bluuebunny/crossref_metadata_2025 huggingface dataset:
Shaking table testing of a U-shaped plan building model
Authors: Lu Xilin , Zhang Huiyun , Hu Zhili , Lu Wensheng
Published: April 2011

In this paper, the dynamic response of a very complex structure which has U-shaped floors and specially shaped slant columns is described. Shaking table tests of a scale model of the building were carried out to verify the safety of the structure and to confirm the results of a finite element analysis of the building. The elastic finite element analysis was done with the help of Super-SAP 93, a well-known structural analysis program. From the shaking table test and the finite element analysis, the dynamic characteristics of the building and its maximum responses were evaluated. In the elastic region, the analytical results were in good agreement with the test results. At the end of this paper, some suggestions are given for engineering design of this type of structures.Key words: shaking table test, structural model, slant column, U-shaped plan, finite element analysis, seismic response.
Computational stereoscopy for three-dimensional scour depth measurement in channels
Authors: Ankamuthu Sathish , Balachandar Ram , Wood Hugh
Published: April 2011

Local channel scour is a problem of substantial importance in hydraulic engineering practice. Scour occurring around hydraulic structures has serious consequences in terms of damage to the structure and to human safety. The mechanisms of scour are complex and dynamic. This has resulted in most of the studies dealing with local scour to be experimental in nature. The amount of erosion has generally been quantified by noting the depth of scour at various locations in the bed. When the flow is unsteady or quasi-steady, determining the instantaneous depth is not a trivial task and involves a three-dimensional analysis of the scour field. Furthermore, the depth measurement has to be non-intrusive. The present study aims at developing a practical video image analysis for use in three-dimensional scour depth measurement. To acquire images, two cameras with similar specifications were mounted over the model of the eroded bed and the two images were simultaneously captured to simulate stereoscopic viewing. The computational scheme involved the use of an epipolar constraint and a relaxation technique to match corresponding points in the pair of stereo images. A correlation technique was developed to eliminate false matches. The depth of scour was determined using the parallax between the matched points. Following the successful application of the stereoscopy procedure to grayscale images, the scheme was applied to colour images using the pixel hue, saturation, and intensity values.Key words: stereoscopy, scour, epipolar constraint, relaxation technique, correlation technique.
Modélisation de l'évolution de l'état structural des réseaux d'égout : application à une municipalité du Québec
Authors: Mailhot Alain , Duchesne Sophie , Musso Emmanuelle , Villeneuve Jean-Pierre
Published: April 2011

Many sanitary and storm sewer networks are old and deteriorating rapidly. Water and sanitation managers are becoming more and more aware of the negative impact of poorly maintained sewer networks, especially since they are asked to comply with stricter environmental standards under already challenging budget constraints. The best way to improve the structural condition of a sewer network is to replace failed pipe sections. Planning replacement works necessitates knowledge of the present structural state of the network and of the evolution of this state in the near future. Towards this goal, a predictive modelling strategy was developed for the structural state of a sewer network. A case study in a Quebec municipality is presented to illustrate how the modelling strategy developed can be used to simulate the evolution, over the next 20 years, of the length of a sewer network in poor state, and to assess the impact of different replacement strategies on the global state of the network. Key words: urban infrastructures, sewers, structural state, modelling.
Experience with alkali-aggregate reaction in British Columbia
Authors: Shrimer Fred H
Published: April 2011

In British Columbia, alkali-aggregate reaction (AAR) in concrete has not historically been a well-studied, and thus well-understood, mechanism. This paper provides a synopsis of the current state of what is known of AAR in British Columbia. The foci of the paper are (i) locations of AAR in concrete, (ii) the AAR characteristics of aggregates in British Columbia, (iii) the relationship between geology and AAR, and (iv) preventative methods and prescriptions used to prevent or mitigate AAR in British Columbia.Key words: alkali-aggregate reaction, British Columbia, concrete, aggregate, expansion test.
Dissolved air flotation model for drinking water treatment
Authors: Shawwa Ayman R , Smith Daniel W
Published: April 2011

In this study, a kinetic model that describes bubble-particle transport and attachment in the contact zone of dissolved air flotation (DAF) process is presented. The kinetic model, which is based on the assumption that the contact zone is analogous to a chemical reactor, describes the particle removal rate as a first-order reaction with respect to the concentration of particles. It identified important parameters, such as the bubble-particle attachment efficiency (αPB). The theoretical first-order particle removal rate constant (kP), based on the mathematical model, was determined by varying αPB from 0.1 to 1.0. On the other hand, the experimental kP value was determined by measuring the mean residence time, the degree of mixing of particles, and the particle removal efficiency of the contact zone by conducting pilot-scale DAF experiments at different hydraulic loading rates and recycle ratios. The experimentally determined first-order particle removal rate constant was equal to the theoretical kP value when the bubble-particle attachment efficiency (αPB) was in the range of 0.35 to 0.55, which is considered typical for water treatment applications. The kinetic model can be used to predict DAF removal efficiencies provided that αPB is determined for the system under investigation and that the operating conditions applied in this research are used. However, independent experiments are required to verify the applicability of the proposed model.Key words: algae, bubble, coagulation, dissolved air flotation, flocculation, kinetic model, particle size distribution, water treatment.
Alkali-aggregate reaction in concrete: a review of basic concepts and engineering implications
Authors: Fournier Benoit , Bérubé Marc-André
Published: April 2011

