bluuebunny/crossref_metadata_2025 huggingface dataset:
New thoughts on the role of the βγ subunit in G protein signal transduction
Authors: Vanderbeld Barbara , Kelly Gregory M
Published: April 2011

Heterotrimeric G proteins are involved in numerous biological processes, where they mediate signal transduction from agonist-bound G-protein-coupled receptors to a variety of intracellular effector molecules and ion channels. G proteins consist of two signaling moieties: a GTP-bound α subunit and a βγ heterodimer. The βγ dimer, recently credited as a significant modulator of G-protein-mediated cellular responses, is postulated to be a major determinant of signaling fidelity between G-protein-coupled receptors and downstream effectors. In this review we have focused on the role of βγ signaling and have included examples to demonstrate the heterogeneity in the heterodimer composition and its implications in signaling fidelity. We also present an overview of some of the effectors regulated by βγ and draw attention to the fact that, although G proteins and their associated receptors play an instrumental role in development, there is rather limited information on βγ signaling in embryogenesis.Key words: G protein, βγ subunit, G-protein-coupled receptor, signal transduction, adenylyl cyclase.
A comparative hybridization analysis of yeast DNA withParameciumparafusin- and different phosphoglucomutase-specific probes
Authors: Wyroba Elzbieta , Satir Birgit H
Published: April 2011

Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phospho glyco protein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3  I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.Key words: parafusin, phosphoglucomutase, yeast, hybridization, PCR.
Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2
Authors: Themsche Céline Van , Jacob Michèle , Salesse Christian
Published: April 2011

The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.Key words: phospholipase A2, retinal pigment epithelium, characterization.
Hydroxyl radical adduct of 5-aminosalicylic acid: A potential marker of ozone-induced oxidative stress
Authors: Kumarathasan Prem , Vincent Renaud , Goegan Patrick , Potvin Marc , Guénette Josée
Published: April 2011

The use of 5-aminosalicylic acid in assessment of reactive oxygen species formation was investigated by in vitro Fenton and ozonation reactions, and by in vivo ozone-exposure experiments. Enzymatic hydroxylation was evaluated by a microsomal assay. Fischer 344 male rats (250 g) injected with 5-aminosalicylic acid (100 mg·kg-1i.p.; 30 min) were exposed to ozone (0, 1, 2 ppm; nose only, 2 h); bronchoalveolar lavage, lung homogenates, and plasma were recovered. Oxidation products of 5-aminosalicylic acid were as follows: salicylic acid, by deamination; 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid, from radical or enzymatic hydroxylation; 5-amino-2-hydroxy-N,N'-bis(3-carboxy-4-hydroxyphenyl)-1,4-benzoquinonediimine, a condensation product of oxidized 5-aminosalicylic acid; and 5-amino-2,3,4,6-tetrahydroxybenzoic acid, attributed to hydroxyl radical attack without deamination, identified by HPLC electrochemical (HPLC-EC) detector system analysis and by GC-MS analysis of trimethylsilyl derivatives. 5-Aminotetrahydroxybenzoic acid was not formed enzymatically. 5-Aminotetrahydroxybenzoic acid, but not 5-aminosalicylic acid, was significantly elevated in bronchoalveolar lavage (+86%) and lung homogenates (+56%) in response to 2 ppm ozone (p < 0.05); no significant changes were detected in plasma. The data indicate that hydroxylation of 5-aminosalicylic acid is a potential specific probe for in vivo oxidative stress.Key words: 5-aminosalicylic acid, biomarker, free radical scavenging, hydroxyl radical, ozone.
Characterization of a novel heterodimeric cathepsin L-like protease and cDNA encoding the catalytic subunit of the protease in embryos ofArtemia franciscana
Authors: Butler Ann M , Aiton Andrea L , Warner Alden H
Published: April 2011

Embryos and larvae of the brine shrimp, Artemia franciscana, contain a novel cathepsin L-like cysteine protease (ACP) composed of 28.5- and 31.5-kDa subunits. Both subunits of the ACP are glycosylated, and seven isoforms of the protease were identified by isoelectric focusing with pI values ranging from 4.6 to 6.2. Several clones containing sequences coding for the 28.5-kDa subunit of the ACP were isolated from an Artemia embryo cDNA library in lambda ZAP II. One clone of 1229 bp, with an open reading frame of 1014 bp, was sequenced and found to contain 50-65% amino acid sequence identity with several members of the cathepsin L subfamily of cysteine proteases. The mature protein predicted from this sequence consisted of 217 amino acids with a mass of 23.5 kDa prior to post-translational modifications. The mature protein showed 68.6% amino acid sequence identity with human cathepsin L and 73.9% identity with cathepsin L-like proteases from Sarcophaga. peregrina and Drosophila melanogaster. The full-length cDNA clone analyzed in this study (pCP-3b) was renamed AFCATL1 (A. franciscana Cathepsin L1) and the sequence has been deposited in the Genbank database, accession number AF147207. Northern blot analyses identified a single transcript of about 1.4 kb in both embryos and young larvae of Artemia. Southern blot analyses of Artemia genomic DNA treated with various restriction endonucleases indicated a single gene for the ACP. The catalytic subunit of the ACP was tightly associated with a 31.5-kDa protein, which may localize the protease to nonlysosomal sites in embryos and larvae.Key words: cathepsin L, proteases, embryos, development, Artemia.
Molecular cloning, expression, and regulation of hippocampal amyloid precursor protein of senescence accelerated mouse (SAMP8)
Authors: Kumar Vijaya B , Vyas Kamlesh , Franko Mark , Choudhary Veena , Buddhiraju Chakradhar , Alvarez Jose , Morley John E
Published: April 2011

