bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site‐directed mutagenesis of ornithine site residues
Authors: Rochera Lourdes , Fresquet Vicente , Rubio Vicente , Cervera Javier
Published: October 2002

The role of residues of the ornithine activator site is probed by mutagenesis in Escherichia coli carbamoyl phosphate synthetase (CPS). Mutations E783A, E783L, E892A and E892L abolish ornithine binding, E783D and T1042V decrease 2–3 orders of magnitude and E892D decreased 10‐fold apparent affinity for ornithine. None of the mutations inactivates CPS. E783 mutations hamper carbamate phosphorylation and increase K+ and MgATP requirements, possibly by perturbing the K+‐loop near the carbamate phosphorylation site. Mutation E892A activates the enzyme similarly to ornithine, possibly by altering the position of K891 at the opening of the tunnel that delivers the carbamate to its phosphorylation site. T1042V also influences modulation by IMP and UMP, supporting signal transmission from the nucleotide effector to the ornithine site mediated by a hydrogen bond network involving T1042. Ornithine activation of CPS may be mediated by K+‐loop and tunnel gating changes.
p21‐activated protein kinase γ‐PAK in pituitary secretory granules phosphorylates prolactin
Authors: Tuazon Polygena T , Lorenson Mary Y , Walker Ameae M , Traugh Jolinda A
Published: October 2002

p21‐activated protein kinase γ‐PAK phosphorylates prolactin (PRL) in rat pituitary secretory granules on Ser‐177 and on the equivalent site, Ser‐179, in recombinant human PRL. This is shown by comparison of phosphopeptide maps with the human PRL mutant S179D. γ‐PAK is present in rat and bovine granules as identified by in‐gel phosphorylation of histone H4, and by immunoblotting. Thus, phosphorylation of PRL by γ‐PAK in granules produces the PRL molecule that has been shown to antagonize the growth‐promoting activity of unmodified PRL, and is consistent with the identified role of γ‐PAK in the induction and maintenance of cytostasis.
Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens‐mediated transient expression and in transgenic tobacco plant
Authors: Merle C , Perret S , Lacour T , Jonval V , Hudaverdian S , Garrone R , Ruggiero F , Theisen M
Published: October 2002

Potential contamination of animal‐derived collagen with pathogens has led to the demand for safe recombinant sources of this complex molecule. In continuation of our previous work [Ruggiero et al. (2000) FEBS Lett. 469, 132–136], here we show that it is possible to produce recombinant hydroxylated homotrimeric collagen in tobacco plants that are co‐transformed with a human type I collagen and a chimeric proline‐4‐hydroxylase (P4H). This is to our knowledge the first time that transient expression in tobacco was used to improve the quality of a recombinant protein produced in plants through co‐expression with an animal cell‐derived modifying enzyme. We demonstrated the functionality of the new chimeric P4H and thus improved the thermal stability of recombinant collagen I from plants to 37°C.
Reactive oxygen species inhibit the succinate oxidation‐supported generation of membrane potential in wheat mitochondria
Authors: Pastore Donato , Laus Maura Nicoletta , Di Fonzo Natale , Passarella Salvatore
Published: October 2002

In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (ΔΨ) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate‐dependent, but not the NADH‐dependent, ΔΨ generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N‐ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, ΔΨ generation and consequent large‐scale ROS production.
Involvement of tumor necrosis factor α, rather than interleukin‐1α/β or nitric oxides in the heme oxygenase‐1 gene expression by lipopolysaccharide in the mouse liver
Authors: Oguro Takiko , Takahashi Yuko , Ashino Takashi , Takaki Atsushi , Shioda Seiji , Horai Reiko , Asano Masahide , Sekikawa Kenji , Iwakura Yoichiro , Yoshida Takemi
Published: October 2002

Heme oxygenase‐1 (HO‐1) is induced under various oxidative stress conditions, such as lipopolysaccharide (LPS) insult. Induction of HO‐1 by LPS is reported to be mediated through interleukin‐1β (IL‐1β), rather than other inflammatory cytokines in the mouse liver. However, we found that IL‐1α/β knockout (KO) mice responded well to LPS insult, as did wild‐type mice with respect to HO‐1 mRNA induction (about 30‐fold increase). In contrast, tumor necrosis factor α KO (TNFαKO) mice responded very weakly to LPS in the HO‐1 mRNA expression, but not metallothionein mRNA. Recent studies reveal that nitric oxide from Kupffer cells is involved in HO‐1 induction in the liver produced by LPS. Therefore, nitrite and nitrate concentrations in the liver were also measured and these parameters did not increase in either IL‐1KO or TNFαKO. In addition, the phosphorylation of c‐JUN N‐terminal kinase (JNK) and p38, but not extracellular signal‐regulated kinase, was very low in TNFαKO mice due to LPS administration. All of these findings indicate that TNFα is a major candidate to trigger HO‐1 induction in response to LPS stimulation, and that its message is likely transduced through JNK and p38 pathways.
Evolutionary relationship between the bacterial HPr kinase and the ubiquitous PEP‐carboxykinase: expanding the P‐loop nucleotidyl transferase superfamily
Authors: Russell Robert B. , Márquez José Antonio , Hengstenberg Wolfgang , Scheffzek Klaus
Published: October 2002

