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Results for “Functional characterization”

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At least 433 records · Page 24Linked to original sources

PubMed2000

Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59.

CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.

Amino Acid Sequence↗

PubMed1992

Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate.

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2004

Molecular cloning and functional characterization of a Lepidopteran insect beta4-N-acetylgalactosaminyltransferase with broad substrate specificity, a functional role in glycoprotein biosynthesis, and a potential functional role in glycolipid biosynthesis.

A degenerate PCR approach was used to isolate a lepidopteran insect cDNA encoding a beta4-galactosyl-transferase family member. The isolation and initial identification of this cDNA was based on bioinformatics, but its identification as a beta4-galactosyltransferase family member was experimentally confirmed. The newly identified beta4-galactosyltransferase family member had unusually broad donor and acceptor substrate specificities in vitro, as transferred galactose, N-acetylglucosamine, and N-acetylgalactosamine to carbohydrate, glycoprotein, and glycolipid acceptors. However, the enzyme preferentially utilized N-acetylgalactosamine as the donor for all three acceptors, and its derived amino acid sequence was closely related to a known N-acetylgalactosaminyltransferase. These data suggested that the newly isolated cDNA encodes a beta4-N-acetylgalactosaminyltransferase that functions in insect cell glycoprotein biosynthesis, glycolipid biosynthesis, or both. The remainder of this study focused on the role of this enzyme in N-glycoprotein biosynthesis. The results showed that the purified enzyme transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to a synthetic N-glycan in vitro. The structure of the reaction product was confirmed by chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses. Co-expression of the new cDNA product in insect cells with an N-glycoprotein reporter showed that it transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to this N-glycoprotein in vivo. Confocal microscopy showed that a GFP-tagged version of the enzyme was localized in the insect cell Golgi apparatus. In summary, this study demonstrated that lepidopteran insect cells encode and express a beta4-N-acetylgalactosaminyltransferase that functions in N-glycoprotein biosynthesis and perhaps in glycolipid biosynthesis, as well. The isolation and characterization of this gene and its product contribute to our basic understanding of insect protein N-glycosylation pathways and to the growing body of evidence that insects can produce glycoproteins with complex N-glycans.

Acetylgalactosamine↗

PubMed2002

Identification and functional characterization of the p66/p68 components of the MeCP1 complex.

Methylation of cytosine at CpG dinucleotides is a common feature of many higher eukaryotic genomes. A major biological consequence of DNA methylation is gene silencing. Increasing evidence indicates that recruitment of histone deacetylase complexes by methyl-CpG-binding proteins is a major mechanism of methylated DNA silencing. We have previously reported that the MeCP1 protein complex represses transcription through preferential binding, remodeling, and deacetylation of methylated nucleosomes. To understand the molecular mechanism of the functioning of the MeCP1 complex, the individual components of the MeCP1 complex need to be characterized. In this paper, we report the identification and functional characterization of the p66 and p68 components of the MeCP1 complex. We provide evidence that the two components are different forms of the same zinc finger-containing protein. Analysis of the p66 homologs from different organisms revealed two highly conserved regions, CR1 and CR2. While CR1 is involved in the association of p66 with other MeCP1 components, CR2 plays an important role in targeting p66 and MBD3 to specific loci. Thus, our study not only completes the identification of the MeCP1 components but also reveals the potential function of p66 in MeCP1 complex targeting. The identification and characterization of all the MeCP1 components set the stage for reconstitution of the MeCP1 complex.

Amino Acid Sequence↗

PubMed1988

In vitro functional characterization of SAG-3--a naturally occurring monoclonal antibody in NZB mice: specificity toward functional T cell subsets.