This paper presents theoretical and applied state-of-the-art information in the field of alkali-aggregate reactivity (AAR) in concrete. The aspects discussed include basic concepts of the reaction and expansion mechanisms, conditions conducive to the development and the sustainability of AAR in concrete, field and laboratory investigation programs for evaluating the potential alkali-reactivity of concrete aggregates, selection of preventive measures against AAR, and the management of structures affected by AAR. The management section includes the diagnosis of AAR in existing concrete structures, evaluation of the potential for future distress due to AAR, and mitigation and repair approaches used on such structures. This is an introductory paper and sets the stage for a special review of the current AAR situation in the various regions of Canada that is presented in seven papers as part of this issue.Key words: alkali-aggregate reaction, alkali-silica reaction, alkali-carbonate reaction, petrography, testing of concrete aggregates, performance of concrete, survey, remedial measures.
Overtopping risk assessment in river diversion facility design
Authors: Yanmaz A Melih
Published: April 2011

Diversion canals supplemented with upstream and downstream cofferdams are constructed to divert river flow for the construction of diversion weirs. Insufficient canal capacity leads to overtopping of water from the crest of the upstream cofferdam and sides of the diversion canal, which is regarded as the dominant risk mode in the system. A dynamic reliability model, which is based on a resistance-loading methodology with random independent loading following a Poisson process and random fixed resistance, may be used to assess the risk levels for various return periods and construction duration of a diversion weir. The system resistance is considered as the maximum canal capacity described by a lognormal probability density function. The river flow rate corresponding to a certain return period is considered as the external loading on the system. In a case study, a dynamic reliability analysis is performed for a diversion facility. From a flood flow frequency analysis, log-Pearson type 3 distribution is selected to describe the loading. The relationships between the total cost of the diversion facility, its reliability, safety, and duration of construction are examined in a decision making framework.Key words: dynamic reliability, diversion facility, resistance, loading.
Air-water flow in bottom outlets
Authors: Speerli Jürg , Hager Willi H
Published: April 2011

Bottom outlets involve high-velocity air-water flow. Depending on the tunnel length, both air entrainment and air detrainment processes are significant. These processes are evaluated using a hydraulic model consisting of a long rectangular tunnel with a bottom slope larger than the critical slope. Expressions are presented for the maximum air concentration and its streamwise development along the tunnel. A prediction of mixture flow depth along the tunnel is developed. Design guidelines are presented relating to the flow pattern required, the air supply system, and the downstream submergence. The effect of tunnel length on the development of mixture flow characteristics is also outlined. A typical example shows the design procedure for bottom outlets.Key words: air entrainment, air-water flow, high-velocity flow, tunnel flow.
Development of glass fiber reinforced plastic poles for transmission and distribution lines
Authors: Ibrahim Sherif , Polyzois Dimos , Hassan Sherif K
Published: April 2011

An extensive research project is currently being carried out at The University of Manitoba, Canada, to develop lightweight glass fiber reinforced plastic (GFRP) poles for use in transmission and distribution networks. In this paper, results from tests involving full-scale tapered GFRP poles with a hollow circular cross section subjected to cantilever bending are presented. The filament winding process was employed to produce the poles using polyester resin reinforced with E-glass fibers. Cantilever bending tests were conducted on twelve full-scale poles up to failure. Test parameters included fiber orientation and number of layers. Extensive theoretical work preceded the test program and a theoretical model was developed for evaluating the failure load. The results to date indicate that the developed theoretical model can predict quite well the ultimate capacity and behavior performance of GFRP poles. This theoretical model was used in this investigation to determine the optimum cross-sectional dimensions for 6.1 m (20 ft) and 18.3 m (60 ft) Class 1 GFRP poles.Key words: transmission and distribution poles, filament winding, fiber-reinforced plastics.
Seismic shear demand on wall segments of ductile coupled shear walls
Authors: Chaallal O , Gauthier D
Published: April 2011

