bluuebunny/crossref_metadata_2025 huggingface dataset:
Site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide
Authors: Ferris F. G. , Beveridge T. J.
Published: April 2011

The site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide was assessed by collecting high-resolution phosphorus nuclear magnetic resonance spectra in the presence of manganese, a paramagnetic divalent cation. This technique revealed high-affinity interactions between the cation and all of the lipopolysaccharide phosphoryl groups. To ascertain whether the carboxyl groups of 2-keto-3-deoxyoctonate contributed to the metal cation binding, lipopolysaccharide was chemically modified using a glycine ethyl ester – carbodiimide reaction. Of the three available carboxyl groups, only one was neutralized by the exogenously added ligand; the others appeared to be cross-linked within the molecule. By analogy, only one carboxyl group should be freely available for binding metallic ions, while the others are probably neutralized by the close proximity of endogenous amino substituents. Although high-resolution phosphorus nuclear magnetic resonance showed that an intermolecular conformational change had occurred after the carboxyl groups were neutralized, titration with manganese revealed no differences in the apparent strength of the interactions between the cation and the phosphoryl groups. Together, these data suggest that the high affinity of lipopolysaccharide for divalent metallic ions can be attributed primarily to the phosphoryl substituents and not free carboxyl groups.
Function- and agonist-specific Ca2+signalling: The requirement for and mechanism of spatial and temporal complexity in Ca2+signals
Authors: Johnson James D , Chang John P
Published: April 2011

Calcium signals have been implicated in the regulation of many diverse cellular processes. The problem of how information from extracellular signals is delivered with specificity and fidelity using fluctuations in cytosolic Ca2+concentration remains unresolved. The capacity of cells to generate Ca2+signals of sufficient spatial and temporal complexity is the primary constraint on their ability to effectively encode information through Ca2+. Over the past decade, a large body of literature has dealt with some basic features of Ca2+-handling in cells, as well as the multiplicity and functional diversity of intracellular Ca2+stores and extracellular Ca2+influx pathways. In principle, physiologists now have the necessary information to attack the problem of function- and agonist-specificity in Ca2+signal transduction. This review explores the data indicating that Ca2+release from diverse sources, including many types of intracellular stores, generates Ca2+signals with sufficient complexity to regulate the vast number of cellular functions that have been reported as Ca2+-dependent. Some examples where such complexity may relate to neuroendocrine regulation of hormone secretion/synthesis are discussed. We show that the functional and spatial heterogeneity of Ca2+stores generates Ca2+signals with sufficient spatiotemporal complexity to simultaneously control multiple Ca2+-dependent cellular functions in neuroendocrine systems.Key words: signal coding, IP3receptor, ryanodine receptor, endoplasmic reticulum, Golgi, secretory granules, mitochondria, exocytosis.
Disruption of LT-antigen/p53 complex by heat treatment correlates with inhibition of DNA synthesis during transforming infection with SV40
Authors: Khandjian Edouard W , Rose Timothy M
Published: April 2011

Transforming infection of Go/G1-arrested primary mouse kidney cell cultures with simian virus 40 (SV40) induces cells to re-enter the S-phase of the cell cycle. In Go-arrested cells, no p53 is detected, whereas in cells induced to proliferate by infection, a gradual accumulation of p53 complexed to SV40 large T-antigen is observed in the nucleus. Heat treatment of actively proliferating SV40-infected cells leads to inhibition of DNA synthesis and growth arrest. To determine the fate of p53 after heat treatment, proliferating infected cells were exposed to mild heat (42.5°C) for increasing lengths of time. The results presented here show that after ninety minutes of treatment, the arrest of DNA synthesis by heat correlates with the disruption of the p53/LT-antigen complex. Longer treatments induce, in addition, a reduction in the solubility of p53, which was recovered tightly associated with the nuclear fraction. This contrasted with large T-antigen, whose solubility remained unaffected by heat treatment. Although the total amount of p53 in the nucleus remained constant, as shown by immunoblot analyses, p53 was no longer detectable after immunoprecipitation or by immunofluorescent staining techniques. These results suggest that heat treatment had either induced conformational changes in its antigenic sites, or had sequestered the sites through aggregation or binding to insoluble nuclear components.Key words: p53, heat shock, LT-antigen/p53 complex, S-phase.
Attraction vs. repulsion: The growth cone decides
Authors: McFarlane Sarah
Published: April 2011

Axons are guided through their environment in response to signals provided by extracellular cues. These cues are transduced into motile responses by the tip of the growing axon, the growth cone, and can be either repulsive or attractive in nature. Recent studies have suggested that how an axon responds to any given signal depends on the internal state of the growth cone. This review discusses these studies and their importance for understanding how nerve connections are made in the developing embryo.Key words: growth cone, axon guidance, calcium, cyclic nucleotides.
Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: Evidence for two distinct enzyme forms
Authors: Thatcher Bradley J , Storey Kenneth B
Published: April 2011

