JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010
William J. McDonald
Abstract Mammalian ,4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. ,4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using ,4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of ,4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with ,4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between ,4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. ,4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD,,4 immunocomplex. Furthermore, deletion mutants of ,4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of ,4, independent of the ,4-PP2Ac binding site. Therefore, in addition to PP2Ac, ,4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression. J. Cell. Biochem. 110: 1123,1129, 2010. Published 2010 Wiley-Liss, Inc. [source]

Water Vapor Permeability of Mammalian and Fish Gelatin Films

JOURNAL OF FOOD SCIENCE, Issue 4 2006
R.J. Avena-Bustillos
ABSTRACT:, Water vapor permeability of cold- and warm-water fish skin gelatins films was evaluated and compared with different types of mammalian gelatins. Alaskan pollock and salmon gelatins were extracted from frozen skins, others were obtained from commercial sources. Water vapor permeability of gelatin films was determined considering differences on percent relative humidity (%RH) at the film underside. Molecular weight distribution, amino acid composition, gel strength, viscoelastic properties, pH, and clarity were also determined for each gelatin. Water vapor permeability of cold-water fish gelatin films (0.93 gmm/m2hkPa) was significantly lower than warm-water fish and mammalian gelatin films (1.31 and 1.88 gmm/m2hkPa, respectively) at 25 °C, 0/80 %RH through 0.05-mm thickness films. This was related to increased hydrophobicity due to reduced amounts of proline and hydroxyproline in cold-water fish gelatins. As expected, gel strength and gel setting temperatures were lower for cold-water fish gelatin than either warm-water fish gelatins or mammalian gelatins. This study demonstrated significant differences in physical, chemical, and rheological properties between mammalian and fish gelatins. Lower water vapor permeability of fish gelatin films can be useful particularly for applications related to reducing water loss from encapsulated drugs and refrigerated or frozen food systems. [source]

Expression of SLURP-1, an endogenous ,7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2009
Kazuhide Horiguchi
Abstract Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for ,7 nicotinic acetylcholine (ACh) receptors (,7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP-1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP-1 is postulated to be involved in regulating tumor necrosis factor-, (TNF-,) release from keratinocytes and macrophages via ,7 nAChR-mediated pathways. In the present study, we assessed SLURP-1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP-1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP-1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5-kDa SLURP-1 protein in whole-lung tissue and trachea. In addition, high-affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP-1 and ACh. We also observed direct contact between F4/80-positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1-positive nerve elements. Collectively, these results suggest that SLURP-1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF-, release from macrophages in bronchial tissue. © 2009 Wiley-Liss, Inc. [source]

Body size and human energy requirements: Reduced mass-specific total energy expenditure in tall adults,

AMERICAN JOURNAL OF HUMAN BIOLOGY, Issue 3 2010
Steven B. Heymsfield
Mammalian resting energy expenditure (REE) increases as ,weight0.75 while mass-specific REE scales as ,weight,0.25. Energy needs for replacing resting losses are thus less relative to weight (W) in large compared with small mammals, a classic observation with biological implications. Human weight scales as ,height2 and tall adults thus have a greater weight than their short counterparts. However, it remains unknown if mass-specific energy requirements are less in tall adults; allometric models linking total energy expenditure (TEE) and weight with height (H) are lacking. We tested the hypothesis that mass-specific energy requirements scale inversely to height in adults by evaluating TEE (doubly labeled water) data collected by the National Academy of Sciences. Activity energy expenditure (AEE) was calculated from TEE, REE (indirect calorimetry), and estimated diet-induced energy expenditure. Main analyses focused on nonmorbidly obese subjects ,50 yrs of age with non-negative AEE values (n = 404), although results were directionally similar for all samples. Allometric models, including age as a covariate, revealed significantly (P < 0.05) greater REE, AEE, and TEE as a function of height (range H1.5,1.7) in both men and women. TEE/W scaled negatively to height (,H,0.7, P < 0.01) with predicted mass-specific TEE (kcal/kg/d) at ±2 SD for US height lower in tall compared with short men (40.3 vs. 46.5) and women (37.7 vs. 42.7). REE/W also scaled negatively to height in men (P < 0.001) and women (P < 0.01). Results were generally robust across several different analytic strategies. These observations reveal previously unforeseen associations between human stature and energy requirements that have implications for modeling efforts and provide new links to mammalian biology as a whole. Am. J. Hum. Biol. 2010. © 2009 Wiley-Liss, Inc. [source]