Natural thymocytotoxic autoantibodies (NTA) have been suggested to contribute to the T cell mediated immune dysfunction prevalent in NZB mice. However, direct studies to confirm these suggestions have been difficult to perform due to the absence of large amounts of pure NTA. To overcome this problem, we generated a naturally occurring monoclonal antibody from an NZB mouse which possesses the serological characteristics of serum-NTA. Herein, we report the in vitro functional characteristics of this monoclonal antibody designated SAG-3. In vitro treatment of spleen and lymph node cells from BALB/c mice with SAG-3 and complement eliminated most of the proliferative response to Con-A and moderately reduced the response of these cells to PHA-P and PWM. Complement mediated cytolysis of SAG-3 reactive cells significantly reduced the capacity of unprimed lymph node cells to generate primary CTL responses in vitro against allogeneic cells. However, only a partial decrease in CTL activity was observed when primed secondary CTL effector cells were treated, due primarily to the depletion of CTL's specific towards MHC class I antigens. Furthermore, a moderate reduction in antigen-specific helper T cell function was observed. Pretreatment of responder cells with SAG-3 + C' did not appear to reduce the proliferative MLR response to a wide range of stimulator cells. In addition, contrary to previous reports on serum-NTA, SAG-3 appears not to be reactive against Con-A induced suppressor T cells. Finally, by FACS analysis SAG-3+ thymocytes constitute an overlapping population of Lyt-2+ and L3T4+ thymocytes. These findings are discussed in comparison to previous reports on NTA.

Animals↗

PubMed2005

Cloning, expression and functional characterization of the full-length murine ADAMTS13.

Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.

ADAM Proteins↗

PubMed2002

Inventory and functional characterization of the HAK potassium transporters of rice.

Plants take up large amounts of K(+) from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K(+) from the soil solution to its final destination is mediated by channels and transporters. To better understand K(+) movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativa cv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1-17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K(+) influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K(+) influx in yeast and in roots of K(+)-starved rice seedlings demonstrated the involvement of HAK transporters in root K(+) uptake. We discuss that all HAK transporters may mediate K(+) transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast.

Amino Acid Sequence↗

PubMedDate not supplied

Theoretical model for myocardial functional characterization: application to a group of patients evaluated before and after surgical revascularization.

BACKGROUND: The functional improvements resulting from coronary revascularization (CABG) in patients with depressed ventricular function may be described by the use of a model combining global or local quantification of myocardial perfusion, viability, and contraction. An illustration of this model, with data provided by conventional radionuclide studies as they are performed routinely in many centers, is presented and the limitations of this approach for predicting the results of CABG are discussed. METHODS AND RESULTS: The model is based on three independent variables, which can be approximated in this preliminary study by parameters derived from standard stress and redistribution/reinjection thallium-201 single-photon emission computed tomography (SPECT) acquisitions with quantification of the tracer uptake defects and from a planar gated blood pool left ventricular ejection fraction (LVEF) measurement: Perfusion is assumed to correspond to 100-stress defect (in percentage), viability is 100-redistribution/reinjection defect, and contraction is 100(LVEF/70), assuming that a normal 70% LVEF corresponds to 100% contraction. In a group of 30 patients prospectively evaluated with this protocol and included in the study on the basis of a pre-CABG LVEF <40%, a significant improvement in LVEF was demonstrated (28.2% +/- 8.5% before CABG vs 35.8% +/- 7.3% after CABG), which is accompanied by a significant decrease of the stress thallium defects (34.8% +/- 13.8% vs 25.6% +/- 10.6%), whereas the average (but not the individual) redistribution/reinjection defects remain almost stable (27.7% +/- 10.9% vs 25.7% +/- 10.1%). As reported in the three-dimensional model, pre-CABG and post-CABG representative points clearly demonstrate the functional improvements for the main variables, but there is a large spectrum of responses to revascularization. It appears that the border between reversible and nonreversible thallium defects does not match the limit between ischemic myocardium (with no contraction alteration and therefore without contraction improvement potential) and hibernating myocardium, which is able to recover mechanical function and therefore is responsible for the improvement of global LVEF. CONCLUSIONS: Thallium SPECT is far from ideal for use as an independent characterization of perfusion and viability because hibernating myocardium may be present in both the fixed and reversible parts of thallium defects. Prediction of functional recovery is conditioned by an accurate identification of viable but underperfused and noncontracting myocardium. In the future, with the use of adequate study protocols that are able to measure viability without interference of perfusion and perfusion independent of viability, the proposed model may be able to characterize regional function as a cluster of representative points for each territory and to delineate areas of the theoretical volume corresponding to a potentially recoverable situation.

Adult↗

PubMed1997

Functional characterization of p53 molecules expressed in human squamous cell carcinomas of the head and neck.