Alzheimer's disease (AD) is associated with increased expression of amyloid precursor protein (APP) with a consequent deposition of amyloid beta peptide (Aβ) which forms characteristic senile plaques. We have noticed that the senescence accelerated mouse (SAMP8), a strain of mouse that exhibits age-dependent defects such as loss of memory and retention at an early age of 8-12 months, also produces increased amounts of APP and Aβ similar to those observed in Alzheimer's disease (AD). In order to investigate if this is due to mutations in APP similar to those observed in AD, and to develop molecular probes that regulate its expression, APP cDNA was cloned from the hippocampus of 8-month-old SAMP8 mouse. The nucleotide sequence is 99.7% homologous with that of mouse and rat, 88.7% with monkey, and 89.2% with human homologues. At the amino acid level, the homology was 99.2% and 97.6% with rodent and primate sequences, respectively. A single amino acid substitution of Alanine instead of Valine at position 300 was unique to SAMP8 mouse APP. However, no mutations similar to those reported in human familial AD were observed. When the cDNA was expressed in HeLa cells, glycosylated mature APP could be detected by immunoblotting technique. The expression could be regulated in a time- and concentration-dependent manner by using an antisense oligonucleotide specific to APP mRNA. Such regulation of APP expression may have a therapeutic application in vivo.Key words: cloning, amyloid precursor protein, transfection, expression, and antisense oligo.
Histone modifications and apoptosis: Cause or consequence?
Authors: Th'ng John PH
Published: April 2011

Since the first description of apoptosis, genetic and biochemical studies have led to a greater understanding of the multiple pathways that eukaryotic cells can take to terminate their existence. These findings have also proven useful in understanding the development of various diseases such as AIDS, Alzheimer's, and Parkinson's and have provided potential targets for possible therapies. Despite all these studies, the mechanism of chromatin condensation, a morphological hallmark of apoptosis, remains elusive. This review describes the work to date on the post-translational modifications of histones during apoptosis and discusses the models that have been presented to explain the apoptotic condensation of chromatin.Key words: histones, nucleosomes, chromatin, apoptosis.
Characterization of matrix metalloprotease activities induced in the sea urchin extraembryonic matrix, the hyaline layer
Authors: Sharpe Christopher , Robinson John J
Published: April 2011

Hyaline layers, freshly prepared from one-hour-old embryos, were devoid of gelatin-cleavage activity. However, upon storage at 4°C, gelatin-cleavage activities appeared; three species of apparent mol mass 94[Formula: see text]117-, 90-, and 45-kDa were seen. All three species required zinc for activity. Using gel-exclusion chromatography we separated the 94[Formula: see text]117-, and 90-kDa species from the 45-kDa activity. The two higher mol mass species were inhibited by ethylenebis (oxyethylenenitrilo) tetraacetic acid and the lost activity was restored by calcium. Reconstitution of activity occurred with an apparent dissociation constant (calcium) of 5 µM. The presence of millimolar concentrations of magnesium had a minimal inhibitory effect on activity. The thermal denaturation profile of the higher mol mass gelatin-cleavage activity was significantly different in the presence and absence of calcium. Stabilization of these activities against thermal denaturation at 60°C occurred with an apparent dissociation constant (calcium) of 0.6 mM. Magnesium had no significant effect on the thermal denaturation profile. Collectively, these results suggest at least two different modes of interaction between calcium and the higher mol mass gelatinases. These conclusions are discussed in the context of the high calcium and magnesium concentrations present in the sea water environment of the sea urchin embryo.Key words: sea urchin, embryo, matrix metalloprotease, calcium.
Phosphorylation of histone variant regions in chromatin: Unlocking the linker?
Authors: Green G R
Published: April 2011

Histone variants illuminate the behavior of chromatin through their unique structures and patterns of postsynthetic modification. This review examines the literature on heteromorphous histone structures in chromatin, structures that are primary targets for histone kinases and phosphatases in vivo. Special attention is paid to certain well-studied experimental systems: mammalian culture cells, chicken erythrocytes, sea urchin sperm, wheat sprouts, Tetrahymena, and budding yeast. A common theme emerges from these studies. Specialized, highly basic structures in histone variants promote chromatin condensation in a variety of developmental situations. Before, and sometimes after condensed chromatin is formed, the chromatin is rendered soluble by phosphorylation of the heteromorphous regions, preventing their interaction with linker DNA. A simple structural model accounting for histone variation and phosphorylation is presented.Key words: phosphorylation, histone variants, chromatin, linker DNA.
Linker histone function in chromatin: Dual mechanisms of action
Authors: Georgel Philippe T , Hansen Jeffrey C
Published: April 2011

