bluuebunny/crossref_metadata_2025 huggingface dataset:
Strength of hollow circular steel sections filled with fibre-reinforced concrete
Authors: Campione Giuseppe , Mindess Sidney , Scibilia Nunzio , Zingone Gaetano
Published: April 2011

The strength of hollow circular steel sections filled with normal-strength plain concrete and fibre-reinforced concrete (FRC) was evaluated. First, the case of centrally loaded composite members was considered and the bearing capacity of the columns was calculated using the methods proposed by a European code (EC4) and an American code (LRDF). Some expressions in these codes were validated for the case of FRC by adapting experimental data to introduce the mechanical properties of the FRC. To do this, experimental results of standard tests on FRC (compression and splitting tension) were used as well as data on circular steel columns filled with 2% FRC by volume with different types of fibres (steel, polyolefin). Second, the moment - axial force diagrams for composite members, taking into account the residual tensile strength of FRC, were calculated, showing the advantages of using FRC compared with plain concrete for filling hollow steel sections.Key words: fibre-reinforced concrete, hollow steel columns, composite members, steel fibres, polyolefin fibres.
Collapse of a viewing platform
Authors: Wilson John C
Published: April 2011

In April 1995 a group of young adults were on a school trip to Department of Conservation (DOC) lands on New Zealand's South Island. Moments after 18 of the group had stepped onto a 3 m × 3 m viewing platform it collapsed and fell 30 m. Fourteen people died and the other four were seriously injured. This paper reviews circumstances that contributed to this tragedy. Features of the construction of the timber platform are presented, along with building code requirements that should have been followed but were not. The principal technical reason for the collapse was simple and straightforward; the cantilevered platform was not properly connected to its foundation. More complex however, was the role of non-technical factors that contributed to the collapse. These included failures in project management, provision of qualified engineering input, construction management, compliance with the building code, construction inspection, and posting of load limit signs. The evolution of DOC and project management issues that allowed these failures to occur are examined. Recommendations of the commission of inquiry are presented and resultant changes in DOC's engineering and management procedures are addressed. The issue of accountability and the legal position of DOC present additional interesting twists to this story. The paper concludes with comments by the author that reflect on factors associated with the platform collapse.Key words: platform, collapse, structural failure, building code, timber construction, nailed connections, project management, commission of inquiry, New Zealand.
Selective medium for the isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum
Authors: George Karen L. , Falkinham III Joseph O.
Published: April 2011

A medium for the selective isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) was developed, based upon the ability of these mycobacteria to utilize Tween 80 as sole carbon source and grow optimally at pH 5.5 on a simple mineral salts medium. Representative MAIS strains had higher efficiencies of plating on the Tween 80 medium compared with Middlebrook 7H10. It was shown that nonmycobacterial organisms in natural waters had lower efficiencies of plating on the Tween 80 medium and smaller colonies, thus allowing direct isolation and enumeration of the slowly growing mycobacteria without overgrowth.
Immunovirological studies on human respiratory syncytial virus structural proteins
Authors: Trudel Michel , Nadon Francine , Séguin Cécile , Ghoubril Simone , Payment Pierre , Trépanier Pierre
Published: April 2011

Immunovirological studies suggest that human respiratory syncytial virus may well be composed of five structural proteins as are other members of the Paramyxoviridae family: the two external membrane glycoproteins H (90 000) and Fo (F1, 49 000; F2, 20 000; disulfide linked), the internal membrane protein M (34 000), the nucleoprotein N (42 000), and a protein (78 000) designated P that could be the equivalent of the polymerase of the morbillivirus and paramyxovirus genus. Neutralizing monoclonal antibodies showed, by immunoprecipitation and immunoblotting, that the fusion protein carries neutralizing epitopes. One monoclonal antibody, which shows a high neutralizing titer, immunoblotted directly with the F1 fragment (49 000) of the fusion protein. Analysis in mice of the immunogenicity of the structural proteins separated on sodium dodecyl sulphate gels indicated that, under our conditions, only the fusion protein dimer Fo and its F1 fragment were capable of inducing neutralizing antibodies.
Response of the microflora in outdoor experimental streams to pentachlorophenol: environmental factors
Authors: Pignatello Joseph J. , Johnson LeeAnn K. , Martinson Michael M. , Carlson Robert E. , Crawford Ronald L.
Published: April 2011