ChemInform Abstract: Structure,Activity Relationships of New Inhibitors of Mammalian 2,3-Oxidosqualene Cyclase Designed from Isoquinoline Derivatives

CHEMINFORM, Issue 32 2002
Jean Binet
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source]

Reelin, radial fibers and cortical evolution: Insights from comparative analysis of the mammalian and avian telencephalon

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2009
Tadashi Nomura
The mammalian cerebral cortex has a remarkable laminated structure, which is derived from the pallium, the dorsal part of the embryonic telencephalon. Recent studies indicate that the pallium is developed as a homologous structure in all vertebrate species. However, the cellular and molecular mechanism for making architectural diversity of the pallium is not fully understood. Here we introduce recent progress in comparative analysis of pallial development, and our data on the role of Reelin protein in the developing avian pallium. These experimental approaches to pallial development in non-mammalian species will provide a new insight into evolution of the cerebral cortex. [source]

Wnt11r is required for cranial neural crest migration

DEVELOPMENTAL DYNAMICS, Issue 11 2008
Helen K. Matthews
Abstract wnt11r is a recently identified member of the Wnt family of genes, which has been proposed to be the true Xenopus homologue to the mammalian wnt11 gene. In this study we have examined the role of wnt11r on neural crest development. Expression analysis of wnt11r and comparison with the neural crest marker snail2 and the noncanonical Wnt, wnt11, shows wnt11r is expressed at the medial or neural plate side of the neural crest while wnt11 is expressed at the lateral or epidermal side. Injection of wnt11r morpholino leads to strong inhibition of neural crest migration with no effect on neural crest induction or maintenance. This effect can be rescued by co-injection of Wnt11r but not by Wnt11 mRNA, demonstrating the specificity of the loss of function treatment. Finally, neural crest graft experiments show that wnt11r is required in a non,cell-autonomous manner to control neural crest migration. Developmental Dynamics 237:3404,3409, 2008. © 2008 Wiley-Liss, Inc. [source]

Developmental expression and comparative genomic analysis of Xenopus cardiac myosin heavy chain genes

DEVELOPMENTAL DYNAMICS, Issue 4 2005
Robert J. Garriock
Abstract Myosin heavy chains (MHC) are cytoskeletal motor proteins essential to the process of muscle contraction. We have determined the complete sequences of the Xenopus cardiac MHC genes, ,-MHC and ventricular MHC (vMHC), and have characterized their developmental expression profiles. Whereas ,-MHC is expressed from the earliest stages of cardiac differentiation, vMHC transcripts are not detected until the heart has undergone chamber formation. Early expression of vMHC appears to mark the cardiac conduction system, but expression expands to include the ventricle and outflow tract myocardium during subsequent development. Sequence comparisons, transgenic expression analysis, and comparative genomic studies indicate that Xenopus ,-MHC is the true orthologue of the mammalian ,-MHC gene. On the other hand, we show that the Xenopus vMHC gene is most closely related to chicken ventricular MHC (vMHC1) not the mammalian ,-MHC. Comparative genomic analysis has allowed the detection of a mammalian MHC gene (MyH15) that appears to be the orthologue of vMHC, but evidence suggests that this gene is no longer active. Developmental Dynamics 233:1287,1293, 2005. © 2005 Wiley-Liss, Inc. [source]

Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevis

DEVELOPMENTAL DYNAMICS, Issue 3 2005
Teun P. De Boer
Abstract Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (,62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864,871, 2005. © 2005 Wiley-Liss, Inc. [source]

Two Na,K-ATPase ,2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis

DEVELOPMENTAL DYNAMICS, Issue 2 2002
Johannes R. Rajarao
Abstract We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase ,2 subunit. The ,2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish ,2a subunit. By using a zebrafish meiotic mapping panel, we determined that the ,2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the ,2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. ,2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, ,2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the ,2a and ,2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian ,2 gene may be partitioned between the two zebrafish ,2 orthologs. © 2002 Wiley-Liss, Inc. [source]