This paper presents the results of nonlinear dynamic analyses carried out on ductile coupled shear walls (CSWs) to investigate the seismic shear demand on wall segments. The objectives of the present study were to evaluate the dynamic amplification and establish a code-format force reduction factor for shear, applicable in Canada. The study considered three Canadian seismic zones (4, 5, and 6), five numbers of storeys (6, 10, 15, 20, and 30), three degrees of coupling (low, medium, and high), and 10 historical earthquake records encompassing a broad range of frequency contents. Overall, 450 analyses were performed. Results indicate that the New Zealand amplification factor βv presently used in Canada overestimates the dynamic amplification. Additionally, the use of the overstrength factor for shear γp for tension walls may underestimate their shear resistance and result in a shear failure. Conversely, the use of γp for compression walls provided a reasonable factor of safety. Finally, for the shear design of CSWs, two alternative approaches are suggested. The first involves the use of a force reduction factor for shear, Rv, including the dynamic amplification factor γd and the overstrength factor γp as follows: Rv = 2.0 for Za > Zv, Rv = 1.0 for Za < Zv, and Rv = 1.3 for Za = Zv, where Za and Zv are acceleration- and velocity-related zonal identifiers. The second approach implies the use of the overstrength factor γp of the compression wall for both walls of CSWs and βv = 1.0.Key words: coupled shear walls, reinforced concrete, degree of coupling, seismic, frequency content, shear demand, dynamic amplification factor, force reduction factor for shear.
Effect of easily biodegradable organic compounds on bacterial growth in a bench-scale drinking water distribution system
Authors: Gagnon Graham A , Slawson Robin M , Huck Peter M
Published: April 2011

Many engineered (e.g., disinfectant residual concentration) and environmental (e.g., temperature) factors influence bacterial regrowth in drinking water distribution systems. This paper examines the effect of nutrients, specifically biodegradable organic matter (BOM) composition, BOM concentration, and hydraulic retention time on bacterial growth in an annular reactor (AR). Drinking water that had an alkalinity of 300 mg/L as CaCO3and a free chlorine residual of approximately 0.2 mg/L was used as process water in the ARs. Prior to entering the ARs, the water was filtered through granular activated carbon (GAC) to remove background chlorine and background organic matter. A cocktail of easily biodegradable organic compounds consisting of carboxylic acids, aldehydes, and free amino acids were spiked into the ARs as the primary carbon source. It was found that the influent BOM concentration (p value = 0.013) and the presence of free amino acids in the BOM cocktail (p value = 0.009) significantly increased the number of viable culturable cells in the biofilm, as measured by heterotrophic plate counts (HPCs). The interaction between the BOM concentration and the presence of amino acids also significantly increased the number of biofilm HPCs (p value = 0.021). Alternatively, the BOM concentration and the amino acid fraction did not affect the number of bulk (i.e., suspended) bacteria. The number of biofilm HPCs in the reactor was approximately 10 times greater than the number of bulk HPCs at high influent BOM concentrations and low retention times (i.e., high BOM loading rates). At low loading rates, the ratio of number of biofilm to bulk cells was less than 2. Consequently, it was deduced that the BOM was utilized predominately by the biofilm cells. This indicates that removal of easily biodegradable organic compounds is an important factor for controlling biofilm growth in distribution systems.Key words: drinking water, distribution systems, biofilm, annular reactor, regrowth.
Secretion of α-amylase and multiple forms of glucoamylase by the yeast Trichosporon pullulans
Authors: Mot René De , Verachtert Hubert
Published: April 2011

Trichosporon pullulans IGC 3488 produced extracellular α-amylase and glucoamylase activities when grown in batches in a medium containing corn steep liquor and soluble starch or corn starch, α-Amylase, unlike glucoamylase activity, was secreted biphasically. For both amylases the maximum concentration was found in stationary phase cultures. The amylolytic enzymes, previously concentrated by ammonium sulfate precipitation, were separated into a glucoamylase fraction and an α-amylase fraction by Ultrogel AcA 54 gel filtration. Pullulanase activity was located in the glucoamylase fraction, whereas cyclodextrinase activity was restricted to the α-amylase fraction. Isoamylase and α-glucosidase were not detected. Electrophoretic analysis showed that α-amylase activity was due to a single protein. Glucoamylase, however, occurred in multiple forms. The four glucoamylases and the α-amylase were glycoproteins.
Purification of the thermostable direct hemolysin of Vibrio parahaemolyticus by immunoaffinity column chromatography
Authors: Honda Takeshi , Yoh Myonsun , Narita Ikuyo , Miwatani Toshio , Sima Huilan , Xiegen You , Xiandi Li
Published: April 2011

A simple method involving immunoaffinity column chromatography to purify the thermostable direct hemolysin of Vibrio parahaemolyticus was developed. The thermostable direct hemolysin purified from the culture supernatant of a strain isolated from the first reported case of V. parahaemolyticus infection in China in 1985 was indistinguishable from the hemolysins purified from strains isolated in Japan.
Novel α4-integrin ligands on an endothelial cell line
Authors: Tudor Kim-Sue RS , Deem Tracy L , Cook-Mills Joan M
Published: April 2011

The unique combination of adhesion molecules expressed on endothelial cells is thought to mediate the specificity of leukocyte-endothelial cell interactions. In this study, murine endothelial cell lines were used as a model to identify novel adhesion molecules that participate in these cellular interactions. Lymphocyte adhesion to the continuous endothelial cell lines mHEVa and mHEVc required alpha 4-integrin. Interestingly, lymphocyte alpha 4-integrin bound to VCAM-1 as well as an unknown ligand on the mHEVa cell line. We have demonstrated that this VCAM-1-independent adhesion to the mHEVa cells was not mediated by other known alpha 4-integrin ligands (fibronectin, alpha 4-integrin itself, or MAdCAM-1). Two novel alpha 4-integrin ligands (p50 and p10) were isolated from the mHEVa cell line but not the mHEVc cell line by B cell alpha 4-integrin-specific ligand binding of radiolabeled mHEV cell membrane proteins. These results provide the first direct evidence that novel ligands for alpha 4-integrin exist on membranes from endothelial cells.