The 2nd year of a 2-year study of the fate of pentachlorophenol in outdoor artificial streams focused on details of microbial degradation by a combination of in situ and laboratory measurements. Replicate streams were dosed continuously at pentachlorophenol concentrations of 0, 48, and 144 μg/L, respectively, for an 88-d period during the summer of 1983. Pentachlorophenol was degraded both aerobically and anaerobically. Aerobic degradation was more rapid than anaerobic degradation. Mineralization of pentachlorophenol was concommitant with pentachlorophenol disappearance under aerobic conditions, but lagged behind loss of the parent molecule under anaerobic conditions. Biodegradation in the streams, or in specific stream compartments such as the sediment or water column, was characterized by an adaptation period (3–5 weeks for the stream as a whole, and reproducible from the previous year), which was inversely dependent on the concentration of pentachlorophenol and microbial biomass. The adaptation in the streams could be attributed to the time necessary for selective enrichment of an initially low population of pentachlorophenol degraders on surface compartments. The extent of biodegradation in the streams (percent loss of initial concentration of pentachlorophenol) increased with increasing pentachlorophenol input, which was explicable by an increase in the pentachlorophenol degrader population with increasing pentachlorophenol concentration. The sediment zone most significant to overall pentachlorophenol biodegradation was the top 0.5- to 1-cm layer as shown by pentachlorophenol migration rates and depth profiles of degrader density within the sediment. Pentachlorophenol profiles in sediment cores taken during and after the adaptation period for degradation showed that diffusion of pentachlorophenol into the sediment was rate limiting to degradation in this compartment. Degradation rates were independent of temperature within the temperature range of the streams during the dosing season (19–30 °C), but became increasingly slower below 19 °C. The impact of sudden increases in toxicant level (to 10 or 100 mg/L) on degradation was significant (negative), and was assessed by laboratory experiments with sediments. Total heterotrophic activity of sedimentary communities over a major part of the season was unaffected by pentachlorophenol at all stream concentrations tested.
Identification of ergosterol as a metabolite of Drechslera graminea and D. teres
Authors: Gordon T. R. , Webster R. K.
Published: April 2011

Ergosterol was detected in mycelial extracts of Drechslera graminea and D. teres. Chemical characterization of ergosterol was based on chromatographic behavior, ultraviolet absorptance, and mass spectrum.
The pattern of sex chromosome kinetochore phosphorylation during nonrandom segregation in a flea beetle
Authors: Kupfer Holly , Wise Dwayne
Published: April 2011

In the flea beetle species, Alagoasa bicolor, males have two sex chromosomes, X and Y, each of which is larger than the rest of the genome combined. These large sex chromosomes do not pair at meiosis I, and are therefore not joined at metaphase I. Nevertheless, they always segregate from each other at anaphase I. As prometaphase I progresses, the unpaired X and Y undergo reorientation from a parallel to a linear configuration. Using 3F3/2, an antibody that detects the level of phosphorylation of a kinetochore protein or proteins, we have determined that this reorientation is not accompanied by a change in the level of phosphorylation of the kinetochores of either X or Y. This implies that: i) either the reorientation does not involve the loss or gain of kinetochore microtubules, or ii) if such loss or gain occurs, it does not effect a change in the tension placed on the nonrandomly segregating kinetochores, or iii) the sex chromosomes, as in some other species, have lost the ability to sense kinetochore tension changes. Evolution of nonrandom segregation may necessitate the inability of the participating chromosomes to affect the metaphase checkpoint.

Key words: nonrandom segregation, sex chromosomes, kinetochores.
Effects of Bcl-2 and Bcl-XL protein levels on chemoresistance of hepatoblastoma HepG2 cell line
Authors: Luo Dan , Cheng Samuel Chak-Sum , Xie Hong , Xie Yong
Published: April 2011

The ratio between apoptotic promoters and repressors in the Bcl-2 family determines the chemosensitivity of cells to apoptotic stimuli. This study examines the chemoresistance of a transfected human hepatoblastoma HepG2 cell-line during Taxol and Doxorubicin application. Sense bcl-2, and anti-sense bcl-XL gene fragments were separately inserted into HepG2 cells via stable transfection. The expression profile of the Bcl-2 family proteins was determined by Western blot analysis. Chemosensitivity of the transfected cells was measured by Trypan blue exclusion assay and XTT reduction assay during drug application. In the absence of Bax protein, HepG2 cells with elevated Bcl-2 protein levels did not exhibit any significant increase in chemosensitivity towards the drugs. Transfected cells with reduced Bcl-XL levels became more sensitive to the drugs, and a significant difference in IC50 values was observed. The chemosensitivity of HepG2 cells to Taxol and Doxorubicin was not affected by Bcl-2 levels, while reduction of Bcl-XL levels rendered the cells more sensitive to the drugs. This suggests that the Bcl-2 protein alone could not protect HepG2 cells from drug-induced apoptosis, and that the Bcl-XL protein may be a target for gene therapy in hepatoblastoma treatment.