Aspects pertaining to linker histone structure and function are discussed, including the extent to which these proteins are essential, their ability to regulate specific gene expression, and recent structural data that provides a potential molecular basis for understanding how linker histones can have both repressive and stimulatory effects on genomic functions in vivo.Key words: chromatin, linker histone, higher-order folding.
Histone variants and histone modifications: A structural perspective
Authors: Ausió Juan , Abbott D Wade , Wang Xiaoying , Moore Susan C
Published: April 2011

In this review, we briefly analyze the current state of knowledge on histone variants and their posttranslational modifications. We place special emphasis on the description of the structural component(s) defining and determining their functional role. The information available indicates that this histone "variability" may operate at different levels: short-range "local" or long-range "global", with different functional implications. Recent work on this topic emphasizes an earlier notion that suggests that, in many instances, the functional response to histone variability is possibly the result of a synergistic structural effect.Key words: histone variants, posttranslational modifications, chromatin.
Kinetics of complexing activation by the magnesium ion on green crab (Scylla serrata) alkaline phosphatase
Authors: Park Yong-Doo , Yang Yi , Chen Qing-Xi , Lin Hai-Ning , Liu Qiang , Zhou Hai-Meng
Published: April 2011

As with mammalian enzymes, green crab (Scylla serrata) alkaline phosphatase can be activated by Mg2+through a time-dependent course. The activation is mainly a Vmaxeffect. Tsou's method was used to study the kinetic course of activation. The results show that the enzyme was activated by a complexing scheme that had not been previously identified: the enzyme first reversibly and quickly binds Mg2+and then undergoes a slow reversible course to activation, with a relatively high activation energy (78 ± 4 kJ/mol) and a slow conformational change. The activation reaction is a single molecule reaction, and the apparent activation rate constant is independent of Mg2+concentration if the concentration is sufficiently high. The microscopic rate constants of activation and the association constant were determined from the measurements. The proposed scheme may also be applied to the Mg2+activation mechanism for mammalian enzyme, to explain why the activation rate is time-dependent and not diffusion controlled. Substrate binding was also shown to affect the activation rate constant.Key words: alkaline phosphatase, green crab, kinetics, activation, magnesium ion.
Cytokeratin expression, fibrillar organization, and subtle function in liver cells
Authors: Marceau Normand , Loranger Anne
Published: April 2011

Cytokeratins (CKs) constitute a diverse group of intermediate filament (IF) proteins, expressed as pairs in keratinized and nonkeratinizing epithelial cells. Much is known now about the expression, assembly, and function of CKs in keratinized epithelial cells, the main features being the tight coupling between CK pair switch and cell terminal differentiation (protection barrier) and the vital role of CK IFs in cell mechanical integrity. However, the picture about nonkeratinizing epithelia, like the hepatic tissue, remains quite unclear. The liver forms a multicellular system, where parenchymal cells (i.e., hepatocytes) exert diverse metabolic function(s) and nonparenchymal epithelial cells (e.g., biliary epithelial cells) usually serve structural (or accessory) purposes. In terms of differential CK gene expression, the data accumulated so far demonstrated that parenchymal cells can contain as few as one single CK pair, whereas nonparenchymal cells contain more than two CKs, one of them being a representative of those found in epidermis. Moreover, the distribution of the CK IF networks present in the different cell types varies a lot and can often be linked to the cell specialization. However, the function(s) played by these IF proteins in this multicellular tissue remains a major issue. The use of new experimental approaches, largely based on gene transfer technology, indicates that it is quite subtle.Key words: cytokeratins, liver, expression, organization, function(s).
Tubulin expression and axonal transport in injured and regenerating neurons in the adult mammalian central nervous system
Authors: Fournier Alyson E. , McKerracher Lisa
Published: April 2011

Microtubules are essential components of the cytoskeleton required for axonal growth. To investigate how changes in tubulin transport and expression may affect axon regeneration, injury in the adult mammalian central nervous system was studied. Axotomized retinal ganglion cells (RGCs) that do not regenerate were compared with RGCs that regenerate their axons when the optic nerve is replaced with a peripheral nerve graft. When RGC axons regenerated through peripheral nerve grafts, the rate of slow transport increased but decreased when no regrowth occurred. To investigate the molecular mechanisms that mediate these responses, alterations in tubulin mRNA levels after injury were examined. Total tubulin mRNA levels fell after injury in the optic nerve but increased in those RGCs that regenerated their axons into a peripheral nerve graft. Further, the expression of four separate β-tubulin isotypes in injured rat RGCs was characterized. mRNA levels for all four isotypes decreased in RGCs after injury in the optic nerve. How the autoregulation of tubulin expression may contribute to the changes in β-tubulin isotype expression after injury is discussed.Key words: tubulin, retinal ganglion cell, axotomy, axonal transport, in situ hybridization.
Microfilament organization and wound repair in retinal pigment epithelium
Authors: Kalnins Vitauts. I. , Sandig Martin , Hergott Greg J. , Nagai Haruhiko
Published: April 2011