Key words: B cell, T cell, endothelial cells, integrin, vascular cell adhesion molecule-1, VCAM-1.
Signal transduction mechanisms mediating secretion in goldfish gonadotropes and somatotropes
Authors: Chang John P , Johnson James D , Goor Fredrick Van , Wong Calvin JH , Yunker Warren K , Uretsky Aubrey D , Taylor Daina , Jobin Richard M , Wong Anderson OL , Goldberg Jeffrey I
Published: April 2011

The intracellular signal transduction mechanisms mediating maturational gonadotropin and somatotropin secretion in goldfish are reviewed. Several major signaling mechanisms, including changes in intracellular [Ca2+], arachidonic acid cascades, protein kinase C, cyclic AMP/protein kinase A, calmodulin, nitric oxide, and Na+/H+antiport, are functional in both cell types. However, their relative importance in mediating basal secretion and neuroendocrine-factor-regulated hormone release differs according to cell type. Similarly, agonist- and cell-type-specificity are also present in the transduction pathways leading to neuroendocrine factor-modulated maturational gonadotropin and somatotropin release. Specificity is present not only in the actions of different regulators within the same cell type and with the same ligand in the two cell types, but this also exists between isoforms of the same neuroendocrine factor within a single cell type. Other evidence suggests that function-selectivity of signaling may also result from differential modulation of Ca2+fluxes from different sources. The interaction of different second messenger systems provide the basis by which regulation of maturational gonadotropin and somatotropin release by multiple neuroendocrine factors can be integrated at the target cell level.

Key words: Ca2+signaling, cAMP, PKC, arachidonic acid, NO.
The role of glutamate and nitric oxide in the reproductive neuroendocrine system
Authors: Dhandapani Krishnan M , Brann Darrell W
Published: April 2011

The preovulatory surge of gonadotropin releasing hormone (GnRH) is essential for mammalian reproduction. Recent work has implicated the neurotransmitters glutamate and nitric oxide as having a key role in this process. Large concentrations of glutamate are found in several hypothalamic nuclei known to be important for GnRH release and glutamate receptors are also located in these key hypothalamic nuclei. Administration of glutamate agonists stimulate GnRH and LH release, while glutamate receptor antagonists attenuate the steroid-induced and preovulatory LH surge. Glutamate has also been implicated in the critical processes of puberty, hormone pulsatility, and sexual behavior. Glutamate is believed to elicit many of these effects by activating the release of the gaseous neurotransmitter, nitric oxide (NO). NO potently stimulates GnRH by activating a heme containing enzyme, guanylate cyclase, which in turn leads to increased production of cGMP and GnRH release. Recent work has focused on identifying anchoring and (or) clustering proteins that target glutamate receptors to the synapse and couple the glutamate-NO neurotransmission system. The present review will discuss these new findings, as well as the role of glutamate and nitric oxide in important mammalian reproductive events, with a focus on the hypothalamic control of preovulatory GnRH release.

Key words: glutamate, nitric oxide, GnRH, postsynaptic density, hypothalamus.
Molecular correlates of the action of bis(ethyl)polyamines in breast cancer cell growth inhibition and apoptosis
Authors: Faaland Carol A , Thomas T J , Balabhadrapathruni Srivani , Langer Thierry , Mian Somia , Shirahata Akira , Gallo Michael A , Thomas Thresia
Published: April 2011

Polyamines are known to be involved in cell growth regulation in breast cancer. To evaluate the efficacy of bis(ethyl)polyamine analogs for breast cancer therapy and to understand their mechanism of action we measured the effects of a series of polyamine analogs on cell growth, activities of enzymes involved in polyamine metabolism, intracellular polyamine levels, and the uptake of putrescine and spermidine using MCF-7 breast cancer cells. The IC50values for cell growth inhibition of three of the compounds, N1,N12-bis(ethyl)spermine, N1,N11- bis(ethyl)norspermine, and N1,N14-bis(ethyl)homospermine, were in the range of 1-2 µM. Another group of three compounds showed antiproliferative activity at about 5 µM level. These compounds are also capable of suppressing colony formation in soft agar assay and inducing apoptosis of MCF-7 cells. The highly effective growth inhibitory agents altered the activity of polyamine biosynthetic and catabolic enzymes and down-regulated the transport of natural polyamines, although each compound produced a unique pattern of alterations in these parameters. HPLC analysis showed that cellular uptake of bis(ethyl)polyamines was highest for bis(ethyl)spermine. We also analyzed polyamine analog conformations and their binding to DNA minor or major grooves by molecular modelling and molecular dynamics simulations. Results of these analyses indicate that tetramine analogs fit well in the minor groove of DNA whereas, larger compounds extend out of the minor groove. Although major groove binding was also possible for the short tetramine analogs, this interaction led to a predominantly bent conformation. Our studies show growth inhibitory activities of several potentially important analogs on breast cancer cells and indicate that multiple sites are involved in the mechanism of action of these analogs. While the activity of an analog may depend on the sum of these different effects, molecular modelling studies indicate a correlation between antiproliferative activity and stable interactions of the analogs with major or minor grooves of DNA.Key words: polyamine analogs, breast cancer cells, apoptosis, molecular modelling.
Orexins (hypocretins): novel hypothalamic peptides with divergent functions
Authors: Mondal Muhtashan S , Nakazato Masamitsu , Matsukura Shigeru
Published: April 2011

