ABSTRACT
U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.
ABSTRACT
The role of peripheral adenosine receptors in pain is a controversial issue and seems to be quite different from the roles of spinal and central adenosine receptors. The present study is aimed at clarifying the role of these receptors in peripheral nociception. To clarify this, studies were done on Swiss mice with adenosine receptor agonists and antagonists. Nociceptive behavior was induced by subcutaneous injection of glutamate (10 µmol) into the ventral surface of the hind paw of mice. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Results showed that intraplantar (i.pl.) administration of N6-cyclohexyl-adenosine (CHA), an adenosine A1 receptor agonist, at 1 or 10 µg/paw significantly reduced glutamate-induced nociception (p<0.01 and p<0.001 vs. vehicle, respectively, n=8-10). In contrast, i.pl. injection of hydrochloride hydrate (CGS21680, an adenosine A2A receptor agonist) (1 µg/paw) induced a significant increase in glutamate-induced nociception compared to the vehicle (p<0.05, n=8), while 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, an adenosine A2A receptor antagonist) (20 µg/paw) caused a significant reduction (p<0.05, n=7-8). There were no significant effects on i.pl. administration of four additional adenosine receptor drugs-8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 antagonist, 1-10 µg/paw), N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA, an A2B agonist, 1-100 µg/paw), alloxazine (an A2B antagonist, 0.1-3 µg/paw), and 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) (an A3 agonist, 1-100 µg/paw) (p>0.05 vs. vehicle for all tests). We also found that prior administration of DPCPX (3 µg/paw) significantly blocked the anti-nociceptive effect of CHA (1 µg/paw) (p<0.05, n=7-9). Similarly, ZM241385 (20 µg/paw) administered prior to CGS21680 (1 µg/paw) significantly blocked CGS21680-induced exacerbation of nociception (p<0.05, n=8). Finally, inosine (10 and 100 µg/paw), a novel endogenous adenosine A1 receptor agonist recently reported by our research group, was also able to reduce glutamate-induced nociception (p<0.001 vs. vehicle, n=7-8). Interestingly, as an A1 adenosine receptor agonist, the inosine effect was significantly blocked by the A1 antagonist DPCPX (3 µg/paw) (p<0.05, n=7-9) but not by the A2A antagonist ZM241385 (10 µg/paw, p>0.05). In summary, these results demonstrate for the first time that i.pl administration of inosine induces an anti-nociceptive effect, similar to that elicited by CHA and possibly mediated by peripheral adenosine A1 receptor activation. Moreover, our results suggest that peripheral adenosine A2A receptor activation presents a pro-nociceptive effect, exacerbating glutamate-induced nociception independent of inosine-induced anti-nociceptive effects.
Subject(s)
Glutamates , Nociception/drug effects , Pain/chemically induced , Pain/psychology , Peripheral Nervous System/drug effects , Receptors, Purinergic P1/drug effects , Adenosine A1 Receptor Agonists/pharmacology , Adenosine A1 Receptor Antagonists/pharmacology , Adenosine A2 Receptor Agonists/pharmacology , Adenosine A2 Receptor Antagonists/pharmacology , Animals , Female , Foot , Glutamates/administration & dosage , Injections , Inosine/pharmacology , Male , Mice , Pain Measurement/drug effects , Receptor, Adenosine A2A/drug effectsSubject(s)
Carcinoid Tumor/diagnosis , Laryngeal Neoplasms/diagnosis , Aged , Diagnosis, Differential , Humans , Larynx/pathology , Male , Medical IllustrationABSTRACT
BACKGROUND: It has been shown that chronic pain is able to induce depressive disorders in humans, in part, due to peripheral inflammation that reaches the central nervous system. However, the mechanisms involved remain to be established. The purpose of this study was to investigate whether sciatic nerve crush could produce depression-like behaviors, in addition to pain-related behaviors, in mice. Once confirmed, this model was used to investigate tumor necrosis factor-α (TNF-α) as a key mediator involved in the pathophysiology of both pain and depression. EXPERIMENTAL APPROACH: Male Swiss mice were divided into three groups, naïve, sham and operated. In the operated group, the sciatic nerve was crushed. Following surgery, animals from the operated group were treated daily by oral gavage (p.o.) with saline (10 ml/kg), fluoxetine (20 mg/kg) or thalidomide (10 mg/kg) for 15 days. Mechanical hyperalgesia was evaluated every 3 days by von Frey filaments and depressive-like behavior was assessed at the end of day 15, using the tail suspension test (TST) and the forced swimming test (FST). Then, samples from the prefrontal cortex, hippocampus and sciatic nerve were processed to measure TNF-α levels by enzyme-linked immunosorbent assay (ELISA). RESULTS: Crush caused significant mechanical hyperalgesia and depressive-like behaviors and increased TNF-α levels in the sciatic nerve, prefrontal cortex and hippocampus of operated animals. Treatment with fluoxetine or thalidomide reversed crush-induced mechanical hyperalgesia, depressive-like behaviors and the increased TNF-α levels in the sciatic nerve, prefrontal cortex and hippocampus. CONCLUSIONS: The sciatic nerve crush model represents a good model to study to mechanisms underlying both pain and depressive-like behaviors. Furthermore, inhibitors of TNF-α synthesis, like thalidomide, have a potential to treat depressive disorders associated with neuropathic pain.