Several systems of microfilaments (MF) associated with adherens-type junctions between adjacent retinal pigment epithelial (RPE) cells and between these cells and the substratum play an important role in maintaining the integrity and organization of the RPE. They include prominent, contractile circumferential MF bundles that are associated with the zonula adherens (ZA) junctions. In chick RPE, these junctions are assembled from smaller subunits thus giving greater structural flexibility to the junctional region. Because the separation of the junctions requires trypsin and low calcium, both calcium-dependent and -independent mechanisms are involved in keeping adjacent RPE cells attached to one another. Another system of MF bundles that crosses the cell at the level of ZA junctions can be induced to form by stretching the epithelium. The MF bundles forming this system are oriented in the direction in which the RPE is stretched, thereby preventing the overextension of the cell in any one direction. The system may be useful as an indicator of the direction in which tension is experienced by RPE during development of the eye, in animal models of disease and during repair of experimentally induced wounds. Numerous single-cell wounds resulting from death of RPE cells by apoptosis at various stages of repair are normally present in developing chick and adult mammalian RPE. These wounds are repaired by the spreading of adjacent RPE cells and by the contraction of MF bundles oriented parallel to the wound edge, which develop during this time. As a result of the spreading in the absence of cell proliferation, the RPE cells increase in diameter with age. Experimentally induced wounds made by removing 5–10 RPE cells are repaired by a similar mechanism within 24 h. In repair of larger wounds, over 125 μm in width, the MF bundles oriented parallel to the wound edge characteristic of spreading cells are later replaced by stress fibers (SFs) that run perpendicularly to the wound edge and interact with the substratum at focal contacts (FCs) as RPE cells start to migrate. Cell proliferation is induced in cells along the wound edge only when the wounds are wide enough to require cell migration. In the presence of antibodies to beta-1-integrins, a component of FCs, cell spreading is not prevented but both cell migration and cell proliferation are inhibited. Thus, only the organization of the cytoskeleton characteristic of migrating RPE cells that have SFs that interact with the substratum at FCs, is associated with the induction of cell proliferation.Key words: retinal pigment epithelium, microfilaments, wound repair.
Determination versus differentiation and the MyoD family of transcription factors
Authors: Megeney Lynn A. , Rudnicki Michael A.
Published: April 2011

The myogenic regulatory factors (MRFs) form a family of basic helix–loop–helix transcription factors consisting of Myf-5, MyoD, myogenin, and MRF4. The MRFs play key regulatory roles in the development of skeletal muscle during embryogenesis. Sequence homology, expression patterns, and genetargeting experiments have revealed a two-tiered subclassification within the MRF family. Myf-5 and MyoD are more homologous to one another than to the others, are expressed in myoblasts before differentiation, and are required for the determination or survival of muscle progenitor cells. By contrast, myogenin and MRF4 are more homologous to one another than to the others and are expressed upon differentiation, and myogenin is required in vivo as a differentiation factor while the role of MRF4 remains unclear. On this basis, MyoD and Myf-5 are classified as primary MRFs, as they are required for the determination of myoblasts, and myogenin and MRF4 are classified as secondary MRFs, as they likely function during terminal differentiation.Key words: MyoD, Myf-5, myogenin, MRF4, skeletal muscle.
Schwannomin: new insights into this member of the band 4.1 superfamily
Authors: Belliveau Michael J. , Lutchman Mohini , Claudio Jaime O. , Marineau Claude , Rouleau Guy A.
Published: April 2011

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease characterized by the development of central nervous system tumours. The NF2 gene was recently cloned and found to encode a protein, schwannomin (or merlin), with homology to the band 4.1 superfamily. This superfamily of proteins includes ezrin, moesin, radixin, and talin, as well as several protein tyrosine phosphatases. How does a cytoskeleton-associated protein act as a tumour suppressor? While this fundamental question remains unanswered, recent studies have begun to address key questions regarding the function of schwannomin. In this review, we examine what is known about the band 4.1 superfamily and how this information pertains to schwannomin. In addition, we summarize recent studies of schwannomin itself.Key words: NF2, band 4.1, tumour suppressor, cytoskeleton.
DNA methylation and genome imprinting in the zebrafish, Danio rerio: some evolutionary ramifications
Authors: McGowan R A , Martin C C
Published: April 2011