From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens

ENVIRONMENTAL MICROBIOLOGY, Issue 3 2000
Minireview
It is becoming apparent that several intracellular bacterial pathogens of humans can also survive within protozoa. This interaction with protozoa may protect these pathogens from harsh conditions in the extracellular environment and enhance their infectivity in mammals. This relationship has been clearly established in the case of the interaction between Legionella pneumophila and its protozoan hosts. In addition, the adaptation of bacterial pathogens to the intracellular life within the primitive eukaryotic protozoa may have provided them with the means to infect the more evolved mammalian cells. This is evident from the existence of several similarities, at both the phenotypic and the molecular levels, between the infection of mammalian and protozoan cells by L. pneumophila. Thus, protozoa appear to play a central role in the transition of bacteria from the environment to mammals. In essence, protozoa may be viewed as a ,biological gym', within which intracellular bacterial pathogens train for their encounters with the more evolved mammalian cells. Thus, intracellular bacterial pathogens have benefited from the structural and biochemical conservation of cellular processes in eukaryotes. The interaction of intracellular bacterial pathogens and protozoa highlights this conservation and may constitute a simplified model for the study of these pathogens and the evolution of cellular processes in eukaryotes. Furthermore, in addition to being environmental reservoirs for known intracellular pathogens of humans and animals, protozoa may be sources of emerging pathogenic bacteria. It is thus critical to re-examine the relationship between bacteria and protozoa to further our understanding of current human bacterial pathogenesis and, possibly, to predict the appearance of emerging pathogens. [source]

Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems

EQUINE VETERINARY JOURNAL, Issue 7 2001
E. L. J. McMONAGLE
Summary Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-, production in cells derived from equine lymph nodes. Preincubation of IFN-, inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-, induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-, production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species. [source]

Erythropoiesis and red cell function in vertebrate embryos

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2005
R. Baumann
Abstract All vertebrate embryos produce a specific erythroid cell population , primitive erythrocytes , early in development. These cells are characterized by expression of the specific embryonic haemoglobins. Many aspects of primitive erythropoiesis and the physiological function of primitive red cells are still enigmatic. Nevertheless, recent years have seen intensive efforts to characterize in greater detail the molecular events underlying the initiation of erythropoiesis in vertebrate embryos. Several key genes have been identified that are necessary for primitive and the subsequent definitive erythropoiesis, which differs in several aspect from primitive erythropoiesis. This review gives in its first part a short overview dealing with comparative aspects of primitive and early definitive erythropoiesis in higher and lower vertebrates and in the second part we discuss the physiological function of primitive red cells based mainly on results from mammalian and avian embryos. [source]

The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivation

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006
G. T. Macleod
Abstract Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cacts2 mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cacts2 mutation occurs in the cytoplasmic carboxyl tail of the ,1 -subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+ -imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cacts2 mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Cav2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 °C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Cav2.1 channel inactivation. [source]

SK channels and the varieties of slow after-hyperpolarizations in neurons

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003
Fivos Vogalis
Abstract Action potentials and associated Ca2+ influx can be followed by slow after-hyperpolarizations (sAHPs) caused by a voltage-insensitive, Ca2+ -dependent K+ current. Slow AHPs are a widespread phenomenon in mammalian (including human) neurons and are present in both peripheral and central nervous systems. Although, the molecular identity of ion channels responsible for common membrane potential mechanisms has been largely determined, the nature of the channels that underlie the sAHPs in neurons, both in the brain and in the periphery, remains unresolved. This short review discusses why there is no clear molecular candidate for sAHPs. [source]

The emerging role of neuronal nitric oxide synthase in the regulation of myocardial function

EXPERIMENTAL PHYSIOLOGY, Issue 6 2006
Barbara Casadei
The recent discovery of a NOS1 gene product (i.e. a neuronal-like isoform of nitric oxide synthase or nNOS) in the mammalian left ventricular (LV) myocardium has provided a new key for the interpretation of the complex experimental evidence supporting a role for myocardial constitutive nitric oxide (NO) production in the regulation of basal and ,-badrenergic cardiac function. Importantly, nNOS gene deletion has been associated with more severe LV remodelling and functional deterioration in murine models of myocardial infarction, suggesting that nNOS-derived NO may also be involved in the myocardial response to injury. To date, the mechanisms by which nNOS influences myocardial pathophysiology remain incompletely understood. In particular, it seems over simplistic to assume that all aspects of the myocardial phenotype of nNOS knockout (nNOS,/,) mice are a direct consequence of lack of NO production from this source. Emerging data showing co-localisation of xanthine oxidoreductase (XOR) and nNOS in the sarcoplasmic reticulum of rodents, and increased XOR activity in the nNOS,/, myocardium, suggest that nNOS gene deletion may have wider implications on the myocardial redox state. Similarly, the mechanisms regulating the targeting of myocardial nNOS to different subcellular compartments and the functional consequences of intracellular nNOS trafficking have not been fully established. Whether this information could be translated into a better understanding and management of human heart failure remains the most important challenge for future investigations. [source]