Subject(s)
Depression/etiology , Depression/prevention & control , Hyperalgesia/prevention & control , Thalidomide/administration & dosage , Animals , Depression/metabolism , Hindlimb Suspension , Hyperalgesia/complications , Hyperalgesia/metabolism , Male , Mice , Nerve Crush , Pain Measurement , Pain Threshold/drug effects , Sciatic Nerve/injuries , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolismSubject(s)
Humans , Female , Infant , Chondrodysplasia Punctata/diagnosis , Chondrodysplasia Punctata , Infant , ArgentinaABSTRACT
We describe the case of a 6-year-old boy with a de novo deletion of the long arm of chromosome 1 encompassing band 1q31.1-q32.1, minor facial anomalies, mild developmental delay, and behavioral disorders. His postnatal karyotype was normal. Using array-comparative genomic hybridization, we identified and characterized a de novo 1q interstitial deletion of about 15.6 Mb, which partially overlaps those of other reported cases. We considered the gene content of the deleted region in an attempt to compare the clinical features of our patient with these other cases, even though they were not characterized molecularly in detail. The most remarkable difference was the absence of microcephaly. To the best of our knowledge, this is the first report of a de novo 1q31.1-q32.1 deletion. Moreover, it illustrates how molecular delineation associated with fine clinical characterization can improve the genotype-phenotype correlations of classical cytogenetic abnormalities.
Subject(s)
Child Behavior Disorders/genetics , Chromosome Deletion , Chromosomes, Human, Pair 1 , Developmental Disabilities/genetics , Child , Comparative Genomic Hybridization , Humans , Karyotyping , MaleABSTRACT
Genetic variability of the ovine parasite Haemonchus contortus from the Alpine area was investigated using mitochondrial DNA (nd4 gene), internal transcribed spacers 1 and 2 and microsatellites, in order to assess whether cross-transmission between domestic and wild ruminants occurs. The dataset was composed of 78 individual adult male H. contortus collected from chamois (Rupicapra r. rupicapra), roe deer (Capreolus capreolus), alpine ibex (Capra ibex ibex), domestic goat (Capra hircus) and sheep (Ovis aries) from different alpine areas. The data obtained show low host specificity and high genetic variation within H. contortus populations. The analyses indicate the presence of two mitochondrial haplotype clusters among host species and the absence of cryptic parasite species, confirming H. contortus as a generalist nematode and suggesting that parasite transmission between populations of domestic and wild ruminants normally occurs.
Subject(s)
Genetic Variation , Host Specificity , Ruminants/parasitology , Trichostrongyloidea/genetics , Animals , DNA, Helminth/genetics , DNA, Mitochondrial/genetics , Deer , Europe , Goats , Helminth Proteins/genetics , Male , Molecular Sequence Data , Phylogeny , Sheep , Trichostrongyloidea/classification , Trichostrongyloidea/isolation & purificationABSTRACT
Since the first description of Kabuki syndrome (KS) in 1981, over 350 cases from a variety of countries have been reported. Even though otolaryngological manifestations are common in KS, only a limited number of the reports provide audiological and vestibular data. The aim of the present study was to investigate the vestibular function and describe the audiological findings in KS. The present study reports no audiological and vestibular features in a group of 10 KS patients (7 males, 3 females), with chronological age ranging from 10 to 25 years (mean age = 14.5): a complete otoneurological and audiological work-up was performed for each patient and included where possible, the measurement of vestibular evoked potentials, caloric tests and static posturography. Hearing loss was found in 65% showing a mix or a conductive impairment; moreover the vestibular function was normal in 95% of the examined ears. In conclusion, audiological and vestibular examination should be considered when evaluating KS subjects.