Although methylation has been recognized as an important component in a number of developmental processes in mammals, in zebrafish almost nothing is known about this epigenetic modification. This is despite the fact that the zebrafish is becoming increasingly popular as a developmental model system. The little work that has been done on methylation and development in fish concerns genomic imprinting. In mammals, imprinting results in an inability to reproduce parthenogenetically because a genetic contribution from both parents is necessary to successfully complete development. However, this is not true of zebrafish, and a number of the theories that have been presented to explain the evolution of imprinting are not consistent with imprinting in these fish. A new model is presented that discusses some of the potential evolutionary ramifications of methylation and imprinting and that leads to the suggestion that imprinting may actually be a simple genetic mechanism to enhance the efficient evolution of both individual genetic loci and combinations of loci with related functions, without risking the population as a whole. This model can accommodate all of the information known about imprinting, including its broad phylogenetic range, imprinting by both males and females, and the diverse nature of the genes that are known to be imprinted.
Contribution of zebrafish-mouse cell hybrids to the mapping of the zebrafish genome
Authors: Chevrette Mario , Joly Lucille , Tellis Patricia , Ekker Marc
Published: April 2011

The zebrafish, Danio rerio, is becoming an increasingly popular model for the study of vertebrate development. Indeed, the biology of the fish offers great advantages for such studies. The life cycle of the zebrafish is relatively short (2-3 months) and the embryos develop outside the mother, facilitating the visualization of any mutated phenotype. At present, more than 1000 embryonic mutations have been reported. However, until recently, there was no physical or genetic map for this organism. In an effort to generate such a map, we have produced and characterized a panel of zebrafish-mouse cell hybrids. We have used whole-cell fusion to transfer zebrafish chromosomes from two different zebrafish cell lines into mouse recipient cells, thus generating more than 100 hybrids. Using fluorescence in situ hybridization and polymerase chain reaction analysis, we have determined the zebrafish chromosome composition of these hybrids. Here we report that elements from the 25 linkage groups of the zebrafish genome are present in our hybrids. These hybrids could identify the chromosomal location of genes affected in zebrafish mutants.
Markers of cell polarity during and after nitrogen starvation inSchizosaccharomyces pombe
Authors: Rupes Ivan , Jochová Jana , Young Paul G
Published: April 2011

In Schizosaccharomyces pombe, nitrogen starvation induces transient acceleration of cell division and reduction in cell size with a final arrest in G1. The division size control appears to be impaired by mutations in cdr1/nim1 and cdr2, genes that encode protein kinases mediating nutritional control over the mitotic cycle. cdr-cells arrest after fewer rounds of division and are larger than the wild type. Recent work suggests that long-term nitrogen starvation causes S. pombe wild-type cells to become spherical, which suggests loss of cell polarity. cdr mutants retain the elongated shape, indicating a potential difference in cell polarity control relative to the wild type. We examined several markers related to maintenance of cell polarity in S. pombe following nitrogen starvation including cell division scar pattern and actin and microtubule cytoskeleton. Wild-type cells as well as cdr mutants maintained a normal cell division scar pattern throughout nitrogen starvation but cells dividing under these conditions developed a wall malformation in the center of the septum. In cells arrested by nitrogen starvation, actin patches, normally associated with sites of cell wall deposition, were larger and distributed randomly along the cell surface. Cytoplasmic arrays of microtubules, which are thought to be involved in control of the polarity signal, were not visibly affected. The effects were similar in wild-type cells and in cdr-mutants. Upon refeeding, the new growth always reoccurred at the tip zones and there were only small deviations of its direction from the original axis. The results indicate that cell polarity is preserved both in wild-type cells, which arrest in G1and appear spherical, and in cdr1/nim1 and cdr2 mutants, which arrest in G2and appear polarized throughout the starvation period. Key words: cell polarity, fission yeast, nitrogen starvation, actin, microtubules, cdr1/nim1, cdr2.
Genetic dissection of vertebrate processes in the zebrafish: a comparison of uniparental and two-generation screens
Authors: Cheng Keith C , Moore Jessica L
Published: April 2011

Genetic dissection is used to identify important genes in biological processes and is accomplished through the generation and study of mutations in model organisms. In diploid organisms, recessive mutations must be rendered hemizygous or homozygous for the mutant phenotype to be detected. In zebrafish (Danio rerio), this can be accomplished in two ways: (i) crosses between siblings who are potential carriers (in two-generation screens) and (ii) the generation of uniparental progeny. Two-generation screens have been the most productive to date but require substantial resources. However, uniparental screens involving haploids and half-tetrads (products of meiosis I) have also been productive, and require more modest resources. In genetic screens, the fraction of an average genome that is heterozygous (heterozygosity index) is inversely proportional to the likelihood that separate recessive mutations will be homozygous at the same time. Heterozygosity indices for haploid, half-tetrad, and two-generation screens are 0, 66, and 87.5%, respectively. Family sizes required to minimize bias in half-tetrad screens are also calculated. We conclude that gynogenetic half-tetrad screens are genetically robust and technically accessible to the independent researcher. The increasingly powerful genetic and experimental tools available for work with zebrafish can be used to address a broad range of questions in vertebrate biology.
Mutations resulting in transient and localized degeneration in the developing zebrafish brain
Authors: Rodriguez Michael , Driever Wolfgang
Published: April 2011