The hypothalamus is the most important region in the control of food intake and body weight. The ventromedial "satiety center" and lateral hypothalamic "feeding center" have been implicated in the regulation of feeding and energy homeostasis by various studies of brain lesions. The discovery of orexin peptides, whose neurons are localized in the lateral hypothalamus and adjacent areas, has given us new insight into the regulation of feeding. Dense fiber projections are found throughout the brain, especially in the raphe nucleus, locus coeruleus, paraventricular thalamic nucleus, arcuate nucleus, and central gray. Orexins mainly stimulate food intake, but by the virtue of wide immunoreactive projections throughout the brain and spinal cord, orexins interact with various neuronal pathways to potentate divergent functions. In this review, we summarize recent progress in the physiological, neuroanatomical, and molecular studies of the novel neuropeptide orexins (hypocretins).Key words: orexins (hypocretins), hypothalamus, lateral hypothalamus, feeding behavior, energy homeostasis, neurons.
Divergent responses ofras-transfected and non-ras-transfected human keratinocytes to extracellular calcium
Authors: Shi Biao , Isseroff R Rivkah
Published: April 2011

Raising extracellular calcium (Cao) induces terminal differentiation in cultured epidermal keratinocytes. The introduction of the ras oncogene into keratinocytes results in resistance to Cao-mediated differentiation. To understand the signaling mechanism involved, we examined the Cao-induced formation of inositol triphosphate (IP3) and changes in intracellular Ca2+(Cai) concentration in non-ras-transfected and ras-transfected HaCaT lines of human keratinocytes. When switched from 0.05- to 1.5-mM Caomedium, the non-ras HaCaT line showed a rapid twofold increase in IP3formation, whereas the IP3level in the ras-transfected I-7 line was slightly affected. G-protein-coupled activation of phospholipase was intact in both lines, as evidenced by the generation of similar amounts of IP3in response to addition of bradykinin or guanosine 5'-[γ-thio]-triphosphate. Addition of 1.0 mM Caoevoked similar Cairesponses in both non-ras- and ras-transfected cells: a transient elevation, followed by a sustained lower plateau. However, the two lines differed in their later responses: after being maintained in 1.0 mM Ca2+for 24 h, the Cailevel was significantly lower in ras-transfected cells than in non-ras-transfected HaCaT cells. The Cao-induced increase in Caiin both lines was inhibited by the Ca2+entry blocker SK&F 96365 or depolarization in high K+bathing solution, demonstrating its dependence of calcium influx. The results suggest fundamental differences in the early signal that are generated in response to an increase in Caoin ras-transfected keratinocytes, with the absence of a Cao-induced rise in IP3-a signaling pathway defect that may play a role in the differentiation block the cells exhibit. In addition, the inability of ras-transfected cells to sustain a prolonged Caiplateau may also contribute to their inability to differentiate in response to the Caosignal.Key words: cell differentiation, intracellular Ca2+, IP3, keratinocytes, ras transfection, signal transduction.
Interplay between liganded and orphan nuclear receptors controls reproductive pathways
Authors: Métivier Raphaël , Le Dréan Yves , Salbert Gilles , Pakdel Farzad
Published: April 2011

Nuclear receptors are transcription factors that belong to an evolutionary ancient superfamily. These proteins, which are even present in primitive metazoans, are implicated in all levels of cell fate: proliferation, differentiation, and apoptosis. Some of these nuclear receptors behave as ligand-inducible transcription factors, as they have acquired during evolution the ability to bind ligands. This is the case for some proteins that recognize small hydrophobic signaling molecules, and particularly the estrogen receptor (ER or NR3A1), which regulates the target gene's transcription rate under estrogen binding. It is now known that the ER alone regulates the transcription of many genes, such as those implicated in reproductive functions. However, this ER-mediated signaling pathway could be modulated by other transcription factors. Our work has established that two other orphan nuclear receptors (SF-1 or NR5A1 and the COUP-TFs, NR2F1 and NR2F2) can enhance two ER-regulated genes implicated in salmonid reproductive functions: the ER gene itself, and the sGTHIIβ gene. Moreover, some xenoestrogens could disturb these regulations. Therefore, our data contribute to the concept that interplay between nuclear receptors is an important event for the transcriptional regulation of genes controlling cellular functions.Key words: reproduction, estrogen receptor, SF-1, COUP-TFI, gene transcription, xenobiotics.
Expression of heat shock protein 47(Hsp47) mRNA levels in rabbit connective tissues during the response to injury and in pregnancy
Authors: Hart David A , Reno Carol , Graverand Marie-Pierre Hellio Le , Hoffman Lisa , Kulyk William
Published: April 2011