Subject(s)
Abnormalities, Multiple/diagnosis , Adolescent , Adult , Caloric Tests , Child , Evoked Potentials, Auditory , Face/abnormalities , Female , Hearing Loss , Humans , Male , Syndrome , Vestibular Diseases/diagnosis , Vestibular Function Tests , Young AdultABSTRACT
The aim of the study was to assess various aspects of visual and visuoperceptual function in patients with Noonan syndrome (NS) or LEOPARD syndrome (LS) with mutations affecting the PTPN11, SOS1 and RAF1 genes. Twenty-four patients were assessed with a battery of tests assessing visual function including ophthalmological and orthoptic evaluation and age appropriate behavioural visual tests, including measures of crowding acuity (Cambridge crowding cards), and stereopsis (TNO test). Twenty-one subjects were also assessed with the visuo-motor integration (VMI) test. Twenty of the 24 patients (83%) had abnormalities of visual function on at least one of the tests used to assess visual function or on ophthalmological examination, and 7 of 21 (33%) also had abnormalities on VMI. Ocular movements and stereopsis were most frequently abnormal (50% and 79%, respectively). Our results suggest that visual and visuoperceptual abilities are commonly impaired in patients with Noonan and LEOPARD syndrome and they are probably related to a multifactorial etiology.
Subject(s)
LEOPARD Syndrome/diagnosis , Noonan Syndrome/diagnosis , Perceptual Disorders/diagnosis , Vision Disorders/diagnosis , Visual Perception , Adolescent , Adult , Child , DNA Mutational Analysis , Depth Perception/genetics , Female , Genotype , Humans , LEOPARD Syndrome/genetics , Male , Noonan Syndrome/genetics , Ocular Motility Disorders/diagnosis , Ocular Motility Disorders/genetics , Perceptual Disorders/genetics , Phenotype , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Proto-Oncogene Proteins c-raf/genetics , Psychomotor Disorders/diagnosis , Psychomotor Disorders/genetics , SOS1 Protein/genetics , Vision Disorders/genetics , Vision Tests , Visual Acuity/genetics , Young AdultABSTRACT
MOTIVATION: A whole set of Expressed Sequence Tags (ESTs) from the Sf9 cell line of Spodoptera frugiperda is presented here for the first time. By this way we want to identify both conserved and specific genes of this pest species. We also expect from this analysis to find a class of protein sequences providing a tool to explore genomic features and phylogeny of Lepidoptera. RESULTS: The ESTs display both housekeeping as well as developmentally regulated genes, and a high percentage of sequences with unknown function. Among the identified ORFs, almost all ribosomal proteins (RPs) were found with high EST redundancy and hence sequence accuracy. The codon usage found among RP genes is in average surprisingly much less biased in Lepidoptera than in other organisms. Other Spodoptera genes also displayed a low bias, suggesting a general genome expression feature in this Lepidoptera. We also found that the L35A and L36 RP sequences, respectively, display 40 and 10 amino-acid insertions, both being present only in insects. Sequence analysis suggests that they are probably not subjected to a strong selective pressure and may be good phylogenetic markers for Lepidoptera. Most interestingly, the Lepidoptera sequences of 9 RP genes displayed a specific signature different from the canonical one. We conclude that the RP family allows valuable comparative genomics and phylogeny of Lepidoptera. AVAILABILITY: All EST sequence data are available from the private 'Spodo-Base' upon request.