In a large-scale mutagenesis screen in the zebrafish, Danio rerio, we have identified a heterogeneous group of 30 recessive, embryonic lethal mutations characterized by degeneration in the developing central nervous system that is either transient or initially localized to one area of the brain. Transient degeneration is defined as abnormal cell death occurring during a restricted period of development. Following degeneration, the affected structures do not appear to regenerate. In each case degeneration is identified after somitogenesis is complete and is not associated with visually identified patterning defects. These 30 mutations, forming 21 complementation groups, have been classified into four phenotypic groups: group 1, transient degeneration (13 mutations); group 2, spreading degeneration, early onset, in which degeneration is initially confined to the optic tectum but subsequently spreads to other areas of the central nervous system (7 mutations); group 3, late-onset degeneration, initially identified after 4 days (6 mutations); and group 4, degeneration with abnormal pigmentation (4 mutations). Although apoptotic cells are seen in the retina and tectum of all mutants, the distribution, temporal progression, and severity of degeneration vary between mutations. Several mutations also show pleiotropic effects, with degeneration involving extraneural structures including the pharyngeal arches and pectoral fins. We discuss some of the pathways important for cell survival in the nervous system and suggest that these mutations will provide entry points for identifying genes that affect the survival of restricted neural populations.
Studies of the dynamics of skeletal muscle regeneration: the mouse came back!
Authors: Anderson Judy E
Published: April 2011

Regeneration of skeletal muscle tissue includes sequential processes of muscle cell proliferation and commitment, cell fusion, muscle fiber differentiation, and communication between cells of various tissues of origin. Central to the process is the myosatellite cell, a quiescent precursor cell located between the mature muscle fiber and its sheath of external lamina. To form new fibers in a muscle damaged by disease or direct injury, satellite cells must be activated, proliferate, and subsequently fuse into an elongated multinucleated cell. Current investigations in the field concern modulation of the effectiveness of skeletal muscle regeneration, the regeneration-specific role of myogenic regulatory gene expression distinct from expression during development, the impact of growth and scatter factors and their respective receptors in amplifying precursor numbers, and promoting fusion and maturation of new fibers and the ultimate clinical therapeutic applications of such information to alleviate disease. One approach to muscle regeneration integrates observations of muscle gene expression, proliferation, myoblast fusion, and fiber growth in vivo with parallel studies of cell cycling behaviour, endocrine perturbation, and potential biochemical markers of steps in the disease-repair process detected by magnetic resonance spectroscopy techniques. Experiments on muscles from limb, diaphragm, and heart of the mdx dystrophic mouse, made to parallel clinical trials on human Duchenne muscular dystrophy, help to elucidate mechanisms underlying the positive treatment effects of the glucocorticoid drug deflazacort. This review illustrates an effective combination of in vivo and in vitro experiments to integrate the distinctive complexities of post-natal myogenesis in regeneration of skeletal muscle tissue.Key words: satellite cell, cell cycling, HGF/SF, c-met receptor, MyoD, myogenin, magnetic resonance spectroscopy, mdx dystrophic mouse, deflazacort.
The sea urchin egg yolk granule is a storage compartment for HCL-32, an extracellular matrix protein
Authors: Mayne Janice , Robinson John J
Published: April 2011

We have utilized protein gel blot analysis and immunogold labelling to define the intracellular storage compartment for HCL-32, a 32-kDa protein component of the sea urchin embryonic extracellular matrices, the hyaline layer and basal lamina. Anti-HCL-32 antiserum specifically labelled yolk granules in unfertilized eggs. Cortical granules, mitochondria, sparse granules, and lipid vacuoles were not labelled. Label continued to be detected in the yolk granules through to the blastula stage of development. However, by the gastrula stage no labelling was detected in the yolk granules. In protein gel blot analysis HCL-32 was detected in yolk granules prepared from unfertilized eggs. These results clearly define the yolk granule as a storage compartment for HCL-32, an extracellular matrix protein.Key words: embryo, yolk granule, extracellular matrix.
Comparisons of the effects of temperature on the liver fatty acid binding proteins from hibernator and nonhibernator mammals
Authors: Stewart J M , English T E , Storey K B
Published: April 2011

