bluuebunny/crossref_metadata_2025 huggingface dataset:
A compendium of the histone H1 family of somatic subtypes: An elusive cast of characters and their characteristics
Authors: Parseghian Missag H , Hamkalo Barbara A
Published: April 2011

The last 35 years has seen a substantial amount of information collected about the somatic H1 subtypes, yet much of this work has been overshadowed by research into highly divergent isoforms of H1, such as H5. Reports from several laboratories in the past few years have begun to call into question some of the traditional views regarding the general function of linker histones and their heterogeneity. Hence, the impression in some circles is that less is known about these ubiquitous nuclear proteins as compared with the core histones. The goal of the following review is to acquaint the reader with the ubiquitous somatic H1s by categorizing them and their characteristics into several classes. The reasons for our current state of misunderstanding is put into a historical context along with recent controversies centering on the role of H1 in the nucleus. Finally, we propose a model that may explain the functional role of H1 heterogeneity in chromatin compaction.Key words: histone H1, linker histones, chromatin organization, chromatin compaction, heat shock.
Repeated exposures of human skin equivalent to low doses of ultraviolet-B radiation lead to changes in cellular functions and accumulation of cyclobutane pyrimidine dimers
Authors: Chouinard Nadine , Therrien Jean-Philippe , Mitchell David L , Robert Marielle , Drouin Régen , Rouabhia Mahmoud
Published: April 2011

Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (64) photoproducts and cyclobutane pyrimidine dimers. Although the (64) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.Key words: UVB, keratinocytes, skin structure, DNA damage, photoproducts.
Cell death and survival alterations in Malpighian tubules ofTriatoma infestansfollowing heat shock
Authors: Mello Maria Luiza S , Tavares Maria Cristina H , Dantas Marly M , Rodrigues Vera Lúcia CC , Maria-Engler Sílvya S , Campos Silvana P , Garcia Nancy L
Published: April 2011

In this study, we examined cell survival and cell death in response to heat shock in an insect organ composed of highly polyploid cells no longer capable of cell division. For this, the frequency of nuclear phenotypes in Feulgen-stained Malpighian tubules of the blood-sucking insect, Triatoma infestans, was analyzed at various times after a short heat shock with or without subsequent moderate fasting. Cell death DNA fragmentation was studied immunocytochemically. Normal phenotypes and phenotypes indicative of cell survival (heterochromatin decondensation, nuclear fusion) and death (apoptosis, necrosis) were observed, especially in heat-shocked specimens. While the number of total and normal nuclei decreased following heat shock, the frequency of apoptosis increased during a short period (7 days) after heat shock. During a 30-day period following heat shock, the frequency of necrosis in fasted but not in fully nourished nymphs increased simultaneously with a decrease in the frequency of apoptosis. This finding suggests that the stress promoted by heat shock, but not that associated with heat shock plus fasting, can be dealt with by the apoptosis program. When considering the forms of cell survival, heterochromatin decondensation was more relevant in fully nourished nymphs, whereas nuclear and cell fusions were more important in fasted specimens. The forms of cell survival and cell death reported here may have protected the organ from damage by the stressing agents. In cells with no induction or accumulation of heat-shock proteins, cell death and the forms of cell survival observed here were the probable consequence.Key words: heat shock, fasting, apoptosis, necrosis, cell survival, Triatoma infestans.
The shape of things to come: Regulation of shape changes in endoplasmic reticulum
Authors: Paiement Jacques , Bergeron John
Published: April 2011

Shape changes in the endoplasmic reticulum control fundamental cell processes including nuclear envelope assembly in mitotic cells, calcium homeostasis in cytoplasmic domains of secreting and motile cells, and membrane traffic in the early secretion apparatus between the endoplasmic reticulum and Golgi. Opposing forces of assembly (membrane fusion) and disassembly (membrane fragmentation) ultimately determine the size and shape of this organelle. This review examines some of the regulatory mechanisms involved in these processes and how they occur at specific sites or subcompartments of the endoplasmic reticulum.Key words: rough endoplasmic reticulum, smooth endoplasmic reticulum, shape changes, assembly, membrane fusion, organelle size, vesicle formation.
Dimerization of the RelA protein ofEscherichia coli
Authors: Yang Xiaoming , Ishiguro Edward E
Published: April 2011