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37°C body temperature) and hibernating (torpid, 5°C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5 ± 2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335 ± 5 kDa for euthermic and 320 ± 5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Kmvalues for glutamate, NH4+, and α-ketoglutarate, as well as in Kaand IC50values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5°C). For example, the Kmfor α-ketoglutarate of euthermic GDH was higher at 5°C (3.66 ± 0.34 mM) than at 37°C (0.10 ± 0.01 mM), whereas hibernator GDH had a higher affinity for α-ketoglutarate at 5°C (Kmwas 0.98 ± 0.08 mM at 37°C and 0.43 ± 0.02 mM at 5°C). Temperature effects on KaADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37°C and weakest with hibernator GDH at 5°C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.Key words: mammalian hibernation, amino acid metabolism, temperature-dependent enzyme kinetics, Spermophilus richardsonii.
Addition of a c-myc epitope tag within the VEGF protein does not affect in vitro biological activity
Authors: Chavand Olivier , Spilsbury Katrina , Rakoczy Piroska E
Published: April 2011

The overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in diseases involving neovascularization. VEGF exists in as many as six different isoforms, each showing a unique pattern of tissue distribution and activity. To investigate the effect of individual VEGF isoform overexpression in neovascular disease models, we inserted c-myc epitope tags into the three VEGF isoforms expressed in retinal pigment epithelial cells, VEGF121, VEGF165, and VEGF189. We found that the 12-amino acid insertion between the receptor binding and heparin binding domains did not affect VEGF transcription, translation, or secretion. In addition, VEGF isoforms containing the c-myc epitope tag were able to stimulate endothelial cell proliferation as efficiently as non-tagged VEGF isoforms and they could be individually identified by Western blotting and immunocytochemistry using the c-myc epitope specific monoclonal antibody 9E10.Key words: Vascular endothelial growth factor, VEGF, c-myc epitope tag, immunocytochemistry.
Contact-activated migration of melanoma B16 and sarcoma XC cells
Authors: Madeja Zbigniew , Szymkiewicz Iwona , Żaczek Anna , Sroka Jolanta , Miękus Katarzyna , Korohoda Włodzimierz
Published: April 2011

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 4050 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 µM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 µM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.Key words: cell movement, metastases, contact activation of cell migration, contact inhibition.
Higher-order folding of heterochromatin: Protein bridges span the nucleosome arrays
Authors: Grigoryev Sergei A
Published: April 2011

In interphase eukaryotic nuclei, chromatin is divided into two morphologically distinct types known as heterochromatin and euchromatin. It has been long suggested that the two types of chromatin differ at the level of higher-order folding. Recent studies have revealed the features of chromatin 3D architecture that distinguish the higher-order folding of repressed and active chromatin and have identified chromosomal proteins and their modifications associated with these structural transitions. This review discusses the molecular and structural determinants of chromatin higher-order folding in relation to mechanism(s) of heterochromatin formation and genetic silencing during cell differentiation and tissue development.Key words: heterochromatin, nucleosome, histone, higher-order folding, chromatin 3D structure.
Induction of apoptosis in HT29 human intestinal epithelial cells by the cytotoxic enterotoxin ofAeromonas hydrophila
Authors: Falcón Rosabel M , Carvalho Hernandes F , Joazeiro Paulo Pinto , Gatti Maria S. Viccari , Yano Tomomasa
Published: April 2011

The cytotoxic enterotoxin produced by Aeromonas hydrophila is considered to be the main virulence factor in gastrointestinal infections mediated by this pathogen. In this study, we examined the morphological and apoptotic effects of this toxin on HT29 cells, using light and electron microscopy in situ, as well as agarose gel electrophoresis of cell DNA. Cells treated with the cytotoxic enterotoxin became round and lost their polarity as well as their adhesion to each other and to the substrate. Cytoplasmic blebbing and nuclear condensation also occurred. DNA fragmentation was detected by TUNEL labelling and agarose gel electrophoresis. These results show that the cytotoxic enterotoxin of A. hydrophila can induce apoptosis in human intestinal cells in culture.Key words: Aeromonas hydrophila, cytotoxic enterotoxin, aerolysin, apoptosis, human intestinal cells.
Influence of linker histone H1 on chromatin remodeling
Authors: Hill David A
Published: April 2011

Chromatin-remodeling complexes have been a central area of focus for research dealing with accessing cellular DNA sequestered in chromatin. Although the linker histone H1 plays a major role in promoting and maintaining higher-order chromatin structure, it has been noticeably absent from assays utilizing chromatin-remodeling enzymes. This review focuses on two ATP-dependent chromatin-remodeling complexes, Drosophila ISWI and mammalian SWI/SNF, that have been assayed using chromatin templates containing histone H1.Key words: SWI/SNF, ISWI, chromatin remodeling, histone H1.
Structural features of trinucleotide repeats associated with DNA expansion
Authors: Kovtun Irina V , Goellner Geoffrey , McMurray Cynthia T
Published: April 2011