Mutation of the p53 tumor suppressor gene has been demonstrated in a large proportion of human head and neck squamous cell carcinomas (HNSCCs) and has been assumed to play a role in the pathogenesis of these tumors, although no formal evidence of functional aberration has been demonstrated. In this study, we isolated cDNA clones encoding the entire p53 coding region from six human HNSCC cell lines that showed aberrant patterns of p53 expression in the parental cells, analyzed their nucleotide sequences, and characterized their function in vivo. cDNAs cloned from four cell lines harbored alterations within the p53 coding sequence (one missense mutation, one missense mutation plus in-frame deletion, one splice donor mutation, and a 1-nt insertion). HN30 cells, which contained wild-type p53 nucleotide sequences, showed a high constitutive level of protein expression. HN26 cells contained wild-type coding sequences but did not express the 53-kDa protein, although the mRNA was transcribed and a molecule of increased molecular mass (70 kDa) was observed by western blotting. Functional studies revealed that none of the four proteins encoded by mutant cDNAs were able to transactivate expression of a reporter plasmid containing a wild-type p53 consensus binding site when cotransfected into p53-null cells, whereas molecules encoded by wild-type p53 cDNAs increased reporter gene expression about a hundredfold over uninduced levels. Co-expression of each mutant cDNA with wild-type p53 cDNA and a wild-type p53-responsive reporter gene demonstrated that each of the proteins encoded by mutant cDNAs harbored some degree of inhibitory activity that varied depending on the mutation present. Thus, aberrant p53 function as a result of mutation or altered expression characterizes oral squamous cell carcinomas. The inhibitory activity of these molecules may be a mechanism for deregulation of the function of co-expressed wild-type p53 that may be of importance during the early stages of tumor development.

Blotting, Western↗

PubMed1986

Functional characterization of human IgG, IgM, and IgA antibody directed to the capsule of Haemophilus influenzae type b.

Antibody to the capsular polysaccharide (CP) of Haemophilus influenzae b (Hib) is bactericidal, opsonic, and protective. Minimum protective levels of primarily IgG antibody to Hib CP, calculated from passive immunization studies, have been found to be approximately .06-.15 microgram/ml of serum. The human response to antigenic challenge with the Hib capsule, however, includes production of antibody to Hib CP of different isotypes whose function against Hib is unclear. In order to characterize the function of antibody to Hib CP of different isotypes, we purified human IgG, IgM, and IgA from the serum of an adult donor who had been previously immunized with purified Hib CP vaccine. The globulin preparations were greater than 99% isotypically pure, contained large quantities of anticapsular antibody, and differed in function against Hib. IgG antibody to Hib CP was bactericidal and opsonic for human polymorphonuclear leukocytes (PMNLs) in the presence of complement and protective in infant rats. IgM, although more bactericidal than IgG (P less than .01) and equally protective in rats, opsonized Hib poorly for PMNLs. IgA was not bactericidal or opsonic and did not prevent bacteremia and meningitis in rats challenged with Hib. We conclude that antibody directed against the capsule of Hib differs in antibacterial function depending on class. These data may be important to acurately estimate minimum protective levels of anticapsular antibody after vaccination or natural infection and may have implications for the manner in which the host clears Hib from the circulation.

Animals↗

PubMed2002

Identification and functional characterization of melatonin Mel 1a receptors in pancreatic beta cells: potential role in incretin-mediated cell function by sensitization of cAMP signaling.

Melatonin receptors are expressed within the pancreatic islets of Langerhans, and melatonin induces a direct effect on insulin secretion ex-vivo. Here, we report the endogenous expression of the melatonin Mel 1a receptor in the INS-1 pancreatic beta cell line. Pharmacological characterization of the receptor using a CRE-luciferase reporter gene demonstrated its functional activity in INS-1 cells, displaying the characteristic signaling properties of the G(i/o) coupled receptor. Acute melatonin treatment of INS-1 cells in the presence of either forskolin or the incretin hormone glucagon-like peptide 1 (GLP-1) caused an attenuation of the responses in insulin secretion, insulin promoter activity, and CRE mediated gene expression, consistent with its effects in inhibiting cAMP mediated signal transduction. However, prolonged exposure (12 h) of INS-1 cells to melatonin treatment resulted in a sensitization of cAMP mediated responses to forskolin and GLP-1. Insulin secretion, insulin promoter activity and CRE mediated gene expression levels were augmented compared with responses without melatonin pre-treatment in INS-1 cells. In isolated rat islets, insulin secretion was enhanced following melatonin pre-treatment both in the absence and presence of GLP-1 or forskolin. This phenomenon reflects observations reported in other cell types expressing the melatonin Mel 1a receptor, and may represent the first evidence of a specific physiological role for melatonin-induced sensitization.