The RelA protein of Escherichia coli is a ribosome-associated (p)ppGpp synthetase that is activated by amino acid deprivation. It was recently reported that the activity of RelA is regulated by oligomerization mediated by the C-terminal domain of RelA. The oligomerization of RelA is further characterized in this study. The C-terminal domain consisting of amino acids 455744, designated 'RelA, formed homooligomers as well as heterooligomers with RelA as demonstrated by copurification of RelA and 'RelA and by an affinity blotting assay. Glutaraldehyde-induced cross-linking indicated that the oligomer was a dimer. The functional analysis of 'RelA was based on a combination of yeast two-hybrid analysis, the determination of the effects of overexpression of 'RelA derivatives on the stringent response, and the cellular localization of the overexpressed 'RelA derivatives. These studies indicated that two regions, designated 'RelA-1 (amino acids 455538) and 'RelA-2 (amino acids 550682), were involved in dimerization. The involvement of one of these two regions, RelA-2, is consistent with a previous site-directed mutagenesis study. In addition to dimerization, 'RelA-2 apparently contained the main ribosome-binding domain of RelA. The third region, 'RelA-3 (amino acids 682744), was not involved in either dimerization or ribosome binding. The overexpression of 'RelA-1 and 'RelA-2, but not 'RelA-3, inhibited the stringent response. These results support the previously proposed model which suggests a role for oligomerization in the regulation of (p)ppGpp synthetase.Key words: RelA, Escherichia coli, stringent response.
The actin-depolymerizing factor destrin has an actin-stabilizing domain
Authors: Tokuraku Kiyotaka , Okamoto Shinya , Katsuki Miho , Nakagawa Hiroyuki , Kotani Susumu
Published: April 2011

Destrin is a 19 kDa actin-depolymerizing protein of the ADFcofilin family. Destrin was digested with trypsin to a structurally stable 9.2 kDa fragment that contains the actin-binding sequence. The purified 9.2 kDa fragment has an actin filament stabilizing activity, rather than an actin filament depolymerizing activity. The deleted region is probably essential for the actin filament depolymerizing activity of intact destrin. Surprisingly, the 9.2 kDa fragment also has an assembly-promoting activity in the absence of ATP.Key words: actin, destrin, cofilin, ADF.
A role for neurofilaments in the pathogenesis of amyotrophic lateral sclerosis
Authors: Julien Jean-Pierre
Published: April 2011

Amyotrophic lateral sclerosis (ALS) is a late-onset degenerative disease of motor neurons, characterized by abnormal accumulation of neurofilaments (NFs) in perikarya and proximal axons. Two lines of evidence suggest that neurofilament accumulation can play a crucial role in ALS pathogenesis. First, transgenic mouse models overexpressing NF proteins were found to develop motor neuron degeneration and, second, variant alleles of the NF heavy-subunit (NF-H) gene have been found in some human ALS patients. Our axonal transport studies with transgenic mice overexpressing the human NF-H gene, a model of ALS, revealed defects of intracellular transport not only for neurofilament proteins but also for other cytoskeletal proteins and organelles such as mitochondria. Therefore, we propose that neurofilament accumulation in mice causes neurodegeneration by disrupting axonal transport, a mechanism that may account for the pathogenesis of ALS.Key words: amyotrophic lateral sclerosis, neurofilaments, transgenic mice, axonal transport.
Dystonin transcripts are altered and their levels are reduced in the mouse neurological mutantdt24J
Authors: Bernier Gilbert , Brown Arthur , Dalpé Gratien , Mathieu Martine , Repentigny Yves De , Kothary Rashmi
Published: April 2011

Dystonia musculorum is a hereditary mouse neurodegenerative disorder that primarily affects the sensory arm of the nervous system. We have recently cloned and identified a candidate gene for this disorder and designated it dystonin. The sequence of dystonin predicts a rod-shaped cytoskeletal-associated protein with an actin-binding domain at the N-terminal end and a hemidesmosomal protein sequence (bpag1) at the C-terminal end. Here we show that abnormal dystonin transcripts are present in neural tissues of a spontaneous dystonia musculorum mutant, dt24J. We further show that dystonin transcript levels are reduced 2- to 3-fold in dt24Jmice.Key words: mouse, dystonia musculorum, neurodegeneration, cytoskeleton, bpag1.
Overexpression of human keratin 16 produces a distinct skin phenotype in transgenic mouse skin
Authors: Coulombe Pierre A. , Bravo Nicola S. , Paladini Rudolph D. , Nguyen Diem , Takahashi Kenzo
Published: April 2011

Human cytokeratin 16 (K16; 48 kDa) is constitutively expressed in postmitotic keratinocytes in a variety of stratified epithelial tissues, but it is best known for the marked enhancement of its expression in stratified squamous epithelia showing hyperproliferation or abnormal differentiation. Of particular interest to us, K16 is strongly induced at the wound edge after injury to the epidermis, and its accumulation correlates spatially and temporally with the onset of reepithelialization. To examine the properties of K16 in its natural cellular context, we introduced a wild-type human K16 gene into the germ line of transgenic mice. Several transgenic lines were established and characterized. Under most conditions, the human K16 transgene is regulated tissue specifically in the skin of transgenic mice. Animals that feature low levels of transgene expression are indistinguishable from controls during die first 6–8 months of life. In contrast, transgenic animals expressing the transgene at higher levels develop skin lesions at 1 week after birth, coinciding with the emergence of fur. At a cellular level, alterations begin with the reorganization of keratin filaments and are first seen at the level of the hair follicle outer root sheath (ORS), where K16 expression is known to occur constitutively. The lesions then progressively spread to involve the proximal epidermis, with which the ORS is contiguous. Elevated transgene expression is associated with a marked thickening of these two epithelia, along with altered keratinocyte cytoarchitecture and aberrant keratinization but no keratinocyte lysis. The implications of this phenotype for epithelial differentiation, human genodermatoses, and wound healing in skin are discussed.Key words: cytokeratin, skin, skin disease, transgenic mouse, wound healing.
Effects of taxol on the polymerization and posttranslational modification of class III β-tubulin in P19 embryonal carcinoma cells
Authors: Laferrière Nicole B. , Brown D. L.
Published: April 2011