Hibernating mammals rely heavily on lipid metabolism to supply energy during hibernation. We wondered if the fatty acid binding protein from a hibernator responded to temperature differently than that from a nonhibernator. We found that the Kd for oleate of the liver fatty acid binding protein (1.5 micromolar) isolated from ground squirrel (Spermophilus richardsonii) was temperature insensitive over 5-37°C, while the rat liver fatty acid binding protein was affected with the Kd at 37°C being about half (0.8 micromolar) that found at lower temperatures. This same trend was observed when comparing the specificity of various fatty acids of differing chain length and degree of unsaturation for the two proteins at 5 and 37°C. At the lower temperature, ground squirrel protein bound long-chain unsaturated fatty acids, particularly linoleate and linolenate, at least as well as at the higher temperature and matched requirements for these fatty acids in the diet. The most common long-chain fatty acid, palmitate, was a more effective ligand for ground squirrel liver fatty acid binding protein at 5°C than at 37°C, with the opposite occurring in the eutherm. Rat protein was clearly not adapted to function optimally at temperatures lower than the animal's body temperature.Key words: fatty acid binding protein, temperature, hibernation.
NMR studies of tandem WW domains of Nedd4 in complex with a PY motif-containing region of the epithelial sodium channel
Authors: Kanelis Voula , Farrow Neil A , Kay Lewis E , Rotin Daniela , Forman-Kay Julie D
Published: April 2011

Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we report the assignment of backbone 1Hα, 1HN, 15N, 13C', 13Cα, and aliphatic 13C resonances of a fragment of rat Nedd4 (rNedd4) containing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-containing peptide derived from the β subunit of rat ENaC, the βP2 peptide. The secondary structures of these two WW domains, determined from chemical shifts of 13Cα and 13Cβ resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the peptidyl-prolyl isomerase Pin1. Triple resonance experiments that detect the 1Hα chemical shift were necessary to complete the chemical shift assignment, owing to the large number of proline residues in this fragment of rNedd4. A new experiment, which correlates sequential residues via their 15N nuclei and also detects 1Hα chemical shifts, is introduced and its utility for the chemical shift assignment of sequential proline residues is discussed. Data collected on the WW(II+III)-βP2 complex indicate that these WW domains have different affinities for the βP2 peptide.Key words: WW domain, PY motif, Nedd4, ENaC, NMR.
Polyelectrolyte-induced domains in lipid bilayer membranes: the deuterium NMR perspective
Authors: Macdonald Peter M , Crowell Kevin J , Franzin Carla M , Mitrakos Peter , Semchyschyn Darlene J
Published: April 2011

Domain formation in lipid bilayer membranes can occur through electrostatic interactions between charged lipids and oppositely charged polyelectrolytes, such as proteins or polynucleic acids. This review describes a novel method for examining such domains in lipid bilayers, based on 2H NMR spectroscopy. The 2H NMR spectrum of choline-deuterated phosphatidylcholine is sensitive to, and reports on, lipid bilayer surface charge. When a charged lipid bilayer is exposed to an oppositely charged polyelectrolyte, the latter binds electrostatically to the bilayer surface and attracts charged lipids into its vicinity. The resulting inhomogeneous charge distribution produces overlapping 2H NMR subspectra arising from phosphatidylcholine within charge-enriched versus charge-depleted regions. Such spectral details as the quadrupolar splittings and the relative intensities of the subspectra permit a complete analysis of the domain composition, size, and, within limits, lifetime. Using 2H NMR, domain formation in lipid bilayer membranes can be observed with both cationic and anionic polyelectrolytes, whether of natural or synthetic origin. Domain size and composition prove to be sensitive to the detailed chemical structure of both the polyelectrolyte and the charged lipids. Within the domains there is always a stoichiometric anion/cation binding ratio, indicating that the polyelectrolyte lies flat on the membrane surface. The amount of phosphatidylcholine within the domain varies as a function of its statistical availability, in accordance with the predictions of a recent thermodynamic model of domain formation. When the molecular weight of the polyelectrolyte is varied, the domain size alters in accordance with the predictions of classical polymer physics. As expected for a predominantly electrostatic phenomenon, the observed domains dissipate at high ionic strength.Key words: electrostatic domains, polyelectrolytes, lipid bilayers, deuterium NMR.
Human cerebral neoplasms studied using MR spectroscopy: a review
Authors: Kaibara Taro , Tyson Randy L , Sutherland Garnette R
Published: April 2011

Of primary central nervous system tumors treated each year, the majority are glioma, followed by meningioma and then pituitary adenoma. While the use of magnetic resonance (MR) and computed tomographic imaging is well established in the diagnosis and management of such tumors, these techniques have a limited role in determining the metabolic state, either prior to or following therapy. Multinuclear MR spectroscopy, on the other hand, provides information on tumor metabolism and the effect of therapy on tumor viability. This paper reviews MR spectroscopic studies performed on patients with central nervous system tumors and discusses the impact that such studies have had on tumor diagnosis and management.Key words: CNS neoplasia, MR spectroscopy, metabolism.
NMR evidence for a conformational adaptation of apolipophorin III upon lipid association
Authors: Wang Jianjun , Sahoo Daisy , Sykes Brian D , Ryan Robert O
Published: April 2011