Similarities between protein three‐dimensional structures can reveal evolutionary and functional relationships not apparent from sequence comparison alone. Here we report such a similarity between the metabolic enzymes histidine phosphocarrier protein kinase (HPrK) and phosphoenolpyruvate carboxykinase (PCK), suggesting that they are evolutionarily related. Current structure classifications place PCK and other P‐loop containing nucleotidyl‐transferases into different folds. Our comparison of both HPrK and PCK to other P‐loop containing proteins reveals that all share a common structural motif consisting of an αβ segment containing the P‐loop flanked by an additional β‐strand that is adjacent in space, but far apart along the sequence. Analysis also shows that HPrK/PCK differ from other P‐loop containing structures no more than they differ from each other. We thus suggest that HPrK and PCK should be classified with other P‐loop containing proteins, and that all probably share a common ancestor that probably contained a simple P‐loop motif with different protein segments being added or lost over the course of evolution. We used the structure‐based sequence alignment containing residues specific to HPrK/PCK to identify additional members of this P‐loop containing family.
The YbgC protein encoded by the ybgC gene of the tol‐pal gene cluster of Haemophilus influenzae catalyzes acyl‐coenzyme A thioester hydrolysis
Authors: Zhuang Zhihao , Song Feng , Martin Brian M , Dunaway-Mariano Debra
Published: October 2002

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol‐pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4‐hydroxybenzoyl‐coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl‐CoA and acyl‐N‐acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl‐CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not.
A divergent archaeal member of the alkaline phosphatase binuclear metalloenzyme superfamily has phosphoglycerate mutase activity
Authors: Graham David E. , Xu Huimin , White Robert H.
Published: October 2002

The hyperthermophilic archaeon Methanococcus jannaschii uses several non‐canonical enzymes to catalyze conserved reactions in glycolysis and gluconeogenesis. A highly diverged gene from that organism has been proposed to function as a phosphoglycerate mutase. Like the canonical cofactor‐independent phosphoglycerate mutase and other members of the binuclear metalloenzyme superfamily, this M. jannaschii protein has conserved nucleophilic serine and metal‐binding residues. Yet the substrate‐binding residues are not conserved. We show that the genes at M. jannaschii loci MJ0010 and MJ1612 encode thermostable enzymes with phosphoglycerate mutase activity. Phylogenetic analyses suggest that this gene family arose before the divergence of the archaeal lineage.
A conserved Asn in TM7 of the thyrotropin receptor is a common requirement for activation by both mutations and its natural agonist
Authors: Claeysen Sylvie , Govaerts Cédric , Lefort Anne , Van Sande Jacqueline , Costagliola Sabine , Pardo Leonardo , Vassart Gilbert
Published: October 2002

The wide spectrum of naturally occurring mutations able to activate the thyrotropin (TSH) receptor provides a useful tool to approach the structure of the active state(s) of the glycoprotein hormone receptors. Here we show that the side‐chain of the highly conserved N7.49 (Asn 674) in TM7 is mandatory for activation of the TSH receptor, not only by TSH, but also by a panel of eight natural and two artificial activating mutations. Basal activity levels of the mutants were significantly decreased by suppression of the side‐chain of N7.49 (N7.49A double mutants). In addition, comparative effects of the N7.49A substitution on the ten mutants demonstrate that basal activity and agonist‐ or mutation‐stimulated activity might involve different structural changes.
A cell‐free protein synthesis system as an investigational tool for the translation stop processes
Authors: Kang Taek Jin , Woo Ji Hyoung , Song Hui Kyoung , Ahn Jin Ho , Kum Jae Wook , Han Jin , Choi Cha Yong , Joo Hyun
Published: October 2002

Using Escherichia coli cell‐free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S‐(2‐nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS–PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell‐free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.
Histone H3 phosphorylation during Xenopus oocyte maturation: regulation by the MAP kinase/p90Rsk pathway and uncoupling from DNA condensation
Authors: Schmitt Anja , Gutierrez Gustavo J , Lénárt Péter , Ellenberg Jan , Nebreda Angel R
Published: October 2002

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine‐10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP‐dependent protein kinase that blocks entry into M phase, also leads to massive serine‐10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen‐activated protein kinase/p90Rsk pathway. Our results indicate that in G2‐arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.
Identification of residues important for NAD+binding by theThermotoga maritimaα‐glucosidase AglA, a member of glycoside hydrolase family 4
Authors: Raasch Carsten , Armbrecht Martin , Streit Wolfgang , Höcker Birte , Sträter Norbert , Liebl Wolfgang
Published: October 2002