Undifferentiated P19 embryonal carcinoma cells and P19 cells induced to differentiate along a neuronal pathway by 10−6 M retinoic acid were treated with taxol to examine the effects of this microtubule-stabilizing drug on the subcellular sorting of class III β-tubulin and on neurite outgrowth. P19 cells were grown on cover slips and then treated with taxol at concentrations of 10−6to 10−9 M for 24 h. The microtubule cytoskeleton was examined after double-immunofluorescence labelling with a monoclonal antibody to α-tubulin (YOL 1/34) and a monoclonal neuron-specific class III β-tubulin antibody (TuJ1). Treatment of undifferentiated P19 cells with concentrations of taxol greater than 4 × 10−8 M caused microtubule bundling and multiple aster formation and promoted polymerization of the low levels of class III β-tubulin found in these cells. In neurons, at 2 × 10−8 M taxol, bundling of microtubules at the base of the neurite was apparent. At taxol concentrations greater than 1 × 10−7 M, enhanced assembly of class III β-tubulin was apparent, although long neurites were not observed. Using isoelectric focusing followed by western blotting, we detected an additional isoform of class III β-tubulin after treatment with 10−6 M taxol. These results indicate taxol treatment alters the normal subcellular sorting of tubulin isotypes, promotes the polymerization and posttranslational modification of class III β-tubulin, and interferes with neurite outgrowth.Key words: tubulin, taxol, microtubule, posttranslational modification, neurite outgrowth.
Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish
Authors: Collas Philippe , Aleström Peter
Published: April 2011

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.
Repetitive elements and their genetic applications in zebrafish
Authors: Izsvák Zsuzsanna , Ivics Zoltan , Hackett Perry B
Published: April 2011

Repetitive elements provide important clues about chromosome dynamics, evolutionary forces, and mechanisms for exchange of genetic information between organisms. Repetitive sequences, especially the mobile elements, have many potential applications in genetic research. DNA transposons and retroposons are routinely used for insertional mutagenesis, gene mapping, gene tagging, and gene transfer in several model systems. Once they are developed for the zebrafish, they will greatly facilitate the identification, mapping, and isolation of genes involved in development as well as the investigation of the evolutionary processes that have been shaping eukaryotic genomes. In this review repetitive elements are characterized in terms of their lengths and other physical properties, copy numbers, modes of amplification, and mobilities within a single genome and between genomes. Examples of how they can be used to screen genomes for species and individual strain differences are presented. This review does not cover repetitive gene families that encode well-studied products such as rRNAs, tRNAs, and the like.
Domains of retinoid signalling and neurectodermal expression of zebrafish otx1 and goosecoid are mutually exclusive
Authors: Joore Jos , Timmermans Ans , de van Water Sandra , Folkers Gert E , van der Saag Paul T , Zivkovic Danica
Published: April 2011

Retinoid signalling plays an important role in embryonic pattern formation. Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning. To study retinoid signalling in zebrafish embryos, we developed a novel method to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain of a retinoic acid receptor and a heterologous DNA binding domain. Using this method, we show that retinoid signalling is localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain. To investigate the relationships between the spatial distribution of retinoid signalling and the regulation of retinoid target genes, we studied the downregulation by retinoic acid of two genes expressed in anterior regions of the embryo, goosecoid and otx1. These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected. Interestingly, a significant downregulation of goosecoid expression by retinoic acid was observed only during midgastrulation but not in earlier stages. In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signalling in the late gastrula. Our data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo. Furthermore, our data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo.
Improved purification protocol of the HSV-1 protease catalytic domain, using immunoaffinity
Authors: McKercher G , Bonneau P R , Lagacé L , Thibeault D , Massariol M -J , Krogsrud R , Lawetz C , McDonald P C , Cordingley M G
Published: April 2011

The catalytic domain of herpes simplex virus protease was expressed in baculovirus-infected cells and purified in milligram quantities by ion-exchange and size-exclusion chromatography. The usefulness of this material was limited by the presence of a contaminating proteolytic activity, which caused time-dependent degradation of the protease. As a result we decided to explore an alternative approach to purification. Specific monoclonal antibodies were produced and evaluated by surface plasmon resonance as ligands for immunoaffinity chromatography. One monoclonal antibody, 6H4, was chosen for coupling to an affinity support, and the resulting column allowed us to obtain a pure and stable enzyme. Immunoaffinity chromatography of herpes simplex virus type 1 protease resulted in successful elimination of the contaminating protease activity. Moreover the immunoaffinity column permitted the isolation of stable and pure enzyme in a one-column procedure.