Key words: apoptosis, chemoresistance, Taxol, Doxorubicin, hepatoblastoma.
Prohormone transport through the secretory pathway of neuroendocrine cells
Authors: Kuiper Roland P , Martens Gerard JM
Published: April 2011

En route through the secretory pathway of neuroendocrine cells, prohormones pass a series of membrane-bounded compartments. During this transport, the prohormones are sorted to secretory granules and proteolytically cleaved to bioactive peptides. Recently, progress has been made in a number of aspects concerning secretory protein transport and sorting, particularly with respect to transport events in the early regions of the secretory pathway. In this review we will deal with some of these aspects, including: i) selective exit from the endoplasmic reticulum via COPII-coated vesicles and the potential role of p24 putative cargo receptors in this process, ii) cisternal maturation as an alternative model for protein transport through the Golgi complex, and iii) the mechanisms that may be involved in the sorting of regulated secretory proteins to secretory granules. Although much remains to be learned, interesting new insights into the functioning of the secretory pathway have been obtained.Key words: regulated secretory pathway, p24 family, vesicular transport, POMC, protein sorting, secretory granule, Xenopus laevis.
Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo
Authors: Flood Justin , Mayne Janice , Robinson John J
Published: April 2011

We have identified and partially characterized several gelatinase activities associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generally not found to be associated with isolated hyaline layers but was dissociated from the surface of 1-h-old embryos in the absence of Ca2+and Mg2+. While hyaline layers, freshly prepared from 1-h-old embryos, were devoid of any associated gelatinase activities, upon storage at 4°C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all species were inhibited by ethylenediamine tetraacetic acid but were refractory to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activities. While several of the storage-induced, hyaline-layer-associated gelatinase activities were inhibited, the 41-kDa collagenase/gelatinase was refractory to inhibition as was a second gelatinase species with an apparent molecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and quantitative assays, Ca2+was the most effective activator while Mn2+, Cu2+, Cd2+, and Zn2+were all inhibitory. In contrast, Mg2+had a minimal inhibitory effect on storage-induced gelatinase activities but significantly inhibited the 41-kDa collagenase/gelatinase. These results identify several distinct gelatin-cleavage activities associated with the sea urchin extraembryonic hyaline layer and point to diversity in the biochemical nature of these species.Key words: gelatinase, sea urchin, extracellular matrix.
Lectins from bulbs of the Chinese daffodilNarcissus tazetta(family Amaryllidaceae)
Authors: Ooi L SM , Ng T B , Geng Yunqi , Ooi V EC
Published: April 2011

The isolation of three lectins with similar N-terminal amino acid sequences from the bulbs of the Chinese daffodil Narcissus tazetta was achieved. The isolation protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on mannose-agarose, and fast protein liquid chromatography-gel filtration on Superose 12. The lectins were all adsorbed on mannose-agarose and demonstrated a single band with a molecular weight of 13 kDa in SDS-polyacrylamide gel electrophoresis and a single 26 kDa peak in gel filtration, indicating that they were mannose-binding, dimeric proteins. The lectins differed in hemagglutinating activity, with the magnitude of the activity correlating with the ionic strength of the buffer required to elute the lectin from the DEAE-cellulose column. The bulb lectin did not exert potent cytotoxicity against cancer cell lines or fetal bovine lung cells but inhibited syncytium formation in, and reinstated viability of, fetal bovine lung cells infected with bovine immunodeficiency virus.Key words: lectins, daffodil bulbs, chromatography.
Transforming growth factor β1selectively regulates ferritin gene expression in malignant H-ras-transformed fibrosarcoma cell lines
Authors: Lo James , Hurta Robert AR
Published: April 2011

Transforming growth factor β1is an important growth regulator in many cell types, usually exerting a negative effect on cellular growth. Inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation, and in some cases, tumor cell proliferation is actually stimulated by TGF-β1. The present study demonstrates a novel link between alterations in TGF-β1regulation during malignant conversion, and the expression of ferritin, an important activity involved in a number of biological functions including iron homeostasis and cell-growth control. A series of H-ras-transformed mouse 10 T 1/2 cell lines, exhibiting increasing malignant potential, was investigated for possible TGF-β1-mediated changes in ferritin gene expression. Selective induction of gene expression was observed, since only H-ras-transformed cells with malignant potential exhibited marked elevations in ferritin gene expression, in particular, alterations in H-ferritin gene expression. The regulation of H-ferritin gene expression in response to TGF-β1did not involve alterations in transcription, but occurred through mechanisms of post-transcriptional stabilization of the H-ferritin mRNA. Additionally, evidence was obtained for a cycloheximide-sensitive regulator of H-ferritin gene expression, since the presence of this protein synthesis inhibitor increased H-ferritin message levels, and in combination with TGF-β1, cooperated in an additive manner to augment H-ferritin gene expression. These results show for the first time that TGF-β1can regulate ferritin gene expression in malignant H-ras transformed cells, and suggest a mechanism for growth factor stimulation of malignant cells, in which early alterations in the control of H-ferritin gene expression are important.Key words: TGF-β1, ferritin gene expression, malignant transformation.
Changes in lung-specific molecular expression during differentiation of hamster embryonic M3E3/C3 cell line
Authors: Yoshimi Tatsuya , Takahashi Yuji , Takahashi Shigeru , Miura Takashi
Published: April 2011