The NAD+‐requiring enzymes of glycoside hydrolase family 4 (GHF4) contain a region with a conserved Gly‐XXX‐Gly‐Ser (GXGS) motif near their N‐termini that is reminiscent of the fingerprint region of the Rossmann fold, a conserved structural motif of classical nicotinamide nucleotide‐binding proteins. The function of this putative NAD+‐binding motif in the α‐glucosidase AglA ofThermotoga maritimawas probed by directed mutagenesis. TheKdfor NAD+of the AglA mutants G10A, G12A and S13A was increased by about 300‐, 5‐, and 9‐fold, respectively, while theirKmforp‐nitrophenyl‐α‐glucopyranoside was not seriously affected. The results indicate that the GXGS motif is indeed important for NAD+binding by the glycosidases of GHF4.
Bax expression protects yeast plasma membrane against ethanol‐induced permeabilization
Authors: Marza Esther , Camougrand Nadine , Manon Stéphen
Published: October 2002

The mechanism by which the expression of pro‐apoptotic protein Bax is able to kill yeast was investigated. Ethanol stress induces a permeabilization of the plasma membrane revealed by propidium iodide accumulation. Bax expression, although killing yeast cells, prevents this permeabilization. These effects are modulated by aeration, by manipulation of the unsaturation index of fatty acids and by addition of resveratrol, a known inhibitor of lipid oxidation. These data suggest that lipid oxidation is involved in Bax effects. Taken together, these data show for the first time a direct effect of Bax on plasma membrane permeability properties and suggest that yeast is a powerful tool for investigating the molecular mechanisms underlying this process.
Effect of protein disulfide isomerase on the rate‐determining steps of the folding of bovine pancreatic ribonuclease A
Authors: Shin Hang-Cheol , Song Myeong-Cheol , Scheraga Harold A
Published: October 2002

The effects of protein disulfide isomerase (PDI) on the four structured des species that accumulate in the rate‐determining steps of ribonuclease A folding were investigated at pH 8.0 and 15°C. The results indicate that PDI catalyzes the conversion of the kinetically trapped intermediates, des‐[26–84] and des‐[58–110], by reshuffling them into the on‐pathway intermediate, des‐[40–95], and the formation of native protein, by acting as both a chaperone and an oxidase on this on‐pathway intermediate. These results provide the first strong evidence for the mechanism of PDI in the rate‐determining steps of the oxidative folding pathways of ribonuclease A. Our approach, using PDI and blocked PDI, combined with the fast‐blocking 2‐aminoethyl methanethiosulfonate method, may be generally applicable to the clarification of the effect of PDI on folding intermediates.
ms1, a novel stress‐responsive, muscle‐specific gene that is up‐regulated in the early stages of pressure overload‐induced left ventricular hypertrophy
Authors: Mahadeva Harin , Brooks Gavin , Lodwick David , Chong Nelson W , Samani Nilesh J
Published: October 2002

We have identified and characterised a cDNA encoding a novel gene, designated myocyte stress 1 (ms1), that is up‐regulated within 1 h in the left ventricle following the application of pressure overload by aortic banding in the rat. The deduced ms1 protein of 317 amino acids contains several putative functional motifs, including a region that is evolutionarily conserved. Distribution analysis indicates that rat ms1 mRNA expression is predominantly expressed in striated muscle and progressively increases in the left ventricle from embryo to adulthood. These findings suggest that ms1 may be important in striated muscle biology and the development of pressure‐induced left ventricular hypertrophy.
Regulation of the Werner helicase through a direct interaction with a subunit of protein kinase A
Authors: Nguyen Duy T , Rovira Ilsa I , Finkel Toren
Published: October 2002

Werner syndrome is a hereditary disease characterized by cancer predisposition, genetic instability, and the premature appearance of features associated with normal aging. At the molecular level this syndrome has been related to mutations in the Werner helicase, a member of the RecQ family of DNA helicases which are required to maintain genomic stability in cells. Here we show by a yeast two‐hybrid screen that the Werner helicase can directly interact with the regulatory subunit (RIβ) of cAMP protein kinase A (PKA). We confirm that this interaction occurs in vivo. Interestingly, serum withdrawal causes a redistribution of the Werner helicase within the nucleus of mammalian cells. Raising intracellular cAMP levels or increased expression of the regulatory but not the catalytic subunit of PKA inhibits this nuclear redistribution stimulated by serum deprivation. These results suggest that similar to lower organisms, gene products linked to genomic instability and aging may be directly regulated by growth factor‐sensitive, PKA‐dependent pathways.
The intracellular tyrosine residues of the ATP‐gated P2X1 ion channel are essential for its function
Authors: Toth-Zsamboki Emese , Oury Cecile , Watanabe Hiroyuki , Nilius Bernd , Vermylen Jos , Hoylaerts Marc F
Published: October 2002

The four highly conserved intracellular tyrosine residues of the P2X1 ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non‐functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild‐type P2X1 activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X1 immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X1 endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three‐dimensional structure and function of the intracellular P2X1 domains.
RIO1, an extraordinary novel protein kinase
Authors: Angermayr Michaela , Bandlow Wolfhard
Published: October 2002

