiro , Tsuji Frederick I.
Published: October 2002

The requirement for a proline residue at the C‐terminus of the Ca2+‐binding photoprotein, aequorin, was investigated by measuring luminescence activities of a series of C‐terminal deletion mutants, substitution mutants and an addition mutant. CD spectral measurements of apoaequorin with the C‐terminal proline deleted showed a small change in secondary structure. In all cases studied, the C‐terminal proline was required for bioluminescence activity.
Interaction of a synthetic peptide of the interferon α‐2 C‐terminal part with human blood leukocytes
Authors: Danilkovitch A.V. , Kharitonenkov A.I. , Freze K.V. , Shevalier A.F. , Kolosova O.V. , Bulargina T.V. , Kirkin A.F. , Gusev M.V.
Published: October 2002

A biologically active synthetic peptide, 2438, representing the 124–138 amino acid sequence of the human interferon α‐2 (IFN α‐2) molecule, which is known to possess IFN‐like antiproliferative activity, specifically binds to human blood leukocytes. Scatchard plots reveal two different K
fd values, for the ‘low’ and ‘high’ affinity binding. The interaction of the 125I‐labelled peptide 2438 with the cells is not impaired by human IFN α‐2 or cholera toxin.
Promiscuity of GABAA‐receptor β3 subunits as demonstrated by their presence in α1, α2 and α3 subunit‐containing receptor subpopulations
Authors: Pollard Simon , Duggan Michael J. , Stephenson F.Anne
Published: October 2002

Polyclonal antibodies were raised in rabbits against the GABAA‐receptor β3 subunit peptide sequence, KQSMPREGHGRHMDR‐NH2 coupled to keyhole limpet haemocyanin. These anti‐β3 379–393 antibodies immunoprecipitated in a dose‐dependent manner specific benzodiazepine agonist binding sites from Na+ deoxycholate extracts of bovine cerebral cortex. In immunoblots, anti‐β3 379–393 antibodies recognised two species with M
fr 59 900 and M
fr 57 200 in all preparations tested, which included crude detergent‐solubilised, benzodiazepine affinity chromatography‐purified receptor, anti‐α1 324–341 antibody, anti‐Cys α2 414–424 antibody and anti‐Cys α3 454–467 antibody immunoaffinity‐purified GABAA‐receptor subpopulations. These results provide evidence for the ubiquity and promiscuity of the GABAA‐receptor β3 subunit.
Delayed activation of phospholipase D by gonadotropin‐releasing hormone in a clonal pituitary gonadotrope cell line (αT3‐1)
Authors: Netiv Einat , Liscovitch Mordechai , Naor Zvi
Published: October 2002

Stimulation of cultured pituitary cells from a gonadotrope lineage (αT3‐1) by the gonadotropin‐releasing hormone agonist analog [D‐Trp6]GnRH (GnRH‐A) resulted in a manifold increase in accumulation of phosphatidylethanol, a specific product of phospholipase D phosphatidyl transferase activity when ethanol is the phosphatidyl group acceptor. Levels of the natural lipid product of phospholipase D, phosphatidic acid, were increased 2–3‐fold. Activation of phospholipase D by GnRH‐A was dose‐ and time‐dependent and was blocked by a GnRH receptor antagonist [D‐pClPhe2,D‐Trp3.6]GnRH. GnRH‐A stimulated phospholipase D activity after a lag of 1–2 min. We conclude that in αT3‐1 gonadotropes GnRH receptor occupancy results in delayed activation of phospholipase D which could participate in late phases of gonadotrope regulation by the neurohormone.
Alternatively spliced mRNA of the pX region of human T lymphotropic virus type I proviral genome
Authors: Furukawa Keiko , Furukawa Koichi , Shiku Hiroshi
Published: October 2002

The pX region of human T lymphotropic virus type I (HTLV‐1) is believed to be expressed as a consequence of a 2 step splicing. It is conceivable, however, that a donor site of the 1st splicing and an acceptor site of the 2nd splicing results in the production of an alternatively spliced mRNA which is capable of coding p21X‐III. This possibility was examined by amplifying cDNA derived from HTLV‐I+ cells between the 5′ LTR and pX region. Bands of 2 different sizes were consistently observed. Sequencing of the longer band corresponded to a cDNA derived from a double‐spliced pX mRNA as previously reported. The shorter band was derived from a single‐spliced mRNA. HTLV‐I+ cell lines had both mRNAs to a varying degree. Expression of p40tax and p21X‐III seem to be well correlated with a double‐spliced and a single‐spliced mRNA, respectively.
Inhibition of protein tyrosine phosphatase activity by diamide is reversed by epidermal growth factor in fibroblasts
Authors: Monteiro Hugo P. , Ivaschenko Yuri , Fischer Roland , Stern Arnold
Published: October 2002