To clarify the differentiation mechanisms of bronchiolar epithelial cells, changes at the transcription level of epithelial cell-specific proteins were examined using M3E3/C3, a cell line derived from hamster fetal lung. During a 9-day incubation period with 24 µg/mL of retinol, the cells became attached to each other and formed large bud-like structures which could be detected with periodic acid-Schiff staining. During the incubation period, the mRNA level of surfactant-associated protein-B significantly increased 2.6- and 5.4-fold higher than cells incubated without retinol on days 3 and 9, respectively. The Clara cell-specific secretory protein mRNA level also increased and peaked at 5.1-fold (P < 0.05) on day 5 compared with control cells. In contrast, mRNA for surfactant-associated protein-C, an alveolar type II cell-specific protein, decreased. Moreover, the expression of the gene for hepatocyte nuclear factor 3α, a putative transactivating factor for lung-related genes, was up-regulated resulting in consistently higher levels (2.4- to 5.6-fold) compared with controls, while those for transmembrane-type mucin-1 and glyceraldehyde-3-phosphate dehydrogenase were constantly expressed during the incubation. The present study confirms that at the gene transcription level M3E3/C3 cells differentiate into Clara-like cells with mucus granules in the presence of retinol.Key words: lung, Clara cell, differentiation, gene transcription, surfactant protein.
Effect of oxidative stress on stability and structure of neurofilament proteins
Authors: Gélinas Sylvie , Chapados Camille , Beauregard Marc , Gosselin Isabelle , Martinoli Maria-Grazia
Published: April 2011

Neurofilament proteins are highly phosphorylated molecules in the axonal compartment of the adult nervous system. We report the structural analysis of neurofilament proteins after oxidative damage. SDS-PAGE, immunoblotting, circular dichroism, and Fourier transform infrared spectroscopy were used to investigate the relative sensitivity of neurofilaments to oxidative stress and to identify changes in their molecular organization. An ascorbate-Fe+3-O2 buffer system as well as catechols were used to generate free radicals on a substrate of phosphorylated and dephosphorylated neurofilaments. By Fourier Transform Infrared spectroscopy and circular dichroism, we established that the neurofilament secondary structure is mainly composed of α-helices and that after free radical damage of the peptide backbone of neurofilaments, those helices are partly modified into β-sheet and random coil structures. These characteristic reorganizations of the neurofilament structure after oxidative exposure suggest that free radical activity might play an important role in the biogenesis of the cytoplasmic inclusions found in several neurodegenerative diseases.Key words: neurofilaments, oxidative stress, neurodegeneration, phosphorylation, infrared spectroscopy, circular dichroism.
Regulation and function of FGF8 in patterning of midbrain and anterior hindbrain
Authors: Mason Ivor , Chambers David , Shamim Huma , Walshe Jennifer , Irving Carol
Published: April 2011

In this article, an adjunct to a platform presentation at the Winternational 2000 Symposium, we summarize the recent findings of this group concerning the regulation and functions of FGF8 expressed at the isthmus of the developing brain. We show that several different FGF8 isoforms, ectopically expressed in midbrain or posterior forebrain, are able to mimic the proliferative and patterning functions previously attributed to the isthmus in tissue grafting studies. Moreover, we also show that FGF8 protein is sufficient to induce an ectopic isthmic organiser (Fgf-8+, Gbx2+) in anterior midbrain. We also provide evidence that isthmic FGF8 patterns anterior hindbrain, repressing Hox-a2 expression and setting aside a territory of the brain that includes the cerebellar anlage. We show that these effects of FGF8 are likely to be mediated via FGFR1 and be modulated by the putative FGF antagonist, Sprouty2, identified using a differential display screen. Finally, we provide evidence that the onset of Fgf8 expression is regulated by En1 and that its expression at the isthmus is subsequently maintained by a specific and direct interaction between rhombomere 1 and midbrain.
Androgen responsiveness and intrarenal localization of transcripts coding for the enzymes of polyamine metabolism in the mouse
Authors: Bettuzzi Saverio , Strocchi Paola , Davalli Pierpaola , Marinelli Maria , Furci Luciana , Corti Arnaldo
Published: April 2011

Polyamines, spermidine (SPD), and spermine (SPM) are intracellular polycations required for cell growth and differentiation. Their biosynthetic precursor, the diamine putrescine (PUT), is produced by regulatory ornithine decarboxylase (ODC). Spermidine/spermine N1-acetyltransferase (SSAT) is the ODC counterpart in the degradation pathway which retroconverts SPM and SPD into PUT. Castration of male mice for 7 days resulted in a 40% decrease of the renal levels of both SSAT and ODC transcripts. Administration of 5-α-dihydrotestosterone (DHT) to castrated mice for the last 3 days before sacrifice caused the levels of ODC and SSAT mRNAs to increase by 250% and 180%, respectively. Thus activation of the retroconversion pathway of polyamine metabolism appears to contribute towards the increase in PUT production known to be caused by androgens in the mouse kidney. In situ hybridization histochemistry experiments showed that the SSAT transcript is expressed only by the epithelial cells of the straight and convoluted distal tubules of the nephron, while the expression of the ODC transcript is confined to the epithelium of the convoluted and straight portion of the proximal tubules. The separation of the biosynthetic from the degradation pathway along the nephron suggests that PUT is mostly produced in the distal tubule, where it may play a physiological role, independent of androgen action, in protecting tubular cells from the very low osmolarity to which they are exposed in this nephron segment.Key words: Adenosylmethionine decarboxylase, spermidine/spermine N1-acetyltransferase, ornithine decarboxylase, mouse kidney, polyamines.
Identification of a transcription factor, an 80-kDa protein that interacts with the HLH recognition motif of the rat p53 promoter
Authors: Song Haisun , Lee Minhyung , Yu Sunhee , Park Jong-sang
Published: April 2011