RIO1 and Rio‐related proteins display little similarity of primary sequence with conventional protein kinases. Based on secondary structure alignments, we show that it contains the domain structure (subdomains I–XI) and conserved secondary structure elements found in conventional protein kinases. We show that recombinant wild‐type Rio1p isolated from Escherichia coli displays kinase activity which depends on autophosphorylation and magnesium or manganese as ATP‐activating ions. An initial biochemical characterization of Rio1p is presented.
D‐Amino acid residue in the C‐type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus
Authors: Torres Allan M , Menz Ian , Alewood Paul F , Bansal Paramjit , Lahnstein Jelle , Gallagher Clifford H , Kuchel Philip W
Published: October 2002

The C‐type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D‐amino acid at position 2 in the primary structure. Peptides containing a D‐amino acid have been found in lower forms of organism, but this report is the first for a D‐amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L‐amino acid residue in the protein to the D‐configuration.
Stability for semilinear parabolic equations with decaying potentials in \( ℝ^{N} \) and dynamical approach to the existence of ground states
Authors: Souplet Philippe , Zhang Qi S.
Published: October 2002

Consider the elliptic problem

\tag{1}
\Delta u - V\left(x\right) + u^{p} = 0 \quad \text{in }ℝ^{n}

with

1 < p < \frac{n + 2}{n - 2}

,

n \geq 2

, and

0⩽V(x)\in L^∞

, which may decay to 0 at infinity. We prove that if

V

is radial and satisfies

\frac{a_{1}}{1 + \left|x\right|^{b}} \leq V\left(x\right)a_{2}
\quad \text{and} \quad
0 \leq \text{b} < \frac{\text{2}\left(\text{n} - \text{1}\right)\left(p - 1\right)}{p + 3},

then (1) admits a (ground state) positive solution. We do not use traditional variational methods and the result relies on the study of global solutions of the parabolic problem

\tag{2}
\Delta u - V\left(x\right)u + u^{p} - \partial _{t}u = 0 \quad \text{in }ℝ^{n} \times \left(0,\infty \right), \quad u\left(x,0\right) = u_{0}\left(x\right).

Indeed, we will show that, under suitable conditions on

V

(not necessarily radial), (2) admits global positive solutions and that when

V

and

u_0

are radial some global solutions have

ω

-limit sets containing a positive equilibrium. The method also covers nonlinearities more general than

u^p

, in which case the standard variational method may be hard to apply.

Nous considérons le problème elliptique

\tag{1}
\Delta u - V\left(x\right)u + u^{p} = 0 \quad \text{dans }ℝ^{n}

avec

1 < p < \frac{n + 2}{n - 2},n \geq 2

, et

0⩽V(x)\in L^∞

,

V(x)

pouvant tendre vers 0 à l'infini. Nous prouvons que si

V

est radial et satisfait

\frac{a_{1}}{1 + \left|x\right|^{b}} \leq \left(x\right) \leq a_{2}
\quad \text{et} \quad
0 \leq \text{b} < \frac{\text{2}\left(\text{n} - \text{1}\right)\left(p - 1\right)}{p + 3},

alors (1) admet une solution positive (état fondamental). Nous n'utilisons pas les méthodes variationnelles traditionnelles : le résultat repose sur l'étude des solutions globales du problème parabolique

\tag{2}
\Delta u - V\left(x\right) + u^{p} - \partial _{t}u = 0
\quad \text{dans}\quad
ℝ^{n} \times \left(0,\infty \right), \quad u\left(x,0\right) = u_{0}\left(x\right).

En effet, nous montrons que, sous des conditions appropriées sur

V

(non nécessairement radial), (2) admet des solutions globales positives et que, lorsque

V

et

u_0

sont radiales, l'ensemble

ω

-limite de certaines solutions globales contient un état d'équilibre positif. La méthode s'applique également à des non-linéarités plus générales que

u^p

, pour lesquelles la méthode variationnelle classique pourrait être difficilement applicable.
Immature osteoblastic cells express the pro‐α2(XI) collagen gene during bone formation in vitro and in vivo
Authors: Urabe Ken , Jingushi Seiya , Ikenoue Takashi , Okazaki Ken , Sakai Hiroaki , Li Changyou , Iwamoto Yukihide
Published: October 2002