The mechanism of DNA expansion is not well understood. Recent evidence from genetic, in vivo, and in vitro studies has suggested a link between the formation of alternative DNA secondary structures by trinucleotide repeat tracts and their propensity to undergo expansion. This review will focus on structural features and the mechanism of expansion relevant to human disease.Key words: expansion, hairpin, trinucleotide repeat, polymerase slippage, recombination, repair.
Deglycosylated ceruloplasmin maintains its enzymatic, antioxidant, cardioprotective, and neuronoprotective properties
Authors: Aouffen M'hammed , Paquin Joanne , Grandpré Eric De , Nadeau Réginald , Mateescu Mircea-Alexandru
Published: April 2011

Ceruloplasmin (CP), an important serum antioxidant, is a blue copper glycoprotein with ferroxidase and oxidase activities. Among other physiological actions, plasma CP was shown to protect isolated rat hearts and cultured P19 neurons exposed to oxidative stress conditions, raising the possibility of using this protein in the treatment of cardiac and neuronal diseases related to oxidative damage. However, since therapeutic applications of CP must be compatible with restrictions in the administration of blood derivatives to humans, there is a need to produce the protein by genetic engineering. To help in the choice of adequate expression systems, we undertook this study to determine if the carbohydrate moiety on the protein is essential for its functions. CP was completely deglycosylated using N-glycosidase F under nondenaturing conditions. Deglycosylated CP was found to retain most of the conformational, antioxidant, and enzymatic properties of the native protein in vitro. Moreover, both forms of the protein had similar cardioprotective and neuronoprotective effects against oxidative stress as evaluated with isolated rat hearts undergoing ischemiareperfusion and with cultured P19 neurons exposed to xanthine  xanthine oxidase. The data thus indicate that the carbohydrate moiety of CP is not essential for its enzymatic and protective actions. Accordingly, even the use of expression systems that do not glycosylate mammalian proteins could provide a recombinant CP that retains its therapeutic potential.Key words: copperproteins, protein-linked carbohydrates, ischemia-reperfusion, isolated rat hearts, cultured P19 neurons.
Purification and characterization of cholesterol sulfotransferase from rat skin
Authors: Rearick James I , Calhoun Eric S
Published: April 2011

Previous work has demonstrated that the activity of the enzyme cholesterol sulfotransferase is rapidly and dramatically increased upon squamous differentiation of a variety of epithelial cells in culture, including epidermal keratinocytes. As a step toward understanding the molecular mechanisms underlying this differentiation-related change, we now report the partial purification and characterization of this enzyme activity from rat skin. Supernatant solutions from rat skin homogenates were subjected to a series of column chromatography steps including anion exchange, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The purification procedure resulted in cholesterol sulfotransferase activity purified 2700-fold with a 11% recovery. The most purified preparation yielded a major Coomassie blue-stained band on denaturing polyacrylamide gel electrophoresis of an apparent molecular weight (MW) of 40 000 Da. Photoaffinity labeling with the donor substrate, 3'-phosphoadenosine-5'-phospho-[35S]-sulfate resulted in a single radiolabeled protein band on denaturing polyacrylamide gel electrophoresis, again of apparent MW 40 000 Da, strongly suggesting that the major Coomassie blue-stained band in the most purified preparation is the cholesterol sulfotransferase protein. Among 3β-hydroxysteroids with a Δ5 double bond that were tested, each served as a substrate, while androgens, estrogens, corticosteroids, p-nitrophenol and DOPA did not serve as substrates. Apparent Michaelis constants for the 3β-hydroxysteroid substrates ranged from 0.6 to 8 µM.Key words: sulfotransferase, ichthyosis, cholesterol, skin, enzymology.
Biology of the prion gene complex
Authors: Mastrangelo Peter , Westaway David
Published: April 2011