Characterization of Zebrafish Cx43.4 Connexin and its Channels

EXPERIMENTAL PHYSIOLOGY, Issue 6 2003
T. Desplantez
Connexins (Cx) form intercellular junctional channels which are responsible for metabolic and electrical coupling. We report here on the biochemical and immunohistochemical characterization of zebrafish connexin zfCx43.4, an orthologue of mammalian and avian Cx45, and the electrophysiological properties of junctional channels formed by this protein. The investigations were performed on transfected COS-7 cells or HeLa cells. Using site-directed antibodies, zfCx43.4 cDNA (GenBank accession no. X96712) was demonstrated to code for a protein with a Mr of 45 000. In transfected cells, zfCx43.4 was localized in cell-cell contact areas as expected for a gap junction protein. zfCx43.4 channels were shown to transfer Lucifer Yellow. The multichannel currents were sensitive to the transjunctional voltage (Vj). Their properties were consistent with a two-state model and yielded the following Boltzmann parameters for negative/positive Vj: Vj,0= -38.4/41.9 mV; gj,min= 0.19/0.18; z = 2.6/2.3. These parameters deviate somewhat from those of zfCx43.4 channels expressed in Xenopus oocytes and from those of Cx45, an orthologue of zfCx43.4, expressed in mammalian cells or Xenopus oocytes. Conceivably, the subtle differences may reflect differences in experimental methods and/or in the expression system. The single channel currents yielded two prominent levels attributable to a main conductance state (,j,main= 33.2 ± 1.5 pS) and a residual conductance state (,j,residual= 11.9 ± 0.6 pS). [source]

Voltage-gated sodium channel isoform-specific effects of pompilidotoxins

FEBS JOURNAL, Issue 4 2010
Emanuele Schiavon
Pompilidotoxins (PMTXs, , and ,) are small peptides consisting of 13 amino acids purified from the venom of the solitary wasps Anoplius samariensis (,-PMTX) and Batozonellus maculifrons (,-PMTX). They are known to facilitate synaptic transmission in the lobster neuromuscular junction, and to slow sodium channel inactivation. By using ,-PMTX, ,-PMTX and four synthetic analogs with amino acid changes, we conducted a thorough study of the effects of PMTXs on sodium current inactivation in seven mammalian voltage-gated sodium channel (VGSC) isoforms and one insect VGSC (DmNav1). By evaluating three components of which the inactivating current is composed (fast, slow and steady-state components), we could distinguish three distinct groups of PMTX effects. The first group concerned the insect and Nav1.6 channels, which showed a large increase in the steady-state current component without any increase in the slow component. Moreover, the dose-dependent increase in this steady-state component was correlated with the dose-dependent decrease in the fast component. A second group of effects concerned the Nav1.1, Nav1.2, Nav1.3 and Nav1.7 isoforms, which responded with a large increase in the slow component, and showed only a small steady-state component. As with the first group of effects, the slow component was dose-dependent and correlated with the decrease in the fast component. Finally, a third group of effects concerned Nav1.4 and Nav1.5, which did not show any change in the slow or steady-state component. These data shed light on the complex and intriguing behavior of VGSCs in response to PMTXs, helping us to better understand the molecular determinants explaining isoform-specific effects. [source]

Mouse RS21-C6 is a mammalian 2,-deoxycytidine 5,-triphosphate pyrophosphohydrolase that prefers 5-iodocytosine