Key words: herpes virus protease, immunoaffinity chromatography, purification.
Cross-interactions between two members of the Dlx family of homeobox-containing genes during zebrafish development
Authors: Zerucha T , Muller J -P , Chartrand N , Ekker M
Published: April 2011

The Dlx homeobox genes of vertebrates are transcribed in multiple cells of the embryo with overlapping patterns but often with different onsets of expression. Here we describe the interaction between two dlx genes, dlx3 and dlx4, during zebrafish development. The observation that dlx3 expression precedes that of dlx4 in the otic vesicle led us to investigate whether dlx3 had the ability to control expression of dlx4. Truncated versions of dlx3 were overexpressed in zebrafish embryos and the expression patterns of dlx4 were examined later in development. Overexpression of truncated forms of Dlx3 or of a Dlx3-Dlx2 chimera was found to result in perturbations in dlx4 expression. In addition, cotransfection experiments indicated the ability of Dlx3 to activate transcription through a 1.7-kb fragment of the 5 prime flanking region of dlx4. These results suggest that dlx4 is one of the target genes of dlx3 in embryos and that cross-regulatory interactions between Dlx genes may be one of the mechanisms responsible for their overlapping expression.
Characterization of a cDNA encoding a novel band 4.1-like protein in zebrafish
Authors: Kelly Gregory M , Reversade Bruno
Published: April 2011

Membrane skeleton protein 4.1 and other members of a family of proteins that link the cytoskeleton to the plasma membrane may play an integral role in cell communication during development. The polymerase chain reaction and degenerate oligodeoxynucleotide primers to consensus sequences in the putative membrane-binding domain of the protein 4.1 superfamily were used to isolate cDNAs encoding members of the zebrafish protein 4.1 family. Zebrafish stage- and tissue-specific first strand cDNA was used in the PCR. After the reaction, amplicons of the predicted size were sequenced to confirm their relationship to the protein 4.1 superfamily. One cDNA, with a high degree of similarity to a mouse novel band 4.1-like cDNA, was used to probe a zebrafish adult brain library. A 2.4-kb cDNA was isolated and found to encode a 619 amino acid polypeptide homologous to mouse novel band 4.1-like protein 4. Zebrafish nbl4 mRNA is maternally supplied and is expressed throughout embryogenesis. In adults, nbl4 is found in the ovary, eye, heart, and brain, but not in gut or skeletal muscle. When synthetic nbl4 mRNA is translated in vitro it binds calmodulin in a calcium-dependent manner. These data indicate that zebrafish nbl4 is a maternal transcript owing to its presence before the midblastula transition, and it is present later on in specific adult structures. The ability to bind calmodulin would suggest that the function of nbl4 protein may be potentially regulated via a calcium-calmodulin dependent mechanism.
UV irradiation of a B-cell hybridoma increases expression of alkaline phosphatase: involvement in apoptosis
Authors: Souvannavong Vongthip , Lemaire Christophe , Brown Spencer , Adam Arlette
Published: April 2011

Expression of alkaline phosphatase (APase) by 7TD1 B-cell hybridoma was amplified by ultraviolet irradiation; cell growth was inhibited and cell death by apoptosis was increased. Irradiation induced high levels of APase activity in cycling as well as in apoptotic cells. In contrast, APase activity faded with time in nonirradiated cells and was no longer expressed in spontaneous apoptotic cells appearing after several days in culture. This was demonstrated by cell morphology, DNA fragmentation, and flow cytometry after simultaneous staining of DNA with Hoechst 33342 and APase with naphthol AS-TR phosphate - fast red RC fluorescent reagent. Levamisole, a specific inhibitor of APase activity, almost totally abrogated apoptosis induced by ultraviolet irradiation at doses that failed to affect 7TD1 cell survival. These data suggest that APase could play a role in the signalling cascade that mediates apoptosis in irradiated cells. Key words: alkaline phosphatase, apoptosis, flow cytometry, levamisole, UV irradiation.
Fibroblast growth factors and their receptors
Authors: Galzie Zoya , Kinsella Anne R , Smith John A
Published: April 2011