Diamide (azodicarboxylic acid bis(dimethylamide)) inhibits protein tyrosine phosphatase activity in fibroblasts without altering protein tyrosine kinase activity associated with the epidermal growth factor receptor. The loss of protein tyrosine phosphatase activity caused by diamide is reversed by 2‐mercaptoethanol or epidermal growth factor.
Interaction between heterologous receptor tyrosine kinases
Authors: Tartare Sophie , Ballotti Robert , Van Obberghen Emmanuel
Published: October 2002

To determine whether heterologous receptor tyrosine kinases interact with each other we have investigated the ability of insulin receptors to transphosphorylate and transactivate IGF‐I receptors. Using partially purified receptors we show that hormone‐stimulated insulin receptors induced a 40% increase in IGF‐I receptor phosphorylation. Remarkably, this transphosphorylation of IGF‐I receptors by insulin receptors resulted in a 2.5‐fold augmentation of the IGF‐I receptor tyrosine kinase activity for substrates. Our findings demonstrate that transphosphorylation with transactivation can occur between insulin and IGF‐I receptors. We would like to propose that such a phenomenon participates in the insulin‐induced pleiotropic program by mediating the growth promoting effects of the hormone.
Cleavage of translation initiation factor 4AI (eIF4AI) but not eIF4AII by foot‐and‐mouth disease virus 3C protease: identification of the eIF4AI cleavage site
Authors: Li Wei , Ross-Smith Natalie , Proud Christopher G. , Belsham Graham J.
Published: October 2002

The translation initiation factor eIF4A is cleaved within mammalian cells infected by foot‐and‐mouth disease virus (FMDV). The FMDV 3C protease cleaves eIF4AI (between residues E143 and V144), but not the closely related eIF4AII. Modification of eIF4AI, to produce a sequence identical to eIF4AII around the cleavage site, blocked proteolysis. Alignment of mammalian eIF4AI onto the three‐dimensional structure of yeast eIF4A located the scissile bond within an exposed, flexible portion of the molecule. The N‐ and C‐terminal cleavage products of eIF4AI generated by FMDV 3C dissociate. Cleavage of eIF4AI by FMDV 3C is thus expected to inactivate it.
Protein secondary structure: category assignment and predictability
Authors: Andersen Claus A , Bohr Henrik , Brunak Søren
Published: October 2002

In the last decade, the prediction of protein secondary structure has been optimized using essentially one and the same assignment scheme known as DSSP. We present here a different scheme, which is more predictable. This scheme predicts directly the hydrogen bonds, which stabilize the secondary structures. Single sequence prediction of the new three category assignment gives an overall prediction improvement of 3.1% and 5.1% compared to the DSSP assignment and schemes where the helix category consists of α‐helix and 310‐helix, respectively. These results were achieved using a standard feed‐forward neural network with one hidden layer on a data set identical to the one used in earlier work.
Over‐expression of FoxM1 stimulates cyclin B1 expression
Authors: Leung Tommy W.C. , Lin Selma S.W. , Tsang Anthony C.C. , Tong Cindy S.W. , Ching Johannes C.Y. , Leung Wai Ying , Gimlich Robert , Wong Gordon G. , Yao Kwok-Ming
Published: October 2002

FoxM1 (previously named WIN, HFH‐11 or Trident) is a Forkhead box (Fox) transcription factor widely expressed in proliferating cells. Various findings, including a recent analysis of FoxM1 knockout mice, suggest that FoxM1 is required for normal S–M coupling during cell cycle progression. To study the regulatory role of FoxM1 and its downstream regulatory targets, three stably transfected HeLa lines that display doxycycline (dox)‐inducible FoxM1 expression were established. Over‐expression of FoxM1 by dox induction facilitates growth recovery from serum starvation. Quantitation of cyclin B1 and D1 levels using flow cytometric, Western and Northern analyses reveals that elevated FoxM1 levels lead to stimulation of cyclin B1 but not cyclin D1 expression. Transient reporter assays in the dox‐inducible lines and upon co‐transfection with a constitutive FoxM1 expression plasmid suggest that FoxM1 can activate the cyclin B1 promoter.
Role of Flk‐1 in mouse hematopoietic stem cells
Authors: Haruta Hirotaka , Nagata Yuka , Todokoro Kazuo
Published: October 2002