The prion protein gene Prnp encodes PrPSc, the major structural component of prions, infectious pathogens causing a number of disorders including scrapie and bovine spongiform encephalopathy (BSE). Missense mutations in the human Prnp gene, PRNP, cause inherited prion diseases such as familial CreutzfeldtJakob Disease. In uninfected animals, Prnp encodes a GPI-anchored protein denoted PrPC, and in prion infections, PrPCis converted to PrPScby templated refolding. Although Prnp is conserved in mammalian species, attempts to verify interactions of putative PrP-binding proteins by genetic means have proven frustrating in that two independent lines of Prnp gene ablated mice (Prnp0/0mice: ZrchI and Npu) lacking PrPCremain healthy throughout development. This indicates that PrPCserves a function that is not apparent in a laboratory setting or that other molecules have overlapping functions. Shuttling or sequestration of synaptic Cu(II) via binding to N-terminal octapeptide residues and (or) signal transduction involving the fyn kinase are possibilities currently under consideration. A new point of entry into the issue of prion protein function has emerged from identification of a paralog, Prnd, with 25% coding sequence identity to Prnp. Prnd lies downstream of Prnp and encodes the Dpl protein. Like PrPC, Dpl is presented on the cell surface via a GPI anchor and has three α-helices: however, it lacks the conformationally plastic and octapeptide repeat domains present in its well-known relative. Interestingly, Dpl is overexpressed in two other lines of Prnp0/0mice (Ngsk and Rcm0) via intergenic splicing events. These lines of Prnp0/0mice exhibit ataxia and apoptosis of cerebellar cells, indicating that ectopic synthesis of Dpl protein is toxic to CNS neurons: this inference has now been confirmed by the construction of transgenic mice expressing Dpl under the direct control of the PrP promoter. Remarkably, Dpl-programmed ataxia is rescued by wt Prnp transgenes. The interaction between the Prnp and Prnd genes in mouse cerebellar neurons may have a physical correlate in competition between Dpl and PrPCwithin a common biochemical pathway that, when misregulated, leads to apoptosis.Key words: spongiform encephalopathy, neurodegenerative disease, paralogs, scrapie, CJD.
In vivo DNA-protein interactions at hypersensitive site 3.5 of the human β-globin locus control region
Authors: Xu Dong-dong , Liu De-pei , Ji Xin-jun , Lv Xiang , Liang Chih-chuan
Published: April 2011

Using ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the status of DNAprotein interactions at hypersensitive site 3.5 (HS3.5) of the locus control region in K562 and HEL cells, we found that there was protein occupancy in vivo at HS3.5 in both cell lines and the status of DNAprotein interaction was different between K562 and HEL. These data provide direct evidence that specific nuclear factor  DNA complexes form in vivo at functionally important sequence motifs of the HS3.5 in erythroid cells. This indicates that HS3.5 may play an important role in the regulation of the β-globin gene cluster. K562 is a human erythroleukemia cell line in which the embryonic ε-globin gene is predominantly expressed, while the HEL cell line expresses predominantly the fetal β-globin genes. Thus, HS3.5 might also be involved in the regulation of developmental stage-specific expression of β-globin genes. Our results are also consistent with the model that each hypersensitive site acts as a functional unit and HS3.5 may facilitate the formation of the HS3 functional unit.Key words: β-globin gene, hypersensitive site, phylogenetic footprint, differential phylogenetic footprint, in vivo footprinting, developmental regulation.
Abstracts of the 11th Winternational Symposium of the CSBMCB / Résumés du 11e symposium internationeige de la SCBBMC
Authors: / Résumés Abstracts
Published: April 2011

Seventy-nine abstracts are presented from the Canadian Society of Biochemistry and Molecular & Cellular Biology's 11th Winternational Symposium on "Dynamics of Intracellular Organelles and Molecular Machines". The abstracts are representative of the symposium's wide-ranging discussions encompassing topics such as endoplasmic reticulum, protein folding and degradation, protein machines and organelle dynamics, and diseases associated with protein folding and trafficking.
Changes in retinal ganglion cell axons after optic nerve crush: neurofilament expression is not the sole determinant of calibre
Authors: Minzenberg Michael , Berkelaar Michelle , Bray Garth , Mckerracher Lisa
Published: April 2011

After injury in the central nervous system of adult mammals, many of the axons that remain attached to their intact cell bodies degenerate and decrease in calibre. To understand this process better, we have investigated the relationship between axonal loss, cell loss, and the time course of changes in axonal calibre. Optic nerves (ONs) were crushed and the numbers and sizes of axons remaining close to the cell bodies (2 mm from the eye) and near the site of the lesion (6 mm from the eye) were determined for nerves examined between 1 week and 3 months after injury. Comparison with the retinal ganglion cell (RGC) counts from the same animals revealed that axonal loss was concomitant with cell body loss for at least the first 2 weeks after injury. However, there was no significant change in the calibre of the surviving neurons until 1 month after injury. Thereafter, the axonal calibre was decreased equally along the ON. No progressive somatofugal atrophy was observed. These decreases in axonal calibre occur much later than the immediate drop in neurofilament (NF) expression that also follows injury. The late effect of injury on axonal calibre suggests that NF expression is not the sole determinant of axon size of the RGC fibers in the ON. Other factors are likely additional contributing factors, such as the decreased rate of axonal transport that would help maintain the axonal neurofilament content.Key words: axonal calibre, axotomy, neuronal cell death, neurofilaments, retinal ganglion cell, optic nerve.
Mechanics of motility: distinct dynein binding domains on α- and β-tubulin
Authors: Goldsmith Matthew , Kooy Derek van der , Yarbrough Lynwood
Published: April 2011