Fibroblast growth factors (FGFs) represent a group of polypeptide mitogens eliciting a wide variety of responses depending upon the target cell type. The knowledge of the cell surface receptors mediating the effects of FGFs has recently expanded remarkably. The complexity of the FGF family and the FGF-induced responses is reflected in the diversity and redundancy of the FGF receptors. In this review, a number of biochemical characteristics and biological properties of the FGF family and its receptors are described and their expression both in normal tissues and in tumours is discussed. Finally we speculate on the targetting of growth inhibition agents to tumours through FGF receptors. Key words: fibroblast growth factor, FGF receptor, heparan sulphate proteoglycans, tyrosine kinase receptors, FGF in tumour diagnosis.
Purification of intact nuclear lamina and identification of novel laminlike proteins in Raji, a cell line devoid of lamins A and C
Authors: Foisy Sylvain , Joly Erik C , Bibor-Hardy Viviane
Published: April 2011

Research on the structure of the nuclear lamina and the nuclear matrix of cells devoid of lamins A and C has been hampered by the fact that intact residual nuclear structures are difficult to isolate from such cells. In this paper, we show that some extraction parameters, such as buffer composition and the nature of the detergent used to remove nuclear membranes, are critical for achieving isolation of whole nuclear residual structures from the lymphoblastic cell line Raji, used as a model for cells without lamins A and C. Electron microscopic analysis shows that the nuclear lamina of Raji cells is formed by a network of intermediate-sized filaments interrupted with circular discontinuities. Both lamins B1 and B2, and lamin D/E, are present in this structure. In addition, a group of 45-kDa proteins or intermediate filament protein - reacting proteins (IFA-RPs), located uniquely in the lamina, were found to exhibit the same immunological and chemical characteristics as lamins. Although they behave like nuclear lamins, microsequencing analysis of the IFA-RPs has revealed no homology with known lamins. These IFA-RPs may contribute to the formation of the nuclear lamina filament network in the absence of lamins A and C. Key words: nuclear lamina, intermediate filaments, lamin.
Structural and functional aspects of G protein-coupled receptor oligomerization
Authors: Hébert Terence E , Bouvier Michel
Published: April 2011

G protein-coupled receptors (GPCRs) represent the single largest family of cell surface receptors involved in signal transduction. It is estimated that several hundred distinct members of this receptor family in humans direct responses to a wide variety of chemical transmitters, including biogenic amines, amino acids, peptides, lipids, nucleosides, and large polypeptides. These transmembrane receptors are key controllers of such diverse physiological processes as neurotransmission, cellular metabolism, secretion, cellular differentiation, and growth as well as inflammatory and immune responses. GPCRs therefore represent major targets for the development of new drug candidates with potential application in all clinical fields. Many currently used therapeutics act by either activating (agonists) or blocking (antagonists) GPCRs. Studies over the past two decades have provided a wealth of information on the biochemical events underlying cellular signalling by GPCRs. However, our understanding of the molecular interactions between ligands and the receptor protein and, particularly, of the structural correlates of receptor activation or inhibition by agonists and inverse agonists, respectively, is still rudimentary. Most of the work in this area has focused on mapping regions of the receptor responsible for drug binding affinity. Although binding of ligand molecules to specific receptors represents the first event in the action of drugs, the efficacy with which this binding is translated into a physiological response remains the only determinant of therapeutic utility. In the last few years, increasing evidence suggested that receptor oligomerization and in particular dimerization may play an important role in the molecular events leading to GPCR activation. In this paper, we review the biochemical and functional evidence supporting this notion.Key words: G proteins, receptors, dimerization, signal transduction, adrenergic.
Guanine nucleotides protect Rho proteins from endogenous proteolytic degradation in renal membranes
Authors: Desrosiers Richard R , Gauthier France , Lin Wei , Béliveau Richard
Published: April 2011

Purified membrane fractions have been widely used for the study of the factors regulating the functions of Rho small GTP-binding proteins. Using brush border membranes from the rat kidney as a model, we observed that in vitro incubation of these membranes resulted in time- and temperature-dependent proteolytic degradation of Cdc42 and RhoA. Treatment of kidney brush border membranes with various nucleotides showed that GDP and GTP weakly protected Cdc42 but not RhoA and that their nonhydrolyzable counterparts, guanosine 5'-O-[β-thio]diphosphate (GDPβS) and guanosine 5'-O-[γ-thio]triphosphate (GTPγS), were highly efficient in protecting both proteins from endogenous proteolytic activity whereas ADP and ATP were without effect. GTPγS also protected Cdc42 and RhoA from proteolytic degradation in crude cell membranes from several rat tissues including intestine, kidney, liver, and testis. In addition, Cdc42 and RhoA associated with brush border membranes were largely resistant to increased proteolytic degradation induced by membrane treatment with the denaturing reagent urea as well as to added trypsin when incubated in the presence of GTPγS. In brush border membranes, the resistance to endo- and exo-genous proteolytic activity conferred by GTPγS was usually lower for RhoA than for Cdc42. GTPγS also protected recombinant Cdc42 and RhoA from the action of proteases associated with brush border membranes. The only protease inhibitor protecting Cdc42 but not RhoA from proteolytic degradation in brush border membranes was the synthetic peptide acetyl-Tyr-Val-Ala-Asp-aldehyde, a selective inhibitor of interleukin-1β-converting enzyme. This latter result showed that different proteases cleaved the two Rho proteins. Taken together, these results suggest that the GTPγS-bound forms of Cdc42 and RhoA are maintained in a conformation that protects them from proteases found in many cell membranes.Key words: rho proteins, GTP, proteolysis, kidney.
The membrane domain of complex I is not assembled in the stopper mutant E35 ofNeurospora
Authors: Alves Paulo Caseiro , Videira Arnaldo
Published: April 2011