The p53 promoter has been shown to contain a number of potential regulatory motifs. It was previously reported that the upstream stimulating factor (USF) played a central role in regulating the p53 expression. The USF binding site, E-box, is located around 40 bp upstream of the major transcription start site. In this study, it was confirmed that the E-box binds to proteins by DNase I footprinting assay. In the electrophoretic mobility shift assay (EMSA), two retarded bands were detected. One band was abolished by the competition of USF consensus oligonucleotide, but the other band was not. This result indicated that a factor, other than USF, was bound to the E-box. The molecular masses of the binding proteins were determined by a Southwestern-blotting assay. As a result, 46- and 80-kDa proteins were detected. The 46-kDa protein was eliminated by the competition of USF consensus oligonucleotide. Also, the Southwestern-blotting assay with32P-labeled USF consensus oligonucleotide showed only a 46-kDa protein. Therefore, the 46-kDa protein was USF. These results showed that USF and the 80-kDa protein were bound to the E-box. In addition, it was proved by in vitro transcription assay that this 80-kDa protein had a basal transcriptional activity.Key words: E-box, HLH, rat p53 promoter, transcription factor, upstream stimulating factor (USF).
Lipid microspheres incorporated by U937 cells via their specific receptors
Authors: Suzuki Kimihiro , Hidaka Toshihiko , Shinohara Tadashi , Kataharada Kouji , Okada Makoto , Takada Kunio , Ohsuzu Fumitaka
Published: April 2011

Lipid microspheres (LM), currently in clinical use as drug carriers, mainly consist of soybean oil as a core and lecithin as a surfactant. The purpose of our study wass to determine whether or not LM incorporation is receptor-mediated. U937 cells resuspended in a serum-free medium abundantly took up unmodulated LM. A binding study showed that U937 cells had a single binding site for LM (410 sites/cell at 24°C; 100 sites/cell at 4°C). Inhibition assays revealed that lecithin liposome, lysophosphatidylcholines, activated α2-macroglobulin, and HDL did not affect the binding of LM to U937 cells. VLDL strongly, and LDL and AcLDL moderately, inhibited the binding of LM to U937 cells. Ligand blotting analysis revealed that unmodulated LM in an apoprotein-free buffer directly bound to a 40 kDa protein in the cell membrane fraction. These results suggest that LM that is not modulated by any protein is incorporated by specific cells via receptor-mediated processes.Key words: lipid emulsion, drug delivery system, monocyte, free fatty acids.
Array analysis of the genes regulated during neuronal differentiation of human embryonal cells
Authors: Bani-Yaghoub Mahmud , Felker Josh M , Ozog Mark A , Bechberger John F , Naus Christian C.G
Published: April 2011

Recent advances in genetic technology have provided a new platform on which the simultaneous analysis of a large number of genes is possible in a rapid and efficient fashion. To assess the differential expression of human genes during neuronal differentiation, we compared the transcript profiles of undifferentiated, partially differentiated, and fully differentiated NT2/D1 cultures with cDNA expression arrays. Approximately 75 genes (13% of the gene array pool) were differentially expressed during neuronal development of NT2/D1 cells. Genes coding for pyruvate kinase M2 isozyme, clathrin assembly proteins, calmodulin, fibronectin, laminin, thymosin β-10, and many others were upregulated as NT2/D1 cells differentiated into neurons. In contrast, several kinases, phosphatases, and G-protein coupled receptor genes showed downregulation upon neuronal differentiation. The information provided here is an invaluable reference for characterizing the phenotype of these cells. This information can also be used in cell therapy and transplantation in which the graft microenvironment and interaction with the host tissue is crucial.Key words: Atlas cDNA expression arrays, differentiation, neurodevelopment, neuron, NT2/D1 cells.
Helicobacter pylorifrom asymptomatic hosts expressing heptoglycan but lacking Lewis O-chains: Lewis blood-group O-chains may play a role inHelicobacter pyloriinduced pathology
Authors: Monteiro Mario A , Michael Frank St , Rasko David A , Taylor Diane E , Conlan J Wayne , Chan Ken H , Logan Susan M , Appelmelk Ben J , Perry Malcolm B
Published: April 2011