Microtubules (MTs) interact with force-generating proteins to generate a variety of intracellular movements, including intracellular particle transport, ciliary–flagellar beating, and chromosome–spindle movements during mitosis–meiosis. Relatively little is known about the mechanics of these motor-MT interactions, in part because the motor binding domains of the MT and the corresponding MT binding domains of the motor have not been well characterized. Using a flagellar motility assay, we report that the MT subunits, α- and β-tubulin, each contain a dynein binding domain located near the C-termini of their respective tubulin subunits. Blocking either α- or β-tubulin binding domains of dynein attenuates motility in demembranated sea urchin sperm up to 50%. Interestingly, blocking both α- and β-tubulin binding domains on dynein produces much greater decreases in motility. These data suggest that flagellar dynein binds to both subunits of the MT polymer, α- and β-tubulin. In addition, the two subunits appear to contribute equivalent, but functionally separate, roles to flagellar motility.Key words: microtubule, microtubule-based motility, dynein, sperm motility.
Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line
Authors: Kyakumoto Seiko , Nemoto Takayuki , Sato Nobuko , Ota Minoru
Published: April 2011

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha ), a heterodimeric partner of RAR alpha . Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha . In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogenous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta . In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha , RXR beta , and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids. Key words: retinoid X receptor, retinoic acid receptor, retinoic acid response element, COUP-transcription factor (COUP-TF).
The zebrafish's swim to fame as an experimental model in biology
Authors: Vascotto Sandy G , Beckham Y , Kelly Gregory M
Published: April 2011

The zebrafish has long been the favorite organism in many scientific disciplines. Although its attributes as a model were expounded for many years and thus were no secret, the zebrafish sat in the wings while other more popular vertebrates such as chick, amphibians, and mouse were examined at length. We cannot say there was a resurgence in popularity, but more an explosion of research utilizing the zebrafish beginning in the late 1970s when investigators at the University of Oregon began using it as their model in neuroscience. Prior to this reawakening, the zebrafish was one of the significant organisms in the study of teratology and toxicology, development, and, to some extent, behavior. Recently, however, the field of zebrafish genetics has gained immense popularity and success, in part owing to the fact that zebrafish are diploid and are amenable to genetic manipulations. Here we present an overview of the multidisciplinary research that has laid some of the foundation of our present understanding of the biochemical, cell biological, and molecular genetic events accompanying zebrafish development.
Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein
Authors: Kozlowski David J , Murakami Tohru , Ho Robert K , Weinberg Eric S
Published: April 2011

Determination of fate maps and cell lineage tracing have previously been carried out in the zebrafish embryo by following the progeny of individual cells injected with fluorescent dyes. We review the information obtained from these experiments and then present an approach to fate mapping and cell movement tracing utilizing the activation of caged fluorescein-dextran. This method has several advantages over single-cell injections in that it is rapid, allows cells at all depths in the embryo to be marked, can be used to follow cells starting at any time during development, and allows an appreciation of the movements of cells located in a coherent group at the time of uncaging. We demonstrate that the approach is effective in providing additional and complementary information on prospective mesoderm and brain tissues studied previously. We also present, for the first time, a fate map of placodal tissues including the otic vesicle, lateral line, cranial ganglia, lens, and olfactory epithelium. The prospective placodal cells are oriented at the 50% epiboly stage on the ventral side of the embryo with anterior structures close to the animal pole, and posterior structures nearer to the germ ring.
Purification and kinetic characterization of hexokinase and glucose-6-phosphate dehydrogenase fromSchizosaccharomyces pombe
Authors: Tsai C Stan , Chen Q
Published: April 2011

Hexokinase and D-glucose-6-phosphate dehydrogenase (G6PDH) from Schizosaccharomyces pombe have been purified 250-fold by an identical three-step. Both enzymes are dimeric with a molecular mass of 88 kDa for the kinase and 112 kDa for the dehydrogenase. Steady-state kinetic studies were performed on hexokinase and G6PDH, which form the glucose phosphate branch of the oxidative pentose phosphate pathway of S. pombe (fission yeast). Hexokinase promotes Mg2+-activated phosphorylation of D-glucose by the equilibrium random Bi Bi mechanism with formation of the abortive enzyme-ADP-glucose complex. ADP inhibits the kinase competitively versus ATP and noncompetitively versus D-glucose. The Mg2+activation of hexokinase is associated with an increase in the maximal velocity by its interaction with the ternary complex to facilitate the transfer of the phosphoryl group. G6PDH catalyzes NADP+-linked oxidation of D-glucose-6-phosphate by the ordered Bi Bi mechanism with NADP+as the leading reactant. High NADP+concentration inhibits the dehydrogenase by forming the dead-end ternary complex. In addition, G6PDH is also subjected to product inhibition by NADPH and noncompetitive inhibition by A(G)TP. Thus, the oxidative pentose phosphate pathway in S. pombe may be regulated via inhibition of hexokinase by ADP in conjunction with inhibition of G6PDH by NADPH and ATP.Key words: yeast hexokinase, glucose-6-phosphate dehydrogenase.
Low-Kmmannose-6-phosphatase as a criterion for microsomal integrity
Authors: Pederson Bartholomew A , Foster James D , Nordlie Robert C
Published: April 2011