Subject(s)
Expressed Sequence Tags , Gene Expression Profiling/methods , Ribosomal Proteins/genetics , Sequence Alignment/methods , Sequence Analysis, Protein/methods , Spodoptera/genetics , Abstracting and Indexing/methods , Animals , Bias , Cell Line , Codon/genetics , Evolution, Molecular , Information Storage and Retrieval/methods , Phylogeny , Reproducibility of Results , Ribosomal Proteins/metabolism , Sensitivity and Specificity , Sequence Homology, Amino Acid , Spodoptera/metabolismABSTRACT
We present in this work two novel Hyposoter didymator ichnovirus genes expressed in parasitized Spodoptera larvae. These genes, named HdCorfS6 and HdGorfP30, are unrelated and present in two different genome segments, possibly nested, SH-C and SH-G respectively. HdCorfS6 encodes a predicted transmembrane protein, putatively glycosylated. HdCorfS6 transcripts appear to be abundant in lepidopteran host hemocytes compared to the other tissues analyzed. The second gene described, HdGorfP30, is well expressed in hemocytes, but also in other tissues, such as the fat body, nervous system and epidermis. This gene is peculiar since it presents 17 perfectly conserved repeated sequences arranged in tandem arrays. Each of these repeats contains 58% of serine and threonine residues and therefore several potential sites for glycosylation. This mucin-like protein, predicted as highly glycosylated, could be involved in host immune suppression.
Subject(s)
Genes, Viral , Lepidoptera/virology , Polydnaviridae/genetics , Serine/chemistry , Threonine/chemistry , Viral Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , DNA Primers , DNA, Viral , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Viral Proteins/chemistryABSTRACT
Anti-double-stranded DNA monoclonal antibodies against a viral transcriptional regulatory site are capable of discriminating single-base replacements with affinities of 1 x 10(-)(9) M, which were optimized for the length of the duplex used as the immunogen. Their affinity for DNA duplexes of increasing length is lower, but reaches a plateau at 2 x 10(-)(8) M, still a fairly high affinity compared to those of most known natural anti-DNA antibodies. The ability of the antibodies to bind to a 166 bp DNA fragment containing the specific sequence strongly suggests that these have the potential of binding the specific sequence within larger genomic DNA fragments. Electrostatic interactions do not play a significant role, the opposite of what is observed in natural DNA binding interfaces. In addition, the insensitivity of the antibody-DNA interaction to solute effects is indicative of a marginal participation of water molecules at the interface compared to the level of participation at the natural E2-DNA interface. Spectroscopic evidence of base unstacking strongly suggests substantial denaturation of antibody-bound DNA, in agreement with thermodynamic results that show an unusual positive heat capacity change, which could be explained at least in part by the exposure of DNA bases upon binding. Lower local DNA stability cooperates with sequence recognition in producing the highest binding affinity. A slow rate of antibody-DNA association indicates an energy barrier imposed by conformational rearrangements, as opposed to an electrostatically assisted diffusion-controlled collision in the E2 DNA binding domain. While the E2-DNA interaction takes place through a typical direct readout mechanism, the anti-double-stranded DNA monoclonal antibody-DNA interaction could be viewed as a distinctive case of indirect readout with a significant distortion in the DNA conformation. However, the precise mechanism with which the DNA bases are accommodated in the antibody combining site will require structural analysis at atomic resolution. These results constitute a first stage for unveiling the unusual molecular recognition mechanism of a specific DNA sequence by antibodies. This mechanism could represent the strategy with which the immune system tightly and specifically recognizes a DNA antigen.
Subject(s)
Antibodies, Antinuclear/metabolism , DNA/immunology , Animals , Antibodies, Monoclonal/metabolism , Antigen-Antibody Reactions , Base Sequence , Binding Sites , DNA/chemistry , DNA/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/immunology , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , In Vitro Techniques , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , Oncogene Proteins, Viral/immunology , Oncogene Proteins, Viral/metabolism , Papillomaviridae/genetics , Papillomaviridae/immunology , ThermodynamicsABSTRACT
The E2 transcriptional activator of the human papillomavirus regulates the expression of most viral transcripts. Its binding to specific target DNA sequences involves large conformational changes in the interacting macromolecules. The high stability of the E2:DNA complex prompted us to analyze the role of macromolecular interactions and adjuvant emulsions in the appearance of conformation-specific antibodies. We demonstrate that immunization with free or DNA-complexed E2 emulsified in an oil-in-water adjuvant elicits a humoral response shifted to the recognition of discontinuous epitopes. Epitope mapping