Hsp47 (also termed "colligin") is a 47 kDa protein that is localized in the ER and cis-Golgi vesicles of fibrocytes, chondrocytes, and other collagen-secreting cells. Under stress conditions, Hsp47 expression is upregulated as part of the heat shock/stress response that mitigates cell damage from noxious stimuli such as elevated temperature, heavy metals, and oxidative stress. Under non-stress conditions, Hsp47 functions as a collagen-specific molecular chaperone that facilitates intracellular procollagen polypeptide synthesis, and triple helix assembly in connective tissues. Previously it has been shown that levels of collagen-specific gene expression are significantly altered in ligaments, menisci, and other connective tissues of the rabbit following surgically induced injuries (increased), and during pregnancy (decreased). The present study was undertaken to determine whether expression of mRNA for the Hsp47 collagen-binding stress protein was also influenced in these experimental models. Since no sequence information was available on the rabbit Hsp47 gene, a partial cDNA for rabbit Hsp47 was first isolated and cloned using reverse transcriptase PCR (RT-PCR) with degenerate oligonucleotide primers. Rabbit Hsp47 sequence-specific primers then designed enabled analysis of Hsp47 mRNA expression in rabbit connective tissues using semiquantitative RT-PCR. It was found that Hsp47 expression is affected in a complex, tissue-specific manner by injury and pregnancy. Hsp47 transcript levels were elevated in the medial collateral ligament (MCL) of the rabbit knee following surgical induction of a gap injury. Transection of the anterior cruciate ligament (ACL), which leads to chronic progressive damage to menisci of the rabbit knee joint, was accompanied by an upregulation of Hsp47 expression in the medial and lateral menisci. Hsp47 mRNA levels were depressed during pregnancy in the kidney and ACL of primigravid adolescent rabbits, but were not altered in corneal tissue during pregnancy or in the ACL of skeletally mature multiparous females. The changes in Hsp47 transcript levels within these connective tissues following injury/pregnancy often, but not always, paralleled changes in collagen-specific gene expression.Key words: Hsp47 and ligament injury, Hsp47 and meniscal injury, Hsp47 and mRNA levels, Hsp47 expression and pregnancy.
An engineeredloxsequence containing part of a long terminal repeat of HIV-1 permits Cre recombinase-mediated DNA excision
Authors: Lee Young-sam , Kim Sung-tae , Kim Gyoung-won , Lee Minhyung , Park Jong-sang
Published: April 2011

In our previous report, one 34-bp sequence from a long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) clone, loxLTR-1, was proposed as a target site for site-specific excision by modified Cre recombinase. To support this suggestion, an engineered lox sequence, designated loxIL1, was made. This variant lox has the corresponding sequence of loxLTR-1 at the spacer region and the last two bases of inverted repeat sequence. Through in vitro recombination assay, loxIL1 also allowed the wild-type Cre to specifically recombine the sequence. An in vitro DNA binding experiment with mutants CreK244R and CreK244L revealed that lysine 244 of Cre plays an important role in interaction with the engineered lox. This result suggests that loxLTR-1 would be a candidate for antiviral strategy using site-specific recombinase.Key words: Cre/lox recombination system, sequence similarity, protein-DNA interaction, long terminal repeat, HIV-1 therapy.
Genetic and biochemical diversity in thePaxgene family
Authors: Underhill D Alan
Published: April 2011

The mammalian Pax gene family comprises nine members that are characterized by a conserved DNA-binding motif, the paired domain, which was originally described in the Drosophila protein paired. Both loss- and gain-of-function studies reveal that Pax genes carry out essential roles during embryogenesis, and in some instances, may function as master regulatory genes. This review focuses on both genetic and biochemical aspects of the Pax family, and emphasizes important differences in the activity of individual Pax genes and their protein products.Key words: Pax, paired domain, homeodomain, development, gene regulation.
Glycine cleavage enzyme complex: Rabbit H-protein cDNA sequence analysis and comparison to human, cow, and chicken
Authors: Choy Francis , Sharp Lisa , Applegarth Derek A
Published: April 2011