The low-Kmactivity of mannose-6-phosphatase (Man-6-Pase) has been used for many years to measure the structural integrity of microsomes. Recently histone II-A has been shown to activate glucose-6-phosphatase (Glc-6-Pase) and Man-6-Pase activities. However, in contrast to detergents, this compound appears to activate without disrupting microsomal vesicles (J.-F. St-Denis, B. Annabi, H. Khoury, and G. van de Werve. 1995. Biochem. J. 310: 221-224). This suggests that Man-6-Pase latency can be abolished without disrupting microsomal integrity and that even normally microsomes may manifest some low-KmMan-6-Pase activity without being "leaky." We have studied the relationship of Man-6-Pase with microsomal integrity further by measuring the latency of several enzymes reported to reside within the lumen of endoplasmic reticulum. We have also correlated this latency with the microsomal permeability of substrates for these enzymes. We found that (i) lumenal enzymes have different degrees of latency when compared with each other, (ii) permeability, as determined via osmotically induced changes in light scattering, is not always consistent with enzymatic latency, (iii) increases in the hydrolysis of Glc-6-P and Man-6-P were not parallel when microsomes were treated with low but increasing concentrations of detergent, and (iv) kinetic studies suggest that mannose-6-phosphate is hydrolyzed by untreated microsomes by more than a single mechanism. We propose that Man-6-Pase is not a reliable index of the integrity of microsomes.Key words: glucose-6-phosphatase, mannose-6-phosphatase, microsomes, rat liver, intactness.
Isolation and characterization of a mannose-binding lectin from leaves of the Chinese daffodil Narcissus tazetta
Authors: Ooi Linda SM , Wang Hexiang , Ng T B , Ooi Vincent EC
Published: April 2011

A mannose-binding lectin was isolated from leaves of the Chinese daffodil Narcissus tazetta (family Amaryllidaceae) using a procedure that comprised extraction with aqueous buffer, ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel Blue gel and mannose-agarose, and FPLC-gel filtration on Superose 12. The lectin was adsorbed on mannose-agarose and unadsorbed on DEAE-cellulose and Affi-gel Blue gel. It was an unglycosylated homodimer with a molecular mass of 26 kDa. Analysis of the N-terminal sequence of the N. tazetta lectin revealed considerable homology to lectins from the daffodil Narcissus pseudonarcissus, the snowdrop Galanthus nivalis (family Amaryllidaceae), the tulip Tulipa, and Kidachi aloe Aloe arborescens (family Liliaceae), and the orchid lectins (family Orchidaceae). The most striking likeness exists among the Amaryllidaceae lectins. The N. tazetta lectin exhibits hemagglutinating activity toward rabbit erythrocytes.Key words: daffodil, mannose-binding lectin, agglutinin.
The solution conformations of amino acids from molecular dynamics simulations of Gly-X-Gly peptides: comparison with NMR parameters
Authors: van der Spoel David
Published: April 2011

The conformations that amino acids can adopt in the random coil state are of fundamental interest in the context of protein folding research and studies of proteinpeptide interactions. To date, no detailed quantitative data from experimental studies have been reported; only nuclear magnetic resonance parameters such as chemical shifts and J coupling constants have been reported. These experimental nuclear magnetic resonance data represent averages over multiple conformations, and hence they do not provide unique structural information. I have performed relatively long (2.5 ns) molecular dynamics simulations of Gly-X-Gly tripeptides, surrounded by explicit water molecules, where X represents eight different amino acids with long side chains. From the trajectories one can calculate time averaged backbone chemical shifts and 3JNHα coupling constants and compare these with experimental data. These calculated quantities are quite close to the experimental values for most amino acids, suggesting that these simulations are a good model for the random coil state of the tripeptides. On the basis of my simulations I predict 3Jαβ coupling constants and present dihedral distributions for the Φ, Ψ, as well as χ1 and χ2 angles. Finally, I present correlation plots for these dihedral angles.Key words: molecular dynamics (MD), nuclear magnetic resonance (NMR) spectroscopy, J-coupling, chemical shift, dihedral probability distribution.
Evidence of Trolox and some gallates as synergistic protectors of erythrocytes against peroxyl radicals
Authors: Wu J , Sugiyama H , Zeng L -H , Mickle D , Wu T -W
Published: April 2011

The peroxidation of human erythrocytes induced by peroxyl radical initiator and its inhibition by several gallate esters (e.g., propyl, methyl, ethyl) and Trolox (a more polar analogue of vitamin E) have been studied. The antioxidant activity was determined on erythrocytes against hemolysis generated by a thermal activator, 2,2prime-azobis-(2-amidinopropane)dihydrogenchloride. It was found that propyl gallate and its two analogues were more effective than Trolox in preventing cell lysis. However, the combination of gallate esters and Trolox produced a protective effect exceeding the arithmetic sum of their individual contributions. These perceived synergisms occur at more than one level of Trolox at a given level of a gallate ester.Key words: Trolox, gallates, synergism, peroxyl radicals.
Chronic ethanol administration alters hepatic rates of glycerol phosphorylation and glycerol 3-phosphate oxidation: a dynamic in vivo 31P magnetic resonance spectroscopy study
Authors: Brauer Manfred , Lu Wuhua , Ling Mingfu
Published: April 2011