Helicobacter pylori is a widespread Gram-negative bacterium responsible for the onset of various gastric pathologies and cancers in humans. A familiar trait of H. pylori is the production of cell-surface lipopolysaccharides (LPSs; O-chain [Formula: see text] core [Formula: see text] lipid A) with O-chain structures analogous to some mammalian histo-blood-group antigens, those being the Lewis determinants (Lea, Leb, Lex, sialyl Lex, Ley) and blood groups A and linear B. Some of these LPS antigens have been implicated as autoimmune, adhesion, and colonization components of H. pylori pathogenic mechanisms. This article describes the chemical structures of LPSs from H. pylori isolated from subjects with no overt signs of disease. Experimental data from chemical- and spectroscopic-based studies unanimously showed that these H. pylori manufactured extended heptoglycans composed of 2- and 3-linked D-glycero-α-D-manno-heptopyranose units and did not express any blood-group O-antigen chains. The fact that another H. pylori isolate with a similar LPS structure was shown to be capable of colonizing mice indicates that H. pylori histo-blood-group structures are not an absolute prerequisite for colonization in the murine model also. The absence of O-chains with histo-blood groups may cause H. pylori to become inept in exciting an immune response. Additionally, the presence of elongated heptoglycans may impede exposure of disease-causing outer-membrane antigens. These factors may render such H. pylori incapable of creating exogenous contacts essential for pathogenesis of severe gastroduodenal diseases and suggest that histo-blood groups in the LPS may indeed play a role in inducing a more severe H. pylori pathology.Key words: lipopolysaccharide, carbohydrates, glycobiology, Helicobacter pylori, histo-blood groups.
Nuclear envelope dynamics
Authors: Salina Davide , Bodoor Khaldon , Enarson Paul , Raharjo Wahyu Hendrati , Burke Brian
Published: April 2011

The nuclear envelope (NE) provides a semi permeable barrier between the nucleus and cytoplasm and plays a central role in the regulation of macromolecular trafficking between these two compartments. In addition to this transport function, the NE is a key determinant of interphase nuclear architecture. Defects in NE proteins such as A-type lamins and the inner nuclear membrane protein, emerin, result in several human diseases that include cardiac and skeletal myopathies as well as lipodystrophy. Certain disease-linked A-type lamin defects cause profound changes in nuclear organization such as loss of peripheral heterochromatin and redistribution of other nuclear envelope components. While clearly essential in maintenance of nuclear integrity, the NE is a highly dynamic organelle. In interphase it is constantly remodeled to accommodate nuclear growth. During mitosis it must be completely dispersed so that the condensed chromosomes may gain access to the mitotic spindle. Upon completion of mitosis, dispersed NE components are reutilized in the assembly of nuclei within each daughter cell. These complex NE rearrangements are under precise temporal and spatial control and involve interactions with microtubules, chromatin, and a variety of cell-cycle regulatory molecules.Key words: nuclear envelope, lamin, nuclear pore complex, nuclear membranes, mitosis.
Mechanism of substrate recognition by the chaperonin GroEL
Authors: Houry Walid A
Published: April 2011

The bacterial chaperonin GroEL functions with its cofactor GroES in assisting the folding of a wide range of proteins in an ATP-dependent manner. GroELGroES constitute one of the main chaperone systems in the Escherichia coli cytoplasm. The chaperonin facilitates protein folding by enclosing substrate proteins in a cage defined by the GroEL cylinder and the GroES cap where folding can take place in a protected environment. The in vivo role of GroEL has recently been elucidated. GroEL is found to interact with 1015% of newly synthesized proteins, with a strong preference for proteins in the molecular weight range of 2060 kDa. A large number of GroEL substrates have been identified and were found to preferentially contain proteins with multiple αβ domains that have α-helices and β-sheets with extensive hydrophobic surfaces. Based on the preferential binding of GroEL to these proteins and structural and biochemical data, a model of substrate recognition by GroEL is proposed. According to this model, binding takes place preferentially between the hydrophobic residues in the apical domains of GroEL and the hydrophobic faces exposed by the β-sheets or α-helices in the αβ domains of protein substrates.Key words: chaperone, folding, binding, hydrophobic interaction, structure.
Isolation and characterization of the muscle-specific isoform of creatine kinase from the zebrafish,Danio rerio
Authors: Harder Gregory , McGowan Ross
Published: April 2011

We have isolated and characterized a cDNA sequence corresponding to the zebrafish muscle-specific isoform of creatine kinase. The sequence is 1552 bases in length and contains an open reading frame capable of producing a 381 amino acid protein. The sequence is very similar to muscle-specific creatine kinases isolated from other species at both the nucleotide and amino acid levels but contains some differences from a previously reported zebrafish clone.Key words: creatine kinase, muscle isoform, zebrafish, Danio rerio.
Aldolase-localization in cultured cells: Cell-type and substrate-specific regulation of cytoskeletal associations
Authors: Schindler Ralf , Weichselsdorfer Elke , Wagner Oliver , Bereiter-Hahn Jürgen
Published: April 2011

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (202000 µM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 2060 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actinaldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.Key words: aldolase, cytoskeleton, electron microscopy, viscosimetry.
Assembly strategies and GTPase regulation of the eukaryotic andEscherichia colitranslocons
Authors: Legate Kyle R , Andrews David W
Published: April 2011