The H-protein is one of the four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex, the major degradative pathway of glycine. We have isolated the full-length cDNA of the H-protein gene from the rabbit (Oryctolagus caniculus) by reverse transcription of liver poly-A mRNA and determined its nucleotide sequence (GenBank Acc. No. BankIt 318281 AF 231451). Similar to that in human, the rabbit H-protein gene possesses a 519-bp open reading frame that translates a 173-amino-acid (aa) protein. This reading frame is comprised of a 48-aa mitochondrial targeting sequence and a 125-aa residue that constitutes the mature mitochondrial matrix protein. In the mature protein region, there is a 95.5% nucleotide and 98.4% amino-acid sequence similarity to human. This conservation was also noted in the mature protein of the cow (Bos taurus) and chicken (Gallus domesticus), where there are a 94.1% and 85.3% nucleotide similarities, and 95.2% and 85.6% amino-acid sequence similarities, respectively. However, the targeting region is not as well conserved. Comparison of the rabbit targeting sequence to that in human, cow, and chicken reveals 84.0%, 79.2%, and 72.9% nucleotide, and 72.9%, 75.0%, and 54.2% amino-acid sequence similarities, respectively. These findings suggest that within the H-protein gene, the regions encoding the mitochondrial targeting and matrix protein may have evolved differently. Gene diversification in the former may reflect the species specificity in targeting proteins destined for the mitochondria, whereas homology in the latter suggests a very similar structure-function of the mature H-protein among these species. This homology in structure-function likely accounts for the observation that non-human H-protein can replace the human protein in the activity assay of the glycine cleavage enzyme system. This includes the biochemical diagnosis of non-ketotic hyperglycinemia (NKH) resulting from defects other than the H-protein, e.g., mutation(s) in the P-protein.Key words: glycine cleavage enzyme, H-protein, sequence comparison, non-ketotic hyperglycinemia.
A tale of two charges: Distinct roles for an acidic and a basic amino acid in the structure and function of cytochromec
Authors: Parrish Jonathan C , Guillemette J Guy , Wallace Carmichael JA
Published: April 2011

Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria. As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history. Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp. There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined. Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electro statically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular). This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86. Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90.Key words: cytochrome c, structure-function studies, molecular modelling, surface electrostatics.
Antioxidant activity levels and oxidative stress as blood markers of allergic response to drugs
Authors: Pérez-Gómez Cristina , Segura José M , Blanca Miguel , Asenjo Maite , Matés José M
Published: April 2011

Antioxidant enzymes work together in human blood cells against toxic reactive oxygen species. Although their relationship with several pathophysiologic processes has been stated, not much is known about the connection between antioxidant defence and allergy. This study was designed to determine the enzymatic activities and the oxidative indices in the blood and serum proteins in patients suffering from allergy to drugs. We hypothesize that serum and blood reactions may serve as useful clinical marker for the allergic state. We used enzymatic antioxidant activities, thiobarbituric acid reactive substances, and carbonyl contents of proteins as suitable markers. We determined superoxide dismutases, glutathione peroxidase and catalase activities in each cell type. After antihistaminics plus steroids were given as part of a protocol treatment, enzymatic antioxidant activities, thiobarbituric acid reactive substance levels, and carbonyl contents were used as recovering markers for the disease. We found a relationship between antioxidant enzymatic activities, thiobarbituric acid reactive substance levels, and carbonyl contents for allergic reactions belonging to several type I and type IV allergies, as well as cross-reactive intolerance to nonsteroidal anti-inflammatory drugs and an anaphylactoid reaction to a radiocontrast media. A similar pattern also exists for analogous allergic manifestations and disease-like status.Key words: allergy, blood, catalase, glutathione peroxidase, superoxide dismutase.
Human growth factor receptor bound 14 binds the activated insulin receptor and alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins
Authors: Hemming Richard , Agatep Ronald , Badiani Ketan , Wyant Kerrie , Arthur Gilbert , Gietz R Daniel , Triggs-Raine Barbara
Published: April 2011

To identify proteins interacting in the insulin-signaling pathway that might define new pathways or regulate existing ones, we have employed the yeast two-hybrid system. In a two-hybrid screen of a human liver cDNA library, we identified the human growth factor receptor bound 14 (hGrb14) adaptor protein as a partner of the activated insulin receptor. Additional analysis of the insulin receptor - hGrb14 interaction in the yeast two-hybrid system revealed that the SH2 domain of hGrb14 was not the sole region involved in binding the activated insulin receptor. The insulin-stimulated interaction between hGrb14 and the insulin receptor was also observed in different mammalian cultured cell lines. This association was detected at 1 min of insulin stimulation and was maximal at 10 nM and greater concentrations of insulin. Chinese hamster ovary cells stably expressing the insulin receptor (CHO-IR) and hGrb14 were used to examine the effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation of proteins; in general, increasing levels of hGrb14 expression resulted in a reduction in tyrosine phosphorylation. This decrease was demonstrated for the specific proteins src homology-containing and collagen-related protein (Shc), insulin receptor substrate-1 (IRS-1), and Downstream of tyrosine Kinase (Dok). The broad effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation suggest that it acts early in the insulin-signaling pathway.Key words: insulin signaling, growth factor receptor bound 14, Grb14, adaptor protein, insulin receptor.
Tumour promoter mediated altered expression and regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase in H-ras-transformed fibrosarcoma cell lines
Authors: Voskas Daniel , Mader Rene , Lee Janet , Hurta Robert AR
Published: April 2011