Animals↗

PubMed2006

Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels.

Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem, we cloned and characterized genes encoding KirBac orthologs from a wide variety of different prokaryotes and a simple unicellular eukaryote. The functional properties of these KirBacs were then examined by growth complementation in a K+ uptake-deficient strain of Escherichia coli (TK2420). Whereas some KirBac genes exhibited robust growth complementation, others either did not complement or showed temperature-dependent complementation including KirBac1.1 and KirBac3.1. In some cases, KirBac expression was also toxic to the growth of E. coli. The KirBac family exhibited a range of sensitivity to the K+ channel blockers Ba2+ and Cs+ as well as differences in their ability to grow on very low-K+ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of K+ channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterization of a range of prokaryotic K+ channels that are difficult to study by traditional methods.

Bacterial Proteins↗

PubMed2002

Functional characterization of multiple domains involved in the subcellular localization of the hematopoietic Pbx interacting protein (HPIP).

We have previously reported the cloning of the Hematopoietic Pbx Interacting Protein (HPIP), a novel protein discovered through its interaction with Pbx1. HPIP is expressed in early hematopoietic precursors, can bind all members of the Pbx family and can inhibit the transcriptional activation of the oncogene E2A-Pbx. To further understand the function of HPIP, we have analysed its cellular localization and characterized its functional localization domains. Using fluorescence microscopy to follow the distribution of different HPIP sequences fused to GFP, we found that HPIP localizes predominantly to cytoskeletal fibers but has the potential ability to shuttle between the nucleus and the cytosol. The cytoskeletal localization of HPIP is mediated by an N-terminal leucine rich region (between aa 190-218) and can be disrupted by the microtubule destabilizing drug vincristine. The HPIP C-terminal domain (aa 443-731) bears a nuclear export activity that is blocked by the CRM1 inhibitor Leptomycin B. In addition, we found two basic amino acid regions located between aa 485-505 and aa 695-720 that contain nuclear import activities attenuated by nuclear export. These observations support a model in which the constitutive attachment of HPIP to the cytoskeleton could be modified by changes in functional domains implicated in nuclear export, import and cytoskeleton binding sequences, allowing the molecule to shuttle between the nucleus and the cytosol.

3T3 Cells↗

PubMed1997

Cloning and functional characterization of the genes encoding 3-dehydroquinate synthase (aroB) and tRNA-guanine transglycosylase (tgt) from Helicobacter pylori.

The aroB gene from Helicobacter pylori strain P1 was cloned and further characterized by sequence analysis and by functional complementation of the aroB mutation in Escherichia coli. The aroB gene encodes the enzyme 3-dehydroquinate synthase which catalyzes one of the early steps in the shikimate pathway. This pathway, which creates aromatic molecules from sugar precursors, is present in prokaryotes, fungi and plants but is absent from mammalian cells. The predicted amino acid sequence of the H. pylori aroB gene product showed significant homology (30-40% identity and 50-60% similarity) to 3-dehydroquinate synthases from various other prokaryotes and eukaryotes. The single gene on a plasmid was biologically active in E. coli. It suppressed the specific phenotype of aroB mutants by restoring the shikimate pathway-dependent synthesis of aromatic amino acids and the production of the siderophore enterobactin. Two other reading frames were found adjacent to the aroB gene. The first, designated as orf1, had no significant homology to proteins and genes present in databases, whereas the second was found to share a significant degree of homology with the tgt gene encoding tRNA-guanine transglycosylase from a variety of other bacteria (40-50% identity and 60-70% similarity). The function of the tgt gene was confirmed by heterologous complementation. The gene on a plasmid was shown to complement the queuosine biosynthesis defect in a genetically defined tgt- strain of E. coli. The presence of the aroB gene and the putative tgt homologue in unrelated H. pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers.

Amino Acid Sequence↗

PubMed2026

Genomic identification and functional characterization of the nuclear receptor gene family in relation to sex determination and gonad development in the Pacific oyster (Crassostrea gigas).