Type XI collagen is predominantly found in cartilage. However, expression of the pro‐α2(XI) collagen gene (COL11A2) has recently been detected in various non‐cartilaginous tissues. We identified the differentiation stage at which COL11A2 was expressed in cultured fetal rat calvarial (FRC) cells and in rat femoral fracture calluses in order to investigate the involvement of COL11A2 during bone formation in vitro and in vivo. We also studied the alternative splicing of exons 6–8 in FRC cells and fracture calluses. In FRC cells, mineralized nodules stained with von Kossa stain were observed from day 9 after confluence. COL11A2 was highly expressed on days 0 and 5, but the expression levels were rapidly decreased on day 9 by Northern blot analysis. During rat femoral fracture repair, intramembranous ossification proceeded and newly formed woven bone was observed on the cortex on day 7 after fracture. In situ hybridization showed that COL11A2 signals were detected in osteoblastic cells in the newly formed woven bone. According to the maturation and remodeling of the woven bone into the trabecular bone, the distribution of the signal for COL11A2 mRNA was limited to the superficial osteoblastic cells of the newly formed trabecular bone. These results demonstrated that COL11A2 was expressed in relatively immature osteoblastic cells during bone formation in vitro and in vivo. RT‐PCR showed that the shortest band corresponding to mRNA lacking exons 6–8 was clearly detected when using RNA from soft calluses. In contrast, the largest band corresponding to mRNA with exons 6–8 was predominant when using RNA from FRC cells or from hard calluses on days 7 and 14. These results indicate that the splicing pattern of exons 6–8 in osteoblastic cells is different from the pattern in chondrocytes. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Deformation and rupture of the articular surface under dynamic and static compression
Authors: Flachsmann R. , Broom N. D. , Hardy A. E.
Published: October 2002

Cartilage‐on‐bone samples were dynamically and statically compressed at various stress levels to determine the deformation and rupture behaviour of the articular surface (AS). Instantaneous deformations were captured photographically by using a transparent indenter in combination with a ultra high speed flash. Principal strains (PS) were evaluated using large deformation theory. The tensile strains induced indirectly in the AS were a function of the rate at which the direct compressive force was applied. At the same compressive stress the tensile strains induced statically were approximately twice those induced dynamically. Rupture of the AS occurred in about 60% of those specimens tested statically at 15 MPa and followed approximately the split‐line direction. By contrast, no rupture was observed dynamically even at stresses as high as 28 MPa. In terms of joint function the research demonstrates that the AS is considerably more resistant to rupture under dynamic than under static loading. The biomechanical parameter governing rupture appears to be the level of indirectly induced surface strain rather than the directly applied compressive stress. The very different mechanisms controlling the compressive deformation of articular cartilage (AC) at high vs low rates of loading clearly influence the levels of in‐plane strain induced in the AS. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
COX‐1 and COX‐2 expression in osteoid osteomas
Authors: Mungo David V. , Zhang Xinping , O'Keefe Regis J. , Rosier Randy N. , Puzas J. Edward , Schwarz Edward M.
Published: October 2002

Osteoid osteoma is a benign bone forming neoplasm that is characterized by its small size (less than 2 cm), self‐limited growth, and the tendency to cause extensive reactive changes in the adjacent tissue. The lesion classically presents with severe pain at night that is dramatically relieved by NSAIDs. The tumor has been shown to express very high levels of prostaglandins, particularly PGE2 and PG12. The high local levels of these prostaglandins are presumed to be the cause of the intense pain seen in patients with this lesion. One generally accepted form of treatment is the prolonged use of NSAIDs. Since the cyclooxygenases are thought to be the source of these prostaglandins, and the central target of NSAIDs, we evaluated the expression of cyclooxygenase‐1 (COX‐1) and cyclooxygenase‐2 (COX‐2) in osteoid osteoma tissues from patients following surgery. In the 12 specimens examined we found that the tumor osteoblasts had strong immunohistochemical staining for COX‐2, while the staining in the surrounding host osteoblasts in the reactive bone was scant. Significant COX‐1 staining was also detected in both tumor and host osteoblasts. For comparison we examined the COX expression in human fracture callus, fibrous dysplasia, osteoblastoma, osteofibrous dysplasia, and myositis ossificans. With the exception of fracture callus, very limited amounts of COX‐2 could be detected in these tissues. Taken together, we conclude that the increased production of prostaglandins by osteoid osteomas implicates that COX‐2 is one of the mediators of this condition. These findings suggest that the newly selective COX‐2 inhibitors could be used to more safely treat osteoid osteomas. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Calcium signaling is required for ultrasound‐stimulated aggrecan synthesis by rat chondrocytes
Authors: Parvizi Javad , Parpura Vladimir , Greenleaf James F. , Bolander Mark E.
Published: October 2002

Low‐intensity ultrasound accelerates fracture healing in humans. In rat femur fracture models, ultrasound advanced healing is associated with increased proteoglycan expression. Here we report that ultrasound stimulation of primary rat chondrocytes elevated the intracellular concentration of calciums [Ca2+]i. The [Ca2+]i increase was rapid and transient at lower pressures (175–320 kPa), but rapid and sustained at higher ultrasound exposures (350–500 kPa). Chelating internal [Ca2+]i with 1,2‐bis(2‐aminophenoxy) ethane‐N‐N‐N′‐N′‐tetraacetic acid (BAPTA‐AM), stopping the Ca2+/ATP‐ase induced mitochondrial release of [Ca2+]i with Thapsigargin, or removing [Ca2+]i from the medium with EGTA inhibited the stimulatory effects of ultrasound on proteoglycan synthesis. These results imply that ultrasound‐stimulated synthesis of cell matrix proteoglycan, associated with accelerated fracture healing, is mediated by intracellular calcium signaling. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Active range of motion of the head and cervical spine: a three‐dimensional investigation in healthy young adults
Authors: Ferrario Virgilio F. , Sforza Chiarella , Serrao Graziano , Grassi Gianpiero , Mossi Erio
Published: October 2002

Purpose. To define reference values for head–cervical range of motion (ROM) in healthy young adults, to assess the effect of sex, and to quantify the separate contribution of other body districts.