FEBS JOURNAL, Issue 6 2009
Mari Nonaka
Free nucleotides in living cells play important roles in a variety of biological reactions, and often undergo chemical modifications of their base moieties. As modified nucleotides may have deleterious effects on cells, they must be eliminated from intracellular nucleotide pools. We have performed a screen for ITP-binding proteins because ITP is a deaminated product of ATP, the most abundant nucleotide, and identified RS21-C6 protein, which bound not only ITP but also ATP. Purified, recombinant RS21-C6 hydrolyzed several canonical nucleoside triphosphates to the corresponding nucleoside monophosphates. The pyrophosphohydrolase activity of RS21-C6 showed a preference for deoxynucleoside triphosphates and cytosine bases. The kcat/Km (s,1·m,1) values were 3.11 × 104, 4.49 × 103 and 1.87 × 103 for dCTP, dATP and dTTP, respectively, and RS21-C6 did not hydrolyze dGTP. Of the base-modified nucleotides analyzed, 5-I-dCTP showed an eightfold higher kcat/Km value compared with that of its corresponding unmodified nucleotide, dCTP. RS21-C6 is expressed in both proliferating and non-proliferating cells, and is localized to the cytoplasm. These results show that RS21-C6 produces dCMP, an upstream precursor for the de novo synthesis of dTTP, by hydrolyzing canonical dCTP. Moreover, RS21-C6 may also prevent inappropriate DNA methylation, DNA replication blocking or mutagenesis by hydrolyzing modified dCTP. [source]

Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria

FEBS JOURNAL, Issue 13 2008
Tatjana M. Hildebrandt
Hydrogen sulfide is a potent toxin of aerobic respiration, but also has physiological functions as a signalling molecule and as a substrate for ATP production. A mitochondrial pathway catalyzing sulfide oxidation to thiosulfate in three consecutive reactions has been identified in rat liver as well as in the body-wall tissue of the lugworm, Arenicola marina. A membrane-bound sulfide : quinone oxidoreductase converts sulfide to persulfides and transfers the electrons to the ubiquinone pool. Subsequently, a putative sulfur dioxygenase in the mitochondrial matrix oxidizes one persulfide molecule to sulfite, consuming molecular oxygen. The final reaction is catalyzed by a sulfur transferase, which adds a second persulfide from the sulfide : quinone oxidoreductase to sulfite, resulting in the final product thiosulfate. This role in sulfide oxidation is an additional physiological function of the mitochondrial sulfur transferase, rhodanese. [source]

Binding of the volatile general anesthetics halothane and isoflurane to a mammalian ,-barrel protein

FEBS JOURNAL, Issue 2 2005
Jonas S. Johansson
A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic,protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large ,-barrel internal cavity (515 ± 30 Å3) lined predominantly by aromatic and aliphatic residues. Halothane binding to the ,-barrel cavity was determined using fluorescence quenching of Trp16, and a competitive binding assay with 1-aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using isothermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the ,-barrel of porcine odorant binding protein with dissociation constants of 0.46 ± 0.10 mm and 0.43 ± 0.12 mm determined using fluorescence quenching and competitive binding with 1-aminoanthracene, respectively. Isothermal titration calorimetry revealed that halothane and isoflurane bound with Kd values of 80 ± 10 µm and 100 ± 10 µm, respectively. Halothane and isoflurane binding resulted in an overall stabilization of the folded conformation of the protein by ,0.9 ± 0.1 kcal·mol,1. In addition to indicating specific binding to the native protein conformation, such stabilization may represent a fundamental mechanism whereby anesthetics reversibly alter protein function. Because porcine odorant binding protein has been successfully analyzed by X-ray diffraction to 2.25 Å resolution [1], this represents an attractive system for atomic-level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules. [source]

Membrane orientation of laminin binding protein

FEBS JOURNAL, Issue 18 2003
An extracellular matrix bridging molecule of Leishmania donovani
Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m -iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end. [source]

Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation?

FEMS MICROBIOLOGY LETTERS, Issue 2 2001
Jean de Rycke
Abstract Cytolethal distending toxins (CDT) constitute a family of genetically related bacterial protein toxins able to stop the proliferation of numerous cell lines. This effect is due to their ability to trigger in target cells a signaling pathway that normally prevents the transition between the G2 and the M phase of the cell cycle. Produced by several unrelated Gram-negative mucosa-associated bacterial species, CDTs are determined by a cluster of three adjacent genes (cdtA, cdtB, cdtC) encoding proteins whose respective role is not yet fully elucidated. The CDT-B protein presents sequence homology to several mammalian and bacterial phosphodiesterases, such as DNase I. The putative nuclease activity of CDT-B, together with the activation by CDT of a G2 cell cycle checkpoint, strongly suggests that CDT induces an as yet uncharacterized DNA alteration. However, the effective entry of CDT into cells and subsequent translocation into the nucleus have not yet been demonstrated by direct methods. The relationship between the potential DNA-damaging properties of this original family of toxins and their role as putative virulence factors is discussed. [source]