The assembly of mitochondrial NADH : ubiquinone oxidoreductase (complex I) was studied in the E35 stopper mutant of Neurospora crassa at different times during growth in liquid media. Assembly of complex I as well as of its membrane domain is impaired in this strain throughout the growth period. Nevertheless, a structure that resembles the peripheral arm of the enzyme is still formed in the mitochondria of this mutant. The absence of the membrane domain of complex I in E35 can be attributed to the specific deletion of the mitochondrial ND2 and ND3 subunits of the enzyme.Key words: mitochondria, complex I, stopper mutants, Neurospora crassa.
A mutant E2F-1 transcription factor that affects the phenotype of NIH3T3 fibroblasts inefficiently associates with cyclin A - cdk2
Authors: Jordan-Sciutto Kelly L , Hall David J
Published: April 2011

The amino-terminal domain of the E2F1 transcription factor is the site of association with cyclin A - cdk2, mapping to residues 87-94. A mutant of E2F1 lacking the first 87 amino acids (termed E2F1d87) has a number of potent effects on cellular phenotype when constitutively expressed in NIH3T3 fibroblasts. For example, in these fibroblasts the duration of S phase and the sensitivity to S phase chemotherapeutic agents are both increased. Since E2F1d87 only partially truncates the cyclin A - cdk2 binding domain, it was important to determine the level of cyclin A - cdk2 association with this mutant to correlate any reduction in association with the observed effects on the cell cycle. It was found that cyclin A - cdk2 binds E2F1d87 in an in vitro assay but that this binding is reduced approximately 8 fold compared with binding to full-length E2F1, whereas no detectable binding was seen to a mutant E2F1 that lacks the first 117 amino acids. Correspondingly, H1 kinase activity in E2F1d87 immunoprecipitates from E2F1d87-expressing cells was significantly reduced compared with that seen for full-length E2F1. From these data it appears that E2F1 with reduced cyclin A - cdk2 binding activity mediates the alteration in cell cycle parameters seen in these cells.Key words: E2F1, apoptosis, cyclin A, cell cycle.
Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains
Authors: Slupsky Carolyn M , Gentile Lisa N , McIntosh Lawrence P
Published: April 2011

The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY - heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.Key words: NMR assignment, aromatic residue, transcription factor, NOE, dihedral angle.
Quadrupolar metal ion NMR studies of metalloproteins
Authors: Aramini James M , Vogel Hans J
Published: April 2011

We present a summary of the quadrupolar metal ion NMR studies of metalloproteins conducted in our laboratory in recent years. The approaches we employ can be subdivided into two categories: (i) the use of low-frequency metal nuclei to probe metal ion binding sites in small proteins, exemplified by 43Ca NMR studies of alpha-lactalbumins and calcium-binding lysozymes, and (ii) the novel detection of the central transition of half-integer quadrupolar nuclei of moderate frequency bound to large metalloproteins, typified by 27Al, 45Sc, 69,71Ga, and 51V NMR studies of the transferrins. We highlight the chemical information regarding the nature of metal ion binding sites that can be obtained from this technique and emphasize the salient parameters that an investigator must consider to successfully apply quadrupolar NMR to the study of biological macromolecules.Key words: quadrupolar NMR, metalloproteins, transferrins.
A review of in vivo 1H magnetic resonance spectroscopy of cerebral ischemia in rats
Authors: Malisza K L , Kozlowski P , Peeling J
Published: April 2011

A number of metabolic alterations are initiated by cerebral ischemia including dramatic increases in lactate concentration, decreases in N-acetylaspartate, choline, and creatine concentrations, as well as changes in amino acid levels. A review of proton nuclear magnetic resonance spectroscopy studies of focal and global cerebral ischemia in rats is presented here. In particular, studies in neonatal rats have shown that a continued elevation of lactate levels without recovery after hypoxia-ischemia or a decrease in N-acetylaspartate concentration at any time are indicative of deleterious outcome. Studies of the effect of temperature on ischemic damage in a model of focal ischemia showed that outcome improved with mild hypothermia. Again, lack of recovery of lactate upon reperfusion was shown to be indicative of poor outcome. Dichloroacetic acid was used to treat rats with focal ischemic damage. Animals subjected to transient ischemia that were treated with dichloroacetic acid showed significant decreases in lactate concentration.Key words: NMR, in vivo, rat, cerebral ischemia.
The structure and function of HPr
Authors: Waygood E Bruce
Published: April 2011