We used dynamic in vivo 31P magnetic resonance spectroscopy to noninvasively study the metabolism of glycerol by the liver in living rats, as a means of detecting subtle metabolic changes induced by chronic ethanol consumption. Rats subjected to chronic ethanol consumption and their pair-fed controls were given a metabolic load of glycerol (0.75 or 1.3 mL glycerol·kg body mass-1, i.p. or i.v) under normoxic or hyperoxic (98% O2) conditions. Changes in the level of glycerol 3-phosphate were followed in situ by monitoring the hepatic 31P phosphomonoester resonance every 7 or 13 min for up to 330 min. When challenged with a large dose of glycerol, chronic ethanol-treated rats exhibited less accumulation of glycerol 3-phosphate than controls, independent of the route of administration of the glycerol or whether the two groups were fasted or fed. For example, 1.3 mL glycerol·kg-1 i.v. under normoxic conditions resulted in a two-fold increase in phosphomonoester in ethanol-treated rats compared with a five-fold increase in controls. The ethanol-treated rats also showed a slower rate of phosphorylation of glycerol and slower oxidation of glycerol 3-phosphate than controls, indicating decreased activities of the glycerol kinase and glycerol 3-phosphate dehydrogenase steps, and hence slower glycerol utilization. The rate of glycerol utilization was dose and oxygen concentration dependent. Kinetic analysis indicated that the chronic ethanol-induced decrease in the glycerol 3-phosphate dehydrogenase reaction was due to a decreased rate of NADH reoxidation in the liver, likely owing to a decrease in oxygen supply or utilization in the ethanol-treated rats. These observations support the hypothesis of pre-existing hypoxia in rat liver after chronic ethanol administration. This study demonstrates the utility of dynamic in vivo 31P magnetic resonance spectroscopy in following the metabolism of a glycerol load as a sensitive, nonperturbing, and potentially clinically applicable test of liver function.Key words: 31P magnetic resonance spectroscopy, ethanol, liver, glycerol, metabolism.
Protein folding and maturation in a cell-free system
Authors: Hebert Daniel N , Zhang Jian-Xin , Helenius Ari
Published: April 2011

Reduced cellular systems have provided important tools to study complex cellular processes. Here we describe the oxidation, oligomerization, and chaperone binding of the viral glycoprotein influenza hemagglutinin in a cell-free system. The cell-free system, comprised of rough endoplasmic reticulum derived microsomes and a reticulocyte lysate, supported the complete maturation of hemagglutinin from the earliest oxidative intermediate to the mature homo-oligomer. Hemagglutinin disulfide bond formation and oligomerization were found to occur in a time- and temperature-dependent manner. Hemagglutinin's temporal association with the molecular chaperones calnexin and calreticulin was similar to that observed for their association with elongating ribosome-attached nascent chains in live cells. Furthermore, a procedure is described that permits the translocation of protein into microsomes that are depleted of lumenal contents. This cell-free system, therefore, provided an effective means to study the biological maturation processes of a protein that traverses the secretory pathway.Key words: protein folding, endoplasmic reticulum, molecular chaperone.
Mechanism of competition between chloride and stilbenedisulfonates for binding to human erythrocyte band 3 (AE1)
Authors: Salhany James M
Published: April 2011

Stilbenedisulfonates (S) constitute an important class of competitive inhibitors of the anion exchange (AE) function found in plasma membranes of various cell types. I present a brief summary of recent kinetic studies that provide insight into the mechanism of stilbenedisulfonate-chloride competition in binding to human erythrocyte band 3 (AE1) (B), the chloride-bicarbonate exchanger. Reversible stilbenedisulfonate binding follows a two-step mechanism (S + B <–> SB <–> SB*). Several lines of evidence are summarized that show that chloride, stilbenedisulfonates, and band 3 form a ternary complex, with chloride lowering stilbenedisulfonate affinity allosterically, by accelerating the rate of stilbenedisulfonate release. Of particular significance was our evidence demonstrating that extracellular chloride could accelerate stilbenedisulfonate release from its binding site on the outer surface of band 3 in resealed ghosts (i.e., acceleration in the release of a bound competitive inhibitor by a cis substrate). I suggest that the latter result may be consistent with our earlier proposal that band 3 follows a two-site ordered sequential mechanism, where two allosterically linked chloride binding transport sites move back and forth across the membrane together.Key words: band 3, anion transport, red cell membrane, membrane proteins.
Calreticulin, a multifunctional Ca2+ binding chaperone of the endoplasmic reticulum
Authors: Michalak Marek , Mariani Paola , Opas Michal
Published: April 2011