Purification and characterization of a glutathione S -transferase from the fungus Cunninghamella elegans

FEMS MICROBIOLOGY LETTERS, Issue 2 2001
Chang-Jun Cha
Abstract Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S -transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120,000×g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate,polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of Mr 27,000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian ,-, ,-, and ,-class GSTs, although it showed a small degree of cross-reactivity with a ,-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs. [source]

Population size, weight distribution and food in a persistent population of the rare medicinal leech, Hirudo medicinalis

FRESHWATER BIOLOGY, Issue 8 2008
J. M. ELLIOTT
Summary 1. It is important for species recovery and conservation management projects to know the minimum viable population size for rare and endangered species, such as the medicinal leech, Hirudo medicinalis. Therefore, using a catch-removal method, this study estimated every two years (1986, 1988, 1990, 1992) the total number of medicinal leeches in a tarn in the English Lake District, and the number of mature adults in the population. 2. Four samples were taken each year in June and July, when water temperatures exceeded 20 °C. Population size was estimated both by maximum likelihood and regression methods. All leeches were weighed alive and size groups were separated by polymodal frequency analysis. A small sample of the blood meal in each leech gut was taken before the leeches were returned to the tarn, and was used to estimate the proportion of mammalian and non-mammalian blood in the meals. 3. Both methods of estimation produced similar values, increasing confidence in the population estimates. Values for the total population in June and July varied among years from 248 to 288, the maximum value being only 16% higher than the minimum. Values for the number of mature leeches varied from 48 to 58 (19,20% of the total population), and this was an estimate of the effective population size. 4. There were four size groups. The largest mature leeches (live weight >5 g) in group IV formed only 1% of the population, and the smallest (0.02,0.5 g) in group I 14,17%. Most leeches were in two overlapping groups of immature (64,67% of population) and mature (18%) leeches with size ranges of 0.4,3.4 g and 2.5,5 g respectively. The percentage of leeches in each size group was very consistent among years. Blood meals were found in 38,44% of the leeches in group I, 45,50% in group II, 70,75% in group III, and 100% in group IV, but mammalian blood was present only in larger mature leeches (>3.5 g). 5. Medicinal leeches were first detected in the tarn in 1980 and are still present in 2007, so the population has persisted for at least 27 years. Compared with minimum viable population sizes for other species, including many endangered species, values for this medicinal leech population are extremely low, but may be typical of some rare freshwater invertebrates in isolated habitats. [source]

Isolation and functional identification of a novel human hepatic growth factor: Hepatopoietin Cn,

HEPATOLOGY, Issue 3 2008
Chun-Ping Cui
Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [3H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. Conclusion: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration. (HEPATOLOGY 2008;47:986,995.) [source]

Glycolipid targets of CD1-mediated T-cell responses

IMMUNOLOGY, Issue 3 2001
D. Branch Moody
Summary Members of the CD1 family of antigen-presenting molecules bind and present a variety of mammalian and microbial glycolipids for specific recognition by T cells. CD1 proteins accomplish their antigen-presenting function by binding the alkyl chains of the antigens within a deep, hydrophobic groove on the membrane distal surface of CD1, making the hydrophilic elements of the antigen available for contact with the variable regions of antigen-specific T-cell receptors. Most models of CD1-restricted T cells function in infectious, neoplastic, or autoimmune diseases and are based on the premise that CD1-restricted T-cell responses are initiated by alterations in cellular glycolipid content. Although a growing number of self, altered self and foreign glycolipid antigens have been identified, the cellular mechanisms that could lead to the generation of antigenic glycolipids within cells, or control the presentation of particular classes of altered self or microbial glycolipids in disease states have only recently come under investigation. Here we review the structures of known glycolipid antigens for T cells and discuss how the chemical nature of these antigens, which is quite different from that of peptides, influences their recognition by T cells. [source]

Molecular characterization of a peroxiredoxin from the hard tick Haemaphysalis longicornis