Histidine-containing phosphocarrier protein, HPr, was one of the early protein tertiary structures determined by two-dimensional 1H-NMR. Tertiary structures for HPrs from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus have been obtained by 1H NMR and the overall folding pattern of HPr is highly conserved, a beta alpha beta beta alpha beta alpha arrangement of three alpha-helices overlaying a four-stranded beta-sheet. High-resolution structures for HPrs from E. coli and B. subtilis have been obtained using 15N- and 13C-labeled proteins. The first application of NMR to the understanding of the structure and function of HPr was to describe the phosphohistidine isomer, Ndelta1-P-histidine in S. aureus phospho-HPr, and the unusual pKas of the His-15 side chain. The pKa values for the His-15 imidazole from more recent studies are 5.4 for HPr and 7.8 for phospho-HPr from E. coli, for example. A consensus description of the active site is proposed for HPr and phospho-HPr. In HPr, His-15 has a defined conformation and N-caps helix A, and is thus affected by the helix dipole. His-15 undergoes a small conformational change upon phosphorylation, a movement to allow the phosphoryl group to be positioned such that it forms hydrogen bonds with the main chain amide nitrogens of residue 16 (not conserved) and Arg-17. Interactions between residue 12 side chain (not conserved: asparagine, serine, and threonine) and His-15, and between the Arg-17 guanidinium group and the phosphoryl group, are either weak or transitory.Key words: HPr, NMR, phosphoenolpyruvate:sugar phosphotransferase system, phosphohistidine, phosphoserine.
Recent developments in solid-state nuclear magnetic resonance of quadrupolar nuclei and applications to biological systems
Authors: Wu Gang
Published: April 2011

Recent advances in nuclear magnetic resonance (NMR) methodology and improvements in high-field NMR instrumentation have generated a new wave of research interests in the application of solid-state NMR to the study of quadrupolar nuclei. These developments now permit increasingly complex biological systems to be probed by quadrupolar NMR. In this review I describe a few recent developments in NMR studies of quadrupolar nuclei and demonstrate the potential of solid-state quadrupolar NMR in the study of biological systems. In particular, I discuss the application of solid-state NMR of 17O, 67Zn, 59Co, 23Na, and 39K nuclei with a prognosis for future work.Key words: nuclear magnetic resonance, quadrupole, solid state, biological system.
Sequence-specific 1H NMR resonance assignments and secondary structure of human apolipoprotein C-I in the presence of sodium dodecyl sulfate
Authors: Rozek Annett , Sparrow James T , Weisgraber Karl H , Cushley Robert J
Published: April 2011

Apolipoprotein (apo) C-I is a 57-residue exchangeable plasma protein distributed mainly in high and very low density lipoprotein. In this report we present the nuclear magnetic resonance spectra of native apoC-I and synthetic apoC-I, containing selected 15N-labelled amino acids, in the presence of sodium dodecyl sulfate. The proton resonances of apoC-I are assigned and the secondary structure is estimated from the difference of measured alpha-proton chemical shifts to random coil values and the observed NOE interactions. According to these data apoC-I forms two helices, Val-4-Lys-30 and Leu-34-Lys-52, linked by an unstructured region Gln-31-Glu-33. The N-terminal segments of each helix, Val-4-Gly-15 and Leu-34-Met-38, appear to be more flexible than the helical core regions Asn-16-Lys-30 and Arg-39-Lys-52.Key words: 15N-filtered NOESY, chemical shift index, amphipathic helix, lecithin:cholesterol acyltransferase.
Overexpression of calreticulin fails to abolish its induction by perturbation of normal ER function
Authors: Llewellyn David H , Roderick H Llewelyn
Published: April 2011

Along with other endoplasmic reticulum (ER) Ca2+-binding proteins, notably the glucose-response proteins grp78 and grp94, expression of calreticulin is induced in response to perturbation of normal ER function. It has yet to be clearly defined how this stress is signaled from the ER to the nucleus in mammalian cells, particularly with regard to its initiation. Using a GFP-calreticulin fusion protein, we have generated and selected stably transfected HeLa cells that overexpress calreticulin to investigate whether the protein might be involved in signaling its own induction. Basal levels of endogenous calreticulin mRNA and protein were unaffected in these cells, indicating that overexpression alone does not induce a stress response. ER stress induced calreticulin expression in response to either thapsigargin or tunicamycin was equivalent in these cells to that seen in control, nontransfected cells, leading us to conclude that calreticulin is unlikely be involved in its own induction. Levels of the mRNA encoding the fusion protein were also increased by tunicamycin, but not thapsigargin, suggesting that, in agreement with our previous observations, inhibition of N-linked glycosylation may increase the stability of calreticulin mRNA. This indicates that in mammalian cells, there is more than one signaling pathway for the ER stress response.Key words: calreticulin, endoplasmic reticulum stress, signaling.
Mechanisms regulating the vascular smooth muscle Na/H exchanger (NHE-1) in diabetes
Authors: Hannan Katherine M , Little Peter J
Published: April 2011