The translocation of most proteins across the endoplasmic reticulum or bacterial inner membrane occurs through an aqueous pore that spans the membrane. Substrates that are translocated co-translationally across the membrane are directed to the translocation pore via an interaction between the cytosolic signal recognition particle and its membrane-bound receptor. Together the translocation pore and the receptor are referred to as a translocon. By studying the biogenesis of the translocon a number of alternate targeting and membrane-integration pathways have been discovered that operate independently of the signal recognition particle (SRP) pathway. The novel assembly strategies of the translocon and the ways in which these components interact to ensure the fidelity and unidirectionality of the targeting and translocation process are reviewed here.Key words: protein translocation, translocon, SRP receptor, GTPases.
The dynamics of karyopherin-mediated nuclear transport
Authors: Marelli Marcello , Dilworth David J , Wozniak Richard W , Aitchison John D
Published: April 2011

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.Key words: Nucleocytoplasmic transport, nuclear pore complex, nucleoporin, karyopherin, Nup2p.
Purification of developmentally regulated avian 400-kDa intermediate filament associated protein. Molecular interactions with intermediate filament proteins and other cytoskeleton components
Authors: Duval Marie , Ma Xiaoying , Valet Jean-Paul , Vincent Michel
Published: April 2011

IFAPa-400, a 400-kDa developmentally regulated protein thought to be associated with intermediate filaments, has been purified from chick embryo hearts to investigate its interaction with vimentin and other IF proteins and to identify other cellular components to which this cytoskeletal protein associates. Previous studies suggested that this protein was associated with the vimentin-containing intermediate filament lattice of myoblasts and neuroblasts before their terminal differentiation, providing these cells with a particular intermediate filament cytoskeleton that could satisfy specific mechanical requirements during their intense morphogenetic activities. Although IFAPa-400 partially reassociated with vimentin and desmin in disassembly–reassembly experiments using crude IF preparations from chick embryo hearts, in vitro recombination of purified IFAPa-400 with vimentin and desmin failed to demonstrate any direct association. When purified IFAPa-400 was used as a probe in blot overlay assays, however, specific binding to vimentin and desmin was observed, providing the first evidence of a physical association between IFAPa-400 and intermediate filament proteins. The blot overlay experiments also demonstrated that IFAPa-400 binds to two unidentified polypeptides of 19 and 32 kDa. These results are thus consistent with the hypothesis that a structural lattice requiring a vimentin–IFAPa-400 combination constitutes the intermediate filament system of myogenic and neurogenic cells.Key words: cytoskeleton, intermediate filaments, intermediate filament associated proteins, vimentin, IFAPa-400.
Production and utilization of detyrosinated tubulin in developingArtemialarvae: evidence for a tubulin-reactive carboxypeptidase
Authors: Xiang Hui , MacRae Thomas H.
Published: April 2011

The reversible, enzymatically driven removal and readdition of its carboxy-terminal tyrosine are major posttranslational modifications of α-tubulin. To study these processes isoform-specific antibodies were produced and subsequently used to characterize tyrosinated and detyrosinated tubulin in the brine shrimp, Artemia. Tyrosinated tubulin existed in relatively constant amounts on western blots of cell-free protein extracts from Artemia at all developmental stages examined, whereas detyrosinated tubulin was present after 20–24 h of postgastrula growth. In agreement with the blots, the detyrosinated isoform was observed in immunofluorescently stained larvae after 24 h of incubation, appearing first in structures of a transient nature, namely spindles and midbodies. The elongated muscle cells encircling the gut and the epithelium bordering the gut lumen were stained extensively with antibody to detyrosinated tubulin. Detyrosination was accompanied by the appearance of a tubulin-reactive carboxypeptidase, which used both nonpolymerized and polymerized tubulin as substrate. The enzyme bound to microtubules very poorly, if at all, under conditions used in this work. Several inhibitors of carboxypeptidase A had no effect on the carboxypeptidase from Artemia and revealed similarities between this enzyme and others thought to be tubulin specific. The use of inhibitors also indicated that the carboxypeptidase from Artemia recognized aspects of tubulin structure in addition to the carboxyterminal tyrosine. Our results support the idea that detyrosinated tubulin appears in microtubules of varying stability, and they demonstrate that Artemia possess a carboxypeptidase with the potential to detyrosinate tubulin during growth of larvae.Key words: tubulin, detyrosination, carboxypeptidase, Artemia.
Purification and partial characterization of peptidase-1, a sex-linked enzyme inPleurodeles waltl(urodele amphibian)
Authors: Rudolf Etienne , Girardet Jean-Michel , Bautz Anne-Marie , Dournon Christian
Published: April 2011

Peptidase-1 is a sex-linked enzyme, which can be purified from the liver of the amphibian urodele Pleurodeles waltl. We estimated its apparent molecular mass as 170 kDa by gel filtration chromatography. The enzyme is composed of two subunits with apparent molecular masses of 90 and 99 kDa. It is strongly inhibited by ethylenediaminotetraacetic acid, ethylene glycol bis( beta -aminoethyl ether)-N,N-tetraacetic acid, and 1,10-phenanthroline, indicating that peptidase-1 is a metallopeptidase. Peptidase-1 has optimal activity at 55°C and pH 8.5. This acidic enzyme displays two apparent isoelectric points, at 4.9 and 5.2, and is essentially located in the cytosolic subcellular fraction.