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34+KDR+ cell fraction. We found that expression levels of Flk‐1, a mouse homologue of KDR, were low or undetectable in mouse Lin−c‐Kit+Sca‐1+CD34low/− cells as well as Hoechst33342− cells (side population), which have long‐term reconstitution capacity. Furthermore, neither Flk‐1+CD34low/− cells nor Flk‐1+CD34+ cells had long‐term reconstitution capacity in mouse. Taken together with other observations using Flk‐1‐deficient mice, these results indicate that Flk‐1 is essential for the development of hematopoietic stem cells in embryo but not for the function of hematopoietic stem cells in adult mouse bone marrow.
A non‐natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe
Authors: Taki Masumi , Hohsaka Takahiro , Murakami Hiroshi , Taira Kazunari , Sisido Masahiko
Published: October 2002

A small and highly fluorescent non‐natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin. The positions were directed by a CGGG/CCCG four‐base codon/anticodon pair. The non‐natural mutants were obtained in excellent yields and some of them retained strong biotin‐binding activity. The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding. These unique properties indicate that the atnDap is the most suitable non‐natural amino acid for a position‐specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.
HIV‐1 reverse transcriptase and integrase enzymes physically interact and inhibit each other
Authors: Tasara Taurai , Maga Giovanni , Hottiger Michael O. , Hübscher Ulrich
Published: October 2002

Ordered molecular interactions and structural changes must take place within the human immunodeficiency virus type 1 (HIV‐1) preintegration complex at various stages for successful viral replication. We demonstrate both physical and biochemical interactions between HIV‐1 reverse transcriptase and integrase enzymes. This interaction may have implications on the in vivo functions of the two enzymes within the HIV‐1 replication complex. It may be one of the various molecular interactions, which facilitate efficient HIV‐1 replication within the target cells.
The protein phosphatase 2A B′‐regulatory subunit par1p is implicated in regulation of the S. pombe septation initiation network
Authors: Le Goff Xavier , Buvelot Stéphanie , Salimova Ekaterina , Guerry Frédéric , Schmidt Susanne , Cueille Nathalie , Cano Elena , Simanis Viesturs
Published: October 2002

In order to identify regulators of the Schizosaccharomyces pombe septation initiation network (SIN), which signals the onset of cell division, we have isolated extragenic suppressors of mutations in the GTPase spg1p, which is a central element in this pathway. One of these encodes the protein phosphatase 2A (PP2A) B′‐regulatory subunit par1p. Loss of par1p function rescues mutants in cdc11, cdc7, and spg1, but no other SIN mutants. Our data suggest that PP2A‐par1p acts as a negative regulator of SIN signalling.
Identification of a functional site on the type I TGF‐β receptor by mutational analysis of its ectodomain
Authors: Guimond Alain , Sulea Traian , Zwaagstra John C , Ekiel Irena , O'Connor-McCourt Maureen D
Published: October 2002

Six charged amino acid residues located in the ectodomain of the full‐length type I transforming growth factor (TGF)‐β receptor were individually mutated to alanine. Mutation of residues D47, D98, K102 and E104 resulted in functionally impaired receptors as demonstrated by a marked decrease in ligand‐dependent signaling and ligand internalization relative to the wild‐type receptor. The other two mutants (K39A and K87A) exhibited wild‐type‐like activity. Molecular modeling indicates that the four functionally important residues are located on the convex face of the ectodomain structure. Since mutation of these four residues affects signaling and ligand internalization but not ligand binding, we propose that this functional site is an interacting site between type I and II receptors.
A mutant P53 can activate apoptosis through a mechanism distinct from those induced by wild type P53
Authors: He Ming , Rennie Paul S , Dragowska Visia , Nelson Colleen C , Jia William
Published: October 2002