Alterations in cellular growth are important in the progression of malignant disease. Cell growth regulation by tumour promoters can be complex. The present study demonstrates a novel link between alterations in phorbol ester tumour promoter mediated regulation during malignant conversion and the expression of ornithine decarboxylase and S-adenosylmethionine decarboxylase, key rate-limiting and regulatory activities in the biosynthesis of polyamines. H-ras-transformed mouse 10 T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible phorbol ester tumour promoter mediated changes in ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) gene expression. Selective induction of ODC and SAMDC gene expression was observed, since in contrast to nontransformed parental 10 T1/2 cells, ras-transformed cells capable of benign tumour formation (NR3 cells) and ras-transformed cells capable of metastasis formation (C2 cells) exhibited marked alterations in the levels of ODC and SAMDC gene expression. Increased ODC gene and SAMDC gene expression in response to phorbol-12-myristate-13-acetate (PMA) treatment was found to involve transcriptional events in both NR3 cells and in C2 cells. Post-transcriptional events also played a role in the regulation of ODC gene expression in NR3 cells and in C2 cells, and in the regulation of SAMDC gene expression in C2 cells but not in NR3 cells. In NR3 cells, alterations in ODC and in SAMDC gene expression was an event requiring de novo protein synthesis, whereas in highly malignant C2 cells, protein synthesis inhibition following cycloheximide treatment in cooperation with PMA resulted in an augmentation of both ODC and SAMDC gene expression. Evidence is presented to suggest that the PMA-mediated alterations in ODC and in SAMDC gene expression in NR3 cells and in C2 cells involved protein kinase C - mediated events. The status of the cellular polyamine levels was also an important determinant of the PMA-mediated alterations that occurred in ODC and in SAMDC expression in these H-ras transformed cells. Collectively, these results suggest that PMA can modulate ODC and SAMDC expression in H-ras transformed cells and that the mechanisms involved in the PMA- mediated regulation of ODC and SAMDC gene expression changes as a function of H-ras mediated cellular transformation and malignant progression. This study further suggests a mechanism of PMA stimulation of transformed cells wherein early alterations in the regulatory control of ODC and SAMDC gene expression are important and critical.Key words: PMA, ornithine decarboxylase, S-adenosylmethionine decarboxylase, malignant progression.
Identification of nuclear-import and cell-cycle regulatory proteins that bind to prothymosin α
Authors: Freire Javier , Covelo Guillermo , Sarandeses Concepción , Díaz-Jullien Cristina , Freire Manuel
Published: April 2011

Prothymosin α (ProTα) is a nuclear protein that is widely distributed in mammalian tissues, and is thought to play a role in cell proliferation. In an attempt to shed light on this role, affinity chromatography on ProTα-Sepharose columns was used to identify proteins in subcellular extracts of transformed human lymphocytes (NC37 cells) that interact with ProTα in vitro, and thus may interact with ProTα in vivo. Immunoblotting techniques were used to screen the ProTα-binding fractions for histones and other proteins involved in nuclear transport and cell-cycle control. The most abundant ProTα-binding proteins were histones H2A, H2B, H3, and H4. Of the nuclear-transport proteins, karyopherin β1, Rch-1, Ran, and RCC1 were detected at high concentrations; NTF2, nucleoporin p62, and Hsp70 were detected at low concentrations; while tranportin, CAS, and Ran BP1 were not detected. Of the cell-cycle control proteins, PCNA, Cdk2, and cyclin A were detected at high concentrations; cdc2, Cdk4, and cyclin B were detected at very low concentrations; while cyclin D1, cyclin D3, Cip1, and Kip1 were not detected. These results suggest (i) that ProTα is transported into the nucleus by the karyopherin β1- Rch-1 complex, and (ii) that ProTα may interact in the nucleus with proteins involved in DNA metabolism and cell-cycle control.Key words: prothymosin α, histone-binding proteins, nuclear transport proteins, cell-cycle proteins, chromatin remodeling.
Palmitic acid uptake by the rat soleus muscle in vitro
Authors: Górecka M , Synak M , Budohoski L , Langfort J , Moskalewski S , Żernicka E
Published: April 2011

The rate of fatty acid uptake, oxidation, and deposition in skeletal muscles in relation to total and unbound to albumin fatty acids concentration in the medium were investigated in the incubated rat soleus muscle. An immunohistochemical technique was applied to demonstrate whether the albumin-bound fatty acid complex from the medium penetrates well within all areas of the muscle strips. It was found that the percentage of incorporation of palmitic acid into intramuscular lipids was fairly constant, independently of the fatty acid concentration in the medium, and amounted to 6372% for triacylglycerols, 712% for diacylglycerols-monoacylglycerols, and 1926% for phospholipids. Both palmitic acid incorporation into the muscle triacylglycerol stores and its oxidation to CO2closely correlated with an increase in both total and unbound to albumin fatty acid concentrations in the incubation medium. Under conditions of increased total but constant unbound to albumin palmitic acid concentrations, the incorporation of palmitic acid into triacylglycerols and its oxidation to CO2were also increased, but to a lower extent. This supports the hypothesis that the cellular fatty acid metabolism depends not only on the availability of fatty acids unbound to albumin, but also on the availability of fatty acids complexed to albumin.Key words: skeletal muscle, fatty acids, triacylglycerols, phospholipids.