Key words: peptidase-1, amphibian, purification, characterization, sex-linked enzyme.
5'-3' Interactions in regulation of translation in Xenopus early embryos
Authors: Browder Leon W , Wilkes-Johnston Jillian , Fraser Sherri D
Published: April 2011

We have investigated the effects of 3' noncoding elements in enhancing translation of messengers having translation-inhibiting 5' untranslated regions (UTRs). The translation of transcripts bearing the 5' UTRs of either human c-myc or a synthetic hairpin structure upstream of a chloramphenicol acetyltransferase (CAT) reporter sequence is greatly attenuated in early embryos of Xenopus laevis. Translation of transcripts bearing the human c-myc-5' UTR was markedly stimulated by the presence of 3' poly(A). Transcripts bearing the 5' hairpin element were insensitive to the presence of poly(A), but they were extremely sensitive to the composition of the 3' UTR. A GC-rich distal sequence repressed translation, whereas a proximal GGAAU sequence promoted translation of these transcripts. Our results support the concept that long-range interactions between the 5' and 3' ends of transcripts are important in regulating translation in Xenopus embryos.

Key words: translational regulation, oncogenes, c-myc, Xenopus laevis, development, embryo.
Analysis of complex formation between Hsp80 and Hsp70, cytosolic molecular chaperones ofNeurospora crassa, by enzyme-linked immunosorbent assays (ELISA)
Authors: Ouimet P M , Kapoor M
Published: April 2011

A physical association between Hsp70 and Hsp80, the major cytosolic stress proteins of Neurospora crassa, was demonstrated previously by interprotein crosslinking and by binding of Hsp80 to Hsp70 immobilized on ATP-agarose. In the present study, [Hsp70:Hsp80] complex formation was analyzed by enzyme-linked immunosorbent assays (ELISA), using specific antibodies. One protein was fixed onto ELISA plate wells and binding of the second mobile protein was monitored by retention of its cognate IgG. Binding of Hsp70 and Hsp80 to immobilized Hsp80 and Hsp70, respectively, was readily detectable at submicrogram levels. The effect of cations and various nucleotides on [Hsp70:Hsp80] complex was examined by inclusion of KCl, MgCl2, MnCl2, and nucleotides in the interaction mixture. K+stimulated interaction between immobilized Hsp70 and Hsp80 in solution and adenosine nucleotides exerted a stimulatory effect on complexation as well. Similarly, CTP, NAD, and NADH enhanced complex formation between plate-bound Hsp70 and Hsp80 in solution, attesting to the conformational flexibility of Hsp80. Epitope blocking revealed an overlap between protein-protein contact surfaces and antibody recognition sites. Binding to alpha -carboxymethylated lactalbumin showed that Hsp70 and Hsp80 can interact with an unfolded polypeptide, individually and in complex.Key words: molecular chaperones, Neurospora, heat shock proteins, Hsp80:Hsp70 complex.
Cardiac magnetic resonance spectroscopy
Authors: Deslauriers Roxanne , Kupriyanov Valery V
Published: April 2011

The article reviews cardiac magnetic resonance spectroscopy (MRS) in Canada. 31P MRS has been used to study cardiac energetics and intracellular pH in hearts subjected to ischemia-reperfusion and to evaluate the effects of pharmacological interventions. 23Na, 87Rb, and 7Li MRS have provided unique probes to study ion balance and fluxes in intact tissue under normal and stressful physiological conditions. 1H MRS has been used to monitor the accumulation of lactate and lipids in hearts subjected to ischemia-reperfusion and follow the effects of diet on cardiac lipid levels and function. The isolated rat heart has been used most commonly to study the effects of pharmacological agents on energy balance, pH, ion fluxes, and contractile function of the heart subjected to ischemia-reperfusion. The pig heart has been developed as an alternative to the rodent heart because its metabolism is more similar to that of the human heart. Human atrial appendages have been useful in evaluating the effects of preservation strategies (temperature, composition of preservation solutions) on energy levels. The pig heart model has been useful in evaluating the effects of preservation solutions on cardiac function of hearts destined for transplantation. An isolated blood-perfused pig heart model has been developed to assess the effects of cardioplegic strategies on the preservation of contractile function of hearts following surgery on the heart. An in vivo canine model has been used to study myocardial infarction and the effects of therapies to reduce the infarct zones and areas of the heart at risk of infarction. Studies of human hearts in vivo have provided insight into the metabolic adaptations that occur in individuals living at high altitudes.Key words: ion transport, metabolism, heart disease, organ preservation, drug effects.