A characteristic property of amphipathic exchangeable apolipoproteins is an ability to exist alternately in lipid-free and lipid-bound states. In the present study, we have used 1H-15N-heteronuclear single quantum correlation spectroscopy to probe structural changes of apolipophorin III upon lipid association, by monitoring alterations of the chemical shifts of specific amino acids as a function of lipid titration. 15N-valine-, 15N-leucine-, 15N-lysine-, and 15N-glycine-labeled apolipophorin III were used in titration experiments with the micelle-forming lipid dodecylphosphocholine. In the absence of lipid, valine and leucine residues are located in the hydrophobic interior of the apolipophorin III helix bundle and their resonances resist chemical shift changes below the critical micelle concentration of dodecylphosphocholine. At the critical micelle concentration, however, dramatic and abrupt chemical shift changes occur, apparently coincident with formation of a protein-lipid micelle complex, as judged by significant line-width broadening of the crosspeaks. By contrast, apolipophorin III lysine and glycine residues are located on the hydrophilic surfaces of amphipathic alpha-helices or in loop regions, exposed to solvent. Their crosspeaks display either a chemical shift change similar to that seen for hydrophobic residues or a more gradual chemical shift change, beginning at very low dodecylphosphocholine concentrations. These results indicate that an interaction occurs between specific solvent-exposed lysine residues and dodecylphosphocholine below the critical micelle concentration of this lipid, whereas valine and leucine residues are not accessible to monomeric dodecylphosphocholine. At the critical micelle concentration, however, the availability of a newly formed lipid surface induces apolipophorin III binding, concomitant with conformational opening of the helix bundle, exposing its hydrophobic surfaces for binding to the dodecylphosphocholine micellar surface. Subsequently, hydrophobic residues undergo characteristic spectral changes. Subtle differences in behavior of specific hydrophobic residues, in terms of their response to dodecylphosphocholine titration and relative locations in the helix-bundle conformation, suggest that one end of the molecule may initiate contact with the lipid surface, followed by helix bundle opening.Key words: apolipophorin III, apolipoprotein.
Interaction between lipid bilayers and a new class of antineoplastic agents derived from arylchloroethylurea: a 2H solid-state NMR study
Authors: Saint-Laurent Audrey , Boudreau Nadine , C.-Gaudreault René , Poyet Patrick , Auger Michèle
Published: April 2011

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and model membrane of dimyristoylphosphatidylcholine by deuterium nuclear magnetic resonance spectroscopy. The results indicate that the drug incorporates in the bilayer and causes an increase of the lipid acyl chain order, this effect being greater close to the interfacial region of the lipid bilayer. The increase in ordering is dependent on the nature (degree of ramification, length of the alkyl chain, and presence of a sulfur atom) as well as on the position of the R substituent and is correlated with the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky ramified substituent and those for which the ordering effect on the lipid bilayer is the smallest. On the other hand, the ordering effect is greater and seen all along the lipid acyl chains for the long-chain CEUs, such as n-hexadecyl CEU, which have been shown to have very weak cytotoxic activity. Finally, the results obtained as a function of the drug concentration indicate that the ordering effect is seen for lipid to drug molar ratios as low as 20:1.Key words: deuterium, NMR, membrane, arylchloroethylurea, liposome.
NMR structural studies on antifreeze proteins
Authors: Sönnichsen Frank D , Davies Peter L , Sykes Brian D
Published: April 2011

Antifreeze proteins (AFPs) are a structurally diverse class of proteins that bind to ice and inhibit its growth in a noncolligative manner. This adsorption-inhibition mechanism operating at the ice surface results in a lowering of the (nonequilibrium) freezing point below the melting point. A lowering of ~1°C, which is sufficient to prevent fish from freezing in ice-laden seawater, requires millimolar AFP levels in the blood. The solubility of AFPs at these millimolar concentrations and the small size of the AFPs (typically 3-15 kDa) make them ideal subjects for NMR analysis. Although fish AFPs are naturally abundant, seasonal expression, restricted access to polar fishes, and difficulties in separating numerous similar isoforms have made protein expression the method of choice for producing AFPs for structural studies. Expression of recombinant AFPs has also facilitated NMR analysis by permitting isotopic labeling with 15N and 13C and has permitted mutations to be made to help with the interpretation of NMR data. NMR analysis has recently solved two AFP structures and provided valuable information about the disposition of ice-binding side chains in a third. The potential exists to solve other AFP structures, including the newly described insect AFPs, and to use solid-state NMR techniques to address fundamental questions about the nature of the interaction between AFPs and ice.Key words: NMR spectroscopy, antifreeze, ice-binding affinity, review.
Solid-state NMR studies of proteins: the view from static 2H NMR experiments
Authors: Siminovitch David J
Published: April 2011

The application of solid-state 2H NMR spectroscopy to the study of protein and peptide structure and dynamics is reviewed. The advantages of solid-state NMR for the study of proteins are considered, and the particular advantages of solid-state 2H NMR are summarized. Examples of work on the integral membrane protein bacteriorhodopsin, and the membrane peptide gramicidin, are used to highlight the major achievements of the 2H NMR technique. These examples demonstrate that through the use of oriented samples, it is possible to obtain both structural and dynamic information simultaneously.Key words: solid-state NMR, 2H NMR, membrane peptides, membrane proteins, oriented bilayers.