Nuclear receptors (NRs) are a large superfamily of transcription factors that control a wide range of physiological processes by modulating the expression of downstream target genes. Numerous studies have confirmed that NR family members play critical and conserved roles in sex determination and gonadal development across metazoans. However, in mollusks, systematic characterization of NRs and their potential functions in gonadal regulation remain largely unexplored. In this study, 46 NR gene family members in the Pacific oyster (Crassostrea gigas) were identified and assigned to eight subfamilies. All NR family members contain at least one of the two core domains (DNA-binding domain, DBD; ligand-binding domain, LBD), and conserved exon-intron structures were observed within the same subgroup, indicating their evolutionary conservation. Furthermore, expression profiling revealed high expression of CgNR2F, CgNR5A1-1, and CgNR0B1 in undifferentiated gonads, suggesting their potential involvement in sex determination. CgNR1A and CgNR2E5 were specifically expressed in female gonads and exhibited female-biased expression patterns, indicating a putative role in ovarian development. Moreover, CgNR3A and CgNR3B showed high expression levels during the undifferentiated stage and early male development stage, implying their possible participation in male gonadal development and gametogenesis. These results expand the understanding of the NR gene family in C. gigas and help elucidate the potential functions of NR genes in sex determination and gonadal development.

Animals↗

PubMed1999

Functional characterization of equine dendritic cells propagated ex vivo using recombinant human GM-CSF and recombinant equine IL-4.

Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone marrow ex vivo with various cytokines. However, all other model systems, including equine, still require the laborious isolation procedures to obtain DC. In this study, we have adapted the methods developed for the human system to generate large numbers of equine DC from PBMC precursors using recombinant human GM-CSF and recombinant equine IL-4. Our report is the first documentation of ex vivo generated DC from PBMC in a domesticated animal model system. Equine DC derived from PBMC were rigorously characterized by analyzing morphological, phenotypic, and functional properties and were determined to have similar attributes as DC generated from human PBMC. Equine DC appeared stellate with large projectiles and veils and had cell surface antigens at similar levels as those defined on human and murine DC. Furthermore, functional attributes of the DC included rapidly capturing antigens by pinocytosis, receptor-mediated endocytosis, and phagocytosis, activating naive T cells in a mixed leukocyte reaction to a much greater extent than macrophage or lymphoblasts, presenting soluble and particulate antigen 10-100 fold more effectively to T cells on a per cell basis than macrophage or lymphoblasts, and presenting soluble and particulate antigen to both CD4+ and CD8+ T cells. Taken together, our study provides a framework by which equine DC can now be readily produced from PBMC precursors and presents an impetus for and model by which DC can be simply generated in other animal model systems.

Animals↗

PubMed2003

Structural-functional characterization of the cathodic haemoglobin of the conger eel Conger conger: molecular modelling study of an additional phosphate-binding site.

The protein sequence data for the alpha- and beta-chains have been deposited in the SWISS-PROT and TrEMBL protein knowledgebase under the accession numbers P83479 and P83478 respectively. The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the alpha- and beta-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-alpha(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule.

Allosteric Regulation↗

PubMed2003

Molecular, ultrastructural and functional characterization of a Spanish family with Hermansky-Pudlak syndrome: role of insC974 in platelet function and clinical relevance.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, which is genetically heterogeneous. In humans, mutations associated with this syndrome have been identified that affect four genes, most of them located in the HPS-1 gene. We evaluated the clinical, molecular, platelet ultrastructure and platelet function data obtained from one Spanish HPS patient and his relatives. The proband was compound heterozygous for a de novo nonsense mutation (Arg-131Stop), which has not been described previously, and for a common frameshift mutation (insC974). These two mutations were also identified by reverse transcription polymerase chain reaction (RT-PCR) in half the RNA, supporting the premise that they have minor effects on either transcription or RNA stability. The patient had an almost complete absence of platelet-dense granules. Accordingly, his platelets showed a small aggregatory response, reduced CD63 surface expression after platelet activation and minor serotonin uptake. Interestingly, despite the absence of clinical symptoms, two relatives carrying only one HPS-1 mutation (insC974) presented a decreased content of platelet-dense granules and showed significant reductions in platelet aggregation, expression of CD63 after platelet activation and serotonin uptake. Data show that the presence of a single mutation affecting one allele of the HPS-1 gene might have relevance in the organogenesis of platelet-dense granules, affecting platelet function. However, these functional defects were not of a great enough magnitude to have clinical significance and, thus, these subjects were clinically asymptomatic.

Blood Platelets↗