Methods. Thirty women and 30 men performed maximal head and cervical spine flexion–extension, lateral bending, and axial rotation. Movements were detected using a digital optoelectronic instrument. Maximum head–cervical spine and thoracic motions were separated.

Results. Flexion and extension were performed mainly in the sagittal plane. The movement was larger in women (136°) than in men (130°). During flexion, both sexes moved the head–neck and the thorax in the same direction. During extension, men moved only the head–cervical spine, while women moved the two analyzed districts in the opposite directions. Lateral bending was nearly symmetric, associated with head–cervical rotation and extension, and larger in women (91°) than in men (77°). Adjunctive thoracic motion was limited in the sagittal and frontal planes, but larger in the horizontal plane (opposite motions of about 20°). Head–neck rotation was symmetric, and associated with concomitant movements in both the sagittal and frontal planes. It was larger in women (162°) than in men (155°), and performed with limited adjunctive thoracic motions.

Conclusions. The present values can be used as a first group of normative data for head–cervical ROM in young men and women. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Stiff and strong compressive properties are associated with brittle post‐yield behavior in equine compact bone material
Authors: Les C. M. , Stover S. M. , Keyak J. H. , Taylor K. T. , Kaneps A. J.
Published: October 2002

Our hypothesis was that post‐yield mechanical behavior of compact bone material in compression, defined as the stress, strain, or energy absorbed between 0.2% strain‐offset and the point of maximum stress, is correlated with material density, modulus, strength, histomorphometric evidence of remodeling, and post‐failure gross specimen morphology. Post‐yield behavior of compact bone material from the third metacarpal bone of 10 horses, ages 5 months to 20 years, was investigated using single‐load compression‐to‐failure. The post‐yield stress, strain, and absorbed energy were compared with the compressive elastic modulus, yield stress, ash density, post‐failure macroscopic appearance of the specimen, and histologic evidence of remodeling. High values of elastic modulus, yield stress, and ash density were associated with low values of post‐yield mechanical properties (stress, strain, and absorbed energy).

Macroscopic post‐failure morphology was associated with post‐yield mechanical behavior, in that specimens displaying fractures were associated with lower post‐yield mechanical properties, and that those without evidence of frank fracture were associated with higher post‐yield mechanical properties. Microscopic evidence of remodeling activity was associated with high post‐yield mechanical properties, but not with gross post‐failure morphology. There was an abrupt change from relatively high values to extremely low values of post‐yield mechanical properties at intermediate levels of ash density. This feature may serve as a functional upper limit to the maximization of bone material stiffness and strength. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Macrophage exposure to particulate titanium induces phosphorylation of the protein tyrosine kinase lyn and the phospholipases Cγ‐1 and Cγ‐2
Authors: Palmbos Phillip L. , Sytsma Mark J. , Deheer David H. , Bonnema Joy D.
Published: October 2002

A frequent long‐term complication of total joint arthroplasty is aseptic loosening, the end result of wear debris production, synovial macrophage activation, inflammatory mediator release, and osteolysis about the implant–bone or cement–bone interface. To elucidate the mechanisms of particle‐induced macrophage activation and mediator production, we studied early signal transduction events using J774A.1 macrophages and 3 μm titanium particles. Treating macrophages with herbimycin A or genistein, two inhibitors of protein tyrosine kinases (PTKs), inhibited titanium phagocytosis as well as secretion of tumor necrosis factor‐α (TNF‐α) and prostaglandin‐E2 (PGE2) in a dose‐dependent manner. Both processes therefore depend on a PTK signaling cascade. Specifically, macrophage exposure to titanium‐induced phosphorylation of multiple proteins including the Src kinase Lyn and phospholipase Cγ‐1 and Cγ‐2. Phosphorylation peaked within 2 min and returned to baseline within 45 min. Similar but not identical phosphorylation patterns were obtained when cells were stimulated with titanium preincubated with serum or albumin, suggesting distinct signal transduction pathways dependent on particle coating. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Early intramedullary nailing in an animal model of a heavily contaminated fracture of the tibia
Authors: Hill P. F. , Clasper J. C. , Parker S. J. , Watkins P. E.
Published: October 2002