Calreticulin is a ubiquitous endoplasmic reticulum Ca2+ binding chaperone. The protein has been implicated in a variety of diverse functions. Calreticulin is a lectin-like chaperone and, together with calnexin, it plays an important role in quality control during protein synthesis, folding, and posttranslational modification. Calreticulin binds Ca2+ and affects cellular Ca2+ homeostasis. The protein increases the Ca2+ storage capacity of the endoplasmic reticulum and modulates the function of endoplasmic reticulum Ca2+-ATPase. Calreticulin also plays a role in the control of cell adhesion and steroid-sensitive gene expression. Recently, the protein has been identified and characterized in higher plants but its precise role in plant cells awaits further investigation.Key words: calreticulin, endoplasmic reticulum, chaperone, Ca2+ binding protein.
Evidence that the transport-related proteins BAT and 4F2hc are not specific for amino acids: induction of Na+-dependent uridine and pyruvate transport activity by recombinant BAT and 4F2hc expressed in Xenopus oocytes
Authors: Yao Sylvia YM , Muzyka William R , Cass Carol E , Cheeseman Christopher I , Young James D
Published: April 2011

Members of the BAT and 4F2hc gene family have one or, in the case of BAT, up to four transmembane domains and induce amino acid transport systems bo,+ (BAT) and y+L (4F2hc) when expressed in Xenopus oocytes. System bo,+ is a Na+-independent process with a broad tolerance for cationic and zwitterionic amino acids, whereas y+L exhibits Na+-independent transport of cationic amino acids (e.g., lysine) and Na+-dependent transport of zwitterionic amino acids (e.g., leucine). Mutations in the human BAT gene are associated with type I cystinuria, a genetic disease affecting the ability of intestinal and renal brush border membranes to transport cationic amino acids and cystine. An unresolved question is whether BAT and 4F2hc themselves have catalytic (i.e., transporting) activity or whether they operate as activators of other, as yet unidentified, transporter proteins. In this report, we have investigated the transport of representatives of four different classes of organic substrates in Xenopus oocytes following injection with rat BAT or 4F2hc RNA transcripts: leucine (a control amino acid substrate), uridine (a nucleoside), pyruvate (a monocarboxylate), and choline (an amine). Both recombinant proteins induced small, statistically significant Na+-dependent fluxes of uridine and pyruvate but had no effect on choline uptake. In contrast, control oocytes injected with transcripts for conventional nucleoside and cationic amino acid transporters (rat CNT1 and murine CAT1, respectively) showed no induction of transport of either leucine or pyruvate (CNT1) or uridine or pyruvate (CAT1). These findings support the idea that BAT and 4F2hc are transport activators and minimize the possibility that they have intrinsic transport capability. The transport-regulating functions of these proteins may extend to permeants other than amino acids.Key words: amino acid transport, uridine, pyruvate, BAT, 4F2hc, Xenopus oocytes.
Regulation and characterization of the Na+/H+ exchanger
Authors: Fliegel Larry , Murtazina Rakhilya , Dibrov Pavel , Harris Carmen , Moor Andrea , Fernandez-Rachubinski Francoise A
Published: April 2011

The Na+/H+ exchanger is a ubiquitous protein present in all mammalian cell types that functions to remove one intracellular H+ for one extracellular Na+. Several isoforms of the protein exist, which are referred to as NHE1 to NHE6 (for Na+/H+ exchanger one through six). The NHE1 protein was the first isoform cloned and studied in a variety of systems. This review summarizes recent papers on this protein, particularly those that have examined regulation of the protein and its expression and activity.Key words: cation translocation, intracellular pH, membrane protein, Na+/H+ antiporter, protein phosphorylation, protein-protein interaction.
Genetic control of extraembryonic cell lineages studied with tetraploid<–>diploid chimeric concepti
Authors: Kupriyanov Sergey , Baribault Hélène
Published: April 2011

The first differentiation event during mammalian embryogenesis is the commitment of blastomeres to the trophectoderm cell lineage. Much remains to be learned about the genetic control of this first cell lineage commitment and the subsequent events underlying the differentiation of all extraembryonic cell lineages. Because of the unique features of intrauterine embryonic development, the study of embryogenesis in lower organisms has shed little light on mammalian extraembryonic lineage differentiation. Rather, two major methods in developmental genetics have contributed to our understanding of genetic control of extraembryonic cell lineages. First, abnormalities in extraembryonic tissues have been described in many genetically engineered mutant mouse lines. However, the histological description of these abnormalities does not demonstrate whether the observed defect is the primary cause of embryonic lethality. Second, tetraploid<–>diploid aggregation experiments have been used to generate chimeric concepti with distinct genotypes in the extraembryonic tissues and the embryo proper. This experimental approach has provided the definitive demonstration of the crucial role of several transcription factors, growth factors and cytoskeleton proteins in extraembryonic tissue formation. The present review summarizes the origin of tetraploid<–>diploid aggregation experiments and it usefulness for the study the genetic control of extraembryonic cell lineages.Key words: tetraploid, aggregation, chimera, extraembryonic cell lineages, placenta.