INSECT MOLECULAR BIOLOGY, Issue 2 2001
N. Tsuji
Abstract Antioxidant enzymes in eukaryotes play an important role in protection against the oxygen radicals generated during aerobic metabolism. Here we report the cloning and characterization of a cDNA encoding the antioxidant enzyme peroxiredoxin from the hard tick Haemaphysalis longicornis (HlPrx). HlPrx is 939 bp long and contains a 101 bp non-translated sequence at the 5, end and a polyadenylation singnal followed by a poly(A) tail at the 3, end. HlPrx encodes a full-length protein with a predicted molecular mass of 26 kDa that possesses one cysteine residue at amino acid 49 that is conserved among Prx proteins of various species. GenBankÔ analysis showed that the deduced amino acid sequence had significant similarity to mammalian and plant Prxs at the amino acid level. A DNA-nicking assay revealed that Escherichia coli,expressed recombinant HlPrx (rHlPrx) inhibited oxidative-nicking of supercoiled plasmid DNA. Two-dimensional immunoblot analysis with mouse antirHlPrx serum showed reaction with a major constituent protein spot in extracts of adult ticks. In addition, immunoblot analysis showed that rHlPrx was immunoreacted with serum from rabbits repeatedly infested with H. longicornis. Localization analysis using mouse antirHlPrx serum revealed that native HlPrx was highly expressed in the salivary gland of the tick. Moreover, Northern blot analysis showed that the level of HlPrx transcripts was increased during blood sucking. The present results indicate that HlPrx may be an important detoxifying enzyme during the normal life span as well as during blood sucking in ticks. [source]

Prey size and ingestion rate in raptors: importance for sex roles and reversed sexual size dimorphism

JOURNAL OF AVIAN BIOLOGY, Issue 6 2007
Tore Slagsvold
Compared to other birds, most raptors take large prey for their size, and feeding bouts are extended. However, ingestion rate has largely been overlooked as a constraint in raptors, foraging and breeding ecology. We measured ingestion rate by offering avian and mammalian prey to eighteen wild raptors temporarily kept in captivity, representing seven species and three orders. Ingestion rate was higher for small than for large prey, higher for mammalian than for avian prey, higher for large than for small raptors, and higher for wide-gaped than for narrow-gaped raptors. Mammalian prey were ingested faster by raptors belonging to species with mainly mammals in their diet than by raptors with mainly birds in their diet, but the drop in ingestion rate with increasing prey size was more rapid for the former than for the latter. We argue that the separate sex roles found in raptors, i.e. the male hunting and the female feeding the young, is a solution of the conflict between the prolonged feeding bouts at the nest, and the benefit of rapid resumption of hunting in general, and rapid return to the previous capture site in particular (the prey size hypothesis). Thus, the sex roles differ more when prey takes longer to feed, i.e. from insects to mammals to birds. We then argue that the reversed sexual size dimorphism in raptors, i.e. smaller males than females, results from a conflict between the benefit of being small during breeding to capture the smallest items with the highest ingestion rate among these agile prey types (mammals and bird), and the benefit of being large outside the breeding season to ensure survival by being able to include large items in the diet when small items are scarce (the ingestion rate hypothesis). This hypothesis explains the observed variation in reversed sexual size dimorphism among raptors in relation to size and type of prey, i.e. increasing RSD from insects to mammals to birds as prey. [source]

The DEAD-box RNA helicase DDX1 interacts with RelA and enhances nuclear factor kappaB-mediated transcription

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009
Musarat Ishaq
Abstract DEAD-box RNA helicases constitute the largest family of RNA helicases and are involved in many aspects of RNA metabolism. In this study, we identified RelA (p65), a subunit of nuclear factor-kappaB (NF-,B), as a cellular co-factor of DEAD-box RNA helicase DDX1, through mammalian two hybrid system and co-immunoprecipitation assay. Additionally, confocal microscopy and chromatin immunoprecipitation assays confirmed this interaction. In NF-,B dependent reporter gene assay, DDX1 acted as a co-activator to enhance NF-,B-mediated transcription activation. The functional domains involved were mapped to the carboxy terminal transactivation domain of RelA and the amino terminal ATPase/helicase domain of DDX1. The DDX1 trans-dominant negative mutant lacking ATP-dependent RNA helicase activity lost it transcriptional inducer activity. Moreover, depletion of endogenous DDX1 by specific small interfering RNAs significantly reduced NF-,B-dependent transcription. Taken together, the results suggest that DDX1 may play an important role in NF-,B-mediated transactivation, and revelation of this regulatory pathway may help to explore the novel mechanisms for regulating NF-,B transcriptional activity. J. Cell. Biochem. 106: 296,305, 2009. © 2008 Wiley-Liss, Inc. [source]