An ovine model was developed to study the outcome following intramedullary nailing of a heavily contaminated fracture. Animals in the control group received no treatment directed at controlling infection, whereas those in the treatment group received wound debridement, lavage and the use of appropriate systemic antibiotics. Despite this, infection developed at the osteotomy site and along the entire length of the implant in all animals in both groups. It was felt that standard methods of wound management might be inadequate when applied at the time of intramedullary nailing since the spread of contamination following the use of reamers was demonstrated in all animals. Results from this study support the clinical impression that heavily contaminated fractures should not be treated by primary intramedullary nailing. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Cyclic AMP inducibility of the myelin basic protein gene promoter requires the NF1 site
Authors: Clark Robert E. , Miskimins W.Keith , Miskimins Robin
Published: October 2002

In the central nervous system oligodendrocyte differentiation is accompanied by the activation of a specific transcriptional program responsible for the synthesis of myelin genes. One of the signals leading to the expression of myelin components, such as the myelin basic protein (MBP) gene is cyclic AMP (cAMP). Previous work using a cell line in which the endogenous MBP gene can be induced by increased cAMP levels (D6P2T) showed that the region of the MBP gene that was required for induction of the gene by cAMP lay between −248 and −105 in the 5′ flanking region. This region contains numerous transcription factor binding sites, including sites for NF1, Sp1, and MEBA. In order to determine if the NF1 site itself was specifically responsible for the cAMP responsiveness of the MBP promoter, stably transfected cells carrying MBP promoter deletion constructs were used. Deletion of just the NF1 site caused loss of responsiveness to cAMP levels. Furthermore, site‐specific mutations in the NF1 site that interfere with NF1 protein binding, in the context of the full length promoter, abolished cAMP responsiveness and caused derepression of the promoter. Analysis of protein binding to the NF1 site showed that the mutation resulted in loss of binding to the site and that the proteins binding at the site are modified in the presence of cAMP elevating agents. These results demonstrate that the NF1 site is indispensable for cAMP responsiveness of the MBP promoter and, together with other DNA elements, plays a role in controlling MBP gene expression.
Plasma brain natriuretic peptide after long‐term treatment for heart failure in general practice
Authors: McGeoch Graham , Lainchbury John , Town G. Ian , Toop Les , Espiner Eric , Richards A. Mark
Published: October 2002

Aim

Plasma brain natriuretic peptide (BNP) concentrations are known to have high sensitivity and specificity in the diagnosis of heart failure in newly symptomatic patients. The relationship of plasma BNP to cardiac function in stable patients on long‐term established treatment for heart failure is unknown. Plasma BNP was assessed for its ability to predict echocardiographic abnormality in 100 patients receiving long‐term treatment in general practice for a provisional diagnosis of heart failure.

Methods and results

BNP >35 pmol/l had a sensitivity and specificity of 69% and 67%, respectively, for a left ventricular ejection fraction of <45%. However, 19% of patients had an LVEF of below 45% whilst BNP was below 35 pmol/l. These patients, in whom a diagnosis of heart failure had been made years previously (mean 3.9 years), were all clinically stable on treatment.

Conclusion

These findings support the view that BNP can be restored to normal levels in well‐compensated patients despite persisting significant systolic dysfunction and suggest that BNP assays may be helpful for monitoring adequacy of therapy. BNP assays will have limited utility in the diagnosis of cardiac impairment once anti‐failure therapy is well established and symptoms have been abolished.
Discourse, Displacement, and Retail Practice: Some Pointers from the Charity Retail Project
Authors: Gregson Nicky , Crewe Louise , Brooks Kate
Published: October 2002

We develop a particular approach to the analysis of retailing, one in which the importance of retailers' talk/practice and the connections between talk/practice and its displacement within retail organisations is emphasised. Displacement means that executive talk is not necessarily powerful, but must be reworked, forged anew, in the specifics of particular stores. Retail (executive) talk/practices have to be translated, and this is the source of their potential instability. We use a particular example to illustrate this argument: that of charity retailing. The authors examine how charity retailing has been reimagined and reworked in head offices, and how this is displaced through charity retail chains. We show the instabilities of charity retailers' (head office) talk, particularly with respect to implementation, and argue that charity retailers' ability to effect the changes they seek to make are limited by the copresence in charity shops of multiple understandings of charity which, themselves, map into particular in-store zones: back rooms, front sales areas, and shop windows. These are: charity as gift; acting charitably (towards other deserving cases or causes); and charity as fundrasing. We also reflect on the likely implications of these findings for the charity retail project; the significance of these findings with respect to future analyses of conventional retailing, its talk and practice; and on the complex intersections between talk/practice, discourse, and space.
Accurate and rapid viability assessment of Trichoderma harzianum using fluorescence‐based digital image analysis
Authors: Hassan Mainul , Corkidi Gabriel , Galindo Enrique , Flores Celia , Serrano‐Carreón Leobardo
Published: October 2002