Vascular disease is a major component of the complications associated with diabetes. The pathology involves hypertrophy and proliferation of vascular smooth muscle cells and the production and modification of extracellular matrix. The sodium/hydrogen exchanger has been widely implicated in the growth of multiple cell types, including vascular smooth muscle. Increases in sodium/hydrogen exchange activity serve as an effector or at least as an indicator of vascular activation. This article is concerned with the role of the biochemical abnormalities of diabetes exerting their pathological effects on vascular smooth muscle cells via altering sodium/hydrogen exchange activity.Key words: diabetes, sodium/hydrogen exchanger, vascular smooth muscle, complications.
Commitment and differentiation of lung cell lineages
Authors: Warburton David , Wuenschell Carol , Flores-Delgado Guillermo , Anderson Kathryn
Published: April 2011

To form a large diffusible interface capable of conducting respiratory gases to and from the circulation, the lung must undergo extensive cell proliferation, branching morphogenesis, and alveolar saccule formation, to generate sufficient surface area. In addition, the cells must differentiate into at least 40 distinct lung cell lineages. Specific transcriptional factors, peptide growth factor receptor-mediated signaling pathways, extracelluar matrix components, and integrin-signaling pathways interact to direct lung morphogenesis and lung cell lineage differentiation. Branching mutants of the respiratory tracheae in Drosophila have identified several functionally conserved genes in the fibroblast growth factor signaling pathway that also regulate pulmonary organogenesis in mice and probably also in man. Key transcriptional factors including Nkx2.1, hepatocyte nuclear factor family forkhead homologues, GATA family zinc finger factors, pou and homeodomain proteins, as well as basic helix-loop-helix factors, serve as master genes to integrate the developmental genetic instruction of lung morphogenesis and cell lineage determination. Key words: lung branching morphogenesis, lung cell proliferation, lung cell differentiation, alveolization, master genes, peptide growth factor signaling, extracellular matrix signaling, mesenchyme induction, alveolar epithelial cells, pulmonary neuroendocrine cells, stem cells, retinoic acid.
Endothelin signalling in the development of neural crest-derived melanocytes
Authors: Opdecamp Karin , Kos Lidia , Arnheiter Heinz , Pavan William J
Published: April 2011

In both mice and humans, mutations in the genes encoding the endothelin B receptor and its ligand endothelin 3 lead to deficiencies in neural crest-derived melanocytes and enteric neurons. The discrete steps at which endothelins exert their functions in melanocyte development were examined in mouse neural crest cell cultures. Such cultures, kept in the presence of fetal calf serum, gave rise to cells expressing the early melanoblast marker Dct even in the absence of the phorbol ester tetradecanoyl phorbol acetate (TPA) or endothelins. However, these early (Dct+) cells did not proliferate and pigmented cells never formed unless TPA or endothelins were added. In fact, endothelin 2 was as potent as TPA in promoting the generation of both Dct+melanoblasts and pigmented cells, and endothelin 1 or endothelin 3 stimulated the generation of melanoblasts and of pigmented cells to an even greater extent. The inhibition of this stimulation by the selective endothelin B receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-alpha-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine) suggested that the three endothelins all signal through the endothelin B receptor. This receptor was indeed expressed in Dct+melanoblasts, in addition to cells lacking Dct expression. The results demonstrate that endothelins are potent stimulators of melanoblast proliferation and differentiation.Key words: neural crest, melanocyte, endothelin, differentiation.
Plasticity of mammary epithelia during normal development and neoplastic progression
Authors: Lochter André
Published: April 2011

The functional unit of the mammary gland is the epithelium. It consists of luminal epithelial cells and myoepithelial cells that are generated from self-renewing stem and progenitor cells. The latter two cell types are scattered throughout the mammary epithelium and are concentrated in specialized structures, the end buds. In transplantation studies the pluripotency of mammary stem cells has been confirmed by demonstrating that they can regenerate a complete mammary gland. The ability of mammary epithelial cells to produce an elaborate ductal system during puberty and to differentiate into milk-producing alveoli during pregnancy is not only influenced by their genetic make-up, but is also governed by local molecular signals. Recent studies suggest that the transdifferentiation of epithelial cells into tumor cells is under microenvironmental control, despite the prominence of genetic mutations in breast cancer. Consequently, disturbances of tissue homeostasis can alter mammary gland development or result in preneoplastic and neoplastic pathologies. The plasticity of mammary epithelia is not limited to the entry of cells into differentiation and transdifferentiation pathways, but extends to their ability to regain facets of their preceding stage of functionality. Deciphering the molecular cues that determine cell plasticity is prerequisite for establishing a unifying concept of mammary gland development and breast tumor progression.Key words: branching morphogenesis, lactogenic differentiation, stem cells, epithelial-to-mesenchymal transition, cancer.