A common mutation in P53 protein occurs at amino acid residue 281 in the DNA binding domain (P53gly(281)), which results in loss of transcriptional regulation of P53 target genes and has been reported to gain pro‐oncogenic functions. In the present study, we investigated the activity of P53gly(281) in P53‐null PC3 human prostate cancer cells and found that the P53gly(281) induced apoptosis as efficiently as the wild‐type P53 (wtP53). However, in contrast to wtP53‐induced apoptosis, the P53gly(281)‐induced apoptosis was insensitive to overexpression of bcl‐2. Thus, our findings indicate that while a mutation in the DNA binding domain of p53 may result in a more oncogenic form of the protein, it may also paradoxically result in the ‘gain’ of a new, alternative pathway for apoptosis.
A novel striated tropomyosin incorporated into organized myofibrils of cardiomyocytes in cell and organ culture
Authors: Zajdel Robert W. , Sanger Jean M. , Denz Christopher R. , Lee Sung , Dube Syamalima , Poiesz Bernard J. , Sanger Joseph W. , Dube Dipak K.
Published: October 2002

Striated muscle tropomyosin is classically described as consisting of 10 exons, 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b, in both skeletal and cardiac muscle. A novel isoform found in embryonic axolotl heart maintains exon 9a/b of striated muscle but also has a smooth muscle exon 2a instead of exon 2b. Translation and subsequent incorporation into organized myofibrils, with both isoforms, was demonstrated with green fluorescent protein fusion protein construct. Mutant axolotl hearts lack sufficient tropomyosin in the ventricle and this smooth/straited chimeric tropomyosin was sufficient to replace the missing tropomyosin and form organized myofibrils.
Biochemical properties of the human REV1 protein
Authors: Masuda Yuji , Kamiya Kenji
Published: October 2002

It has been proposed that the REV1 protein plays an important role in the induced‐mutagenesis pathway. We show that purified REV1 protein inserts dCMP opposite template G, A, T and C, and dGMP and dTMP opposite template G in the presence of magnesium, while in the presence of manganese the specificity for dCMP was found to be relaxed and the REV1 protein acquired the ability to insert dCMP, dGMP, dAMP and dTMP opposite templates G, A, T, and C. Kinetic analysis provided evidence for high affinity for dCTP with template G, suggesting that the REV1 protein is specialized for dCTP and template G.
cIAP‐1, but not XIAP, is cleaved by caspases during the apoptosis induced by TGF‐β in fetal rat hepatocytes
Authors: Herrera Blanca , Fernández Margarita , Benito Manuel , Fabregat Isabel
Published: October 2002

We have studied the expression of XIAP, cIAP‐1 and cIAP‐2 in fetal rat hepatocytes and its possible regulation by pro‐apoptotic stimuli (transforming growth factor‐β (TGF‐β)) and survival signals (epidermal growth factor (EGF)). The three forms of inhibitor of apoptosis proteins (IAPs) are expressed in fetal hepatocytes and only cIAP‐1, but not XIAP or cIAP‐2, is cleaved during TGF‐β‐induced apoptosis. The pan‐caspase inhibitor Z‐VAD.fmk blocked this effect, which indicates that cIAP‐1 is a caspase substrate. EGF plays a dual role in the regulation of IAPs expression. On one hand, it increases cIAP‐1 and cIAP‐2 basal expression and, on the other hand, it blocks the cleavage of cIAP‐1 by caspases induced by TGF‐β.
A putative nuclear receptor coactivator (TMF/ARA160) associates with hbrm/hSNF2α and BRG‐1/hSNF2β and localizes in the Golgi apparatus
Authors: Mori Katsuhiro , Kato Hiroyuki
Published: October 2002

An ATP‐dependent chromatin remodeling factor, SNF/SWI complex, acts as a coactivator for numerous transcriptional factors. One of the best‐documented examples is nuclear receptors, although the molecular mechanism for this coactivation has not been sufficiently elucidated. Here we show that hbrm/hSNF2α and BRG‐1/hSNF2β, the ATPase subunits of the human SNF/SWI complexes, specifically associate in vitro and in vivo with TATA element modulatory factor (TMF)/ARA160, which has been described as a binding protein to and coactivator for the androgen receptor. This interaction requires highly conserved N‐terminal regions of hbrm/hSNF2α and BRG‐1/hSNF2β and a C‐terminal region of TMF/ARA160. Immunofluorescence and Western blot studies revealed that the TMF isoforms differentially localize in the Golgi apparatus and the nucleus.
Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF‐187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase IIα
Authors: Wessel Irene , Jensen Lars H. , Renodon-Corniere Axelle , Sorensen Tina K. , Nitiss John L. , Jensen Peter B. , Sehested Maxwell
Published: October 2002

Bisdioxopiperazine anti‐cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature‐conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug‐resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co‐transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.
Anti‐neovascular therapy by liposomal DPP‐CNDAC targeted to angiogenic vessels
Authors: Asai Tomohiro , Shimizu Kosuke , Kondo Masami , Kuromi Koichi , Watanabe Koh , Ogino Koichi , Taki Takao , Shuto Satoshi , Matsuda Akira , Oku Naoto
Published: October 2002

We previously reported that liposomalized 5′‐O‐dipalmitoylphosphatidyl 2′‐C‐cyano‐2′‐deoxy‐1‐β‐D‐arabino‐pentofuranosylcytosine (DPP‐CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, is quite useful for cancer therapy. On the other hand, for anti‐neovascular therapy, we recently isolated peptides homing to angiogenic vessels from a phage‐displayed random peptide library, and observed that peptide‐modified liposomal adriamycin strongly suppressed tumor growth, perhaps through damaging angiogenic endothelial cells. In the present study, we modified DPP‐CNDAC‐liposomes with one of the angiogenic homing peptides, APRPG, and examined their antitumor activity. Three doses of APRPG‐modified DPP‐CNDAC‐liposomes (15 mg/kg as CNDAC) strongly inhibited tumor growth compared with the same number of doses of unmodified DPP‐CNDAC‐liposomes. The life span was increased 31.8%, with one completely cured mouse out of the six mice treated. Since the accumulation of liposomes in the tumor tissue was not so much different between APRPG‐liposomes and non‐modified liposomes, the enhanced therapeutic efficacy may be explained as the alteration of targets, i.e. APRPG‐modified DPP‐CNDAC‐liposomes caused tumor growth suppression through damage of angiogenic endothelial cells. Anti‐neovascular therapy promises no drug resistance, and should be effective against essentially any kind of solid tumor; and thus the present results demonstrate another benefit of the therapy, namely, high efficacy of cancer treatment.
Telomere maintenance in telomerase‐positive human ovarian SKOV‐3 cells cannot be retarded by complete inhibition of telomerase
Authors: Gan Yuebo , Mo Yiqun , Johnston Jeffrey , Lu Jie , Wientjes M.Guillaume , Au Jessie L.-S
Published: October 2002

The two known mechanisms for telomere maintenance in eukaryocytes are telomerase in telomerase‐positive cells and alternative lengthening of telomeres (ALT) in telomerase‐negative cells. We report here that telomere maintenance in the telomerase‐positive human ovarian SKOV‐3 cells was not affected by inhibition of telomerase. For comparison, the effect of telomerase inhibitors on telomere maintenance in another telomerase‐positive cell line (i.e. human pharynx FaDu cells) and the telomerase‐negative human osteosarcoma Saos‐2 cells was examined. Telomerase activity was measured using a modified telomeric repeat amplification protocol and telomere length was measured using a solution hybridization‐based method and fluorescence in situ hybridization. A reverse transcriptase inhibitor (3′‐azido‐deoxythymidine or AZT) and an antisense against a component of human telomerase RNA (antisense hTR) were used to inhibit telomerase. FaDu and SKOV‐3 cells showed comparable baseline telomerase activity. Telomerase activity in both cells was inhibited about equally by AZT (maximal inhibition of ∼80%) and by expression of antisense hTR (complete inhibition in SKOV‐3 cells and maximal inhibition of ∼80% in FaDu cells). However, treatment with telomerase inhibitors resulted in ∼50% telomere shortening in FaDu cells but had no effect on SKOV‐3 nor Saos‐2 cells. SKOV‐3 cells did not show the characteristic features of ALT (i.e. heterogeneous telomere length and promyelocytic leukemia bodies), whereas these ALT features were observed in Saos‐2 cells. Collectively, these results suggest the existence of a telomerase‐independent mechanism of telomere maintenance in the telomerase‐positive SKOV‐3 cells.