Fluorescence microscopy and image analysis were evaluated in order to assess the viability of Trichoderma harzianum, an economically important filamentous fungus. After the evaluation of the two most commonly used fluorochromes, acridine orange (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave ambiguous results and therefore FDA was chosen. The lower stability at room temperature and fast fluorescence intensity decay (50% after only 30 s of illumination in UV light) could be overcome by the use of a digital image acquisition system including frame grabber and a video camera. Fresh (live) fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 μg/L), whereas a total absence of fluorescence was observed when using sterilized (dead) fungal cells. Fresh cells were subjected to different lethal and sublethal treatments and the percentage of FDA stained fluorescent hyphae was then measured over the total hyphal area (% of FDA‐stained area) by image analysis. At the same time, samples were cultivated in shake flasks in order to correlate this % of FDA‐stained area with its growth rate, a functional indicator of viability. The linear correlation (r = 0.979) was: growth rate (g/L · h) = 2.25 × 10−3 (% of FDA‐stained area). This method was used to evaluate the viability of the fungus under two different fermentation conditions in a 10‐L bioreactor. Estimated viable biomass during fermentation was strongly influenced by the process conditions. The use of FDA, with computer‐aided quantitative image analysis, has made it possible to rapidly and reliably quantify the viability of T. harzianum. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 677–684, 2002.
Determination of biomass detachment rate coefficients in anaerobic fluidized‐bed‐GAC reactors
Authors: Nakhla George , Suidan Makram T.
Published: October 2002

In this study a simple analytical technique that determines the active biomass in a sample capable of degrading specific substrates anaerobically was developed and used to measure first‐order detachment rate coefficients of acetate and phenol utilizing organisms. Different detachment rate coefficients were measured for the two microbial populations. A general relationship between the detachment rate coefficients and biomass accumulation was developed. The detachment rate coefficient was found to increase sharply after a certain biomass accumulation until it reaches a maximum, after which it decreases continuously with increasing biomass until it levels off at a constant value independent of biomass. Detachment rate coefficients measured in different reactors, operating at different conditions with respect to loading, biological SRT, and attachment media particle size, were in close agreement with each other after being normalized to the shear stress and daily methane production rate per unit biofilm area. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 660–669, 2002.
Double‐decker chemotaxis: no evidence for photonic stimulation of directed locomotion by human blood polymorphonuclear leukocytes
Authors: Kaplow Leonard S. , Chevance Anne de Boisfleury , Henkel Joel , Malawista Stephen E.
Published: October 2002

The study was carried out under direct videomicroscopic control to ascertain whether electromagnetic forces (photons) can initiate directed cell motility of human polymorphonuclear neutrophils (PMN). Cell suspensions containing a mixture of randomly motile white blood cells and erythrocytes (red cells) were placed in a double‐decked preparation created by a glass slide and two cover slips and sealed by paraffin. Erythrocytes in the upper or lower chamber were destroyed by a single burst from a narrow ruby laser beam. Directed locomotion of PMN toward the erythrocyte debris occurred exclusively in the chamber in which the erythrocytes had been destroyed. Only random PMN locomotion was observed in the adjacent chamber. The results indicate that in this experimental model, electromagnetic forces do not initiate directed locomotion. Cell Motil. Cytoskeleton 53:289–292, 2002. © 2002 Wiley‐Liss, Inc.
2‐D finite element analysis of a compressible dynamic viscoelastic cylinder using FFT
Authors: Barrett K. E. , Gotts A. C.
Published: October 2002

An extension to a two‐dimensional compressible dynamic viscoelastic cylinder problem using the finite element method in the Laplace domain coupled with fast Fourier transform is performed. The governing viscoelastic equations of motion are transformed into the Laplace domain via the elastic–viscoelastic correspondence principle. Real and imaginary parts of the nodal displacements are obtained by solving a non‐symmetric matrix equation in the complex Laplace domain. Inversion into the time domain is performed using the discrete inverse Fourier transform. Problems involving both pressure and displacement boundary conditions are analysed using the proposed method. In the case of a quasi‐static cylinder problem, numerical solutions are compared to both the exact Laplace and time domain solutions. Copyright © 2002 John Wiley & Sons, Ltd.
Cost of compliance assessments and the water industry in England and Wales
Authors: McMahon Paul
Published: October 2002

Environmental compliance cost assessments (CCAs) are being increasingly demanded and used in the water industry, in order to allow regulators to balance conflicting objectives. The 1999 periodic review of water company price limits concerned massive environmental expenditures, and consequently major use of CCAs. There were major differences between Ofwat (the economic regulator) and the water companies relating to the compliance costs submitted. The assumptions used by Ofwat vis‐à‐vis future efficiency savings and the cost of capital are notable causes of the differentials. There are a number of other reasons why this differential might have arisen, including gaming. However, the principal cause might be more a real lack of knowledge on the part of companies of future efficiencies and the actual costs of projects. The CCAs produced in the water industry have had massive impact on policy design. A number of specific improvements to CCA are identified. These changes relate to increased collaboration between industry and regulators in working groups to design/approve, inter alia, regulatory methodologies for use in the periodic review. More detailed guidance is required for preparation of a CCA. Further use of compliance cost databases is recommended. The entire process would be facilitated by increased training and awareness raising of economics for the engineers largely responsible for preparation of CCAs. Copyright © 2002 John Wiley & Sons, Ltd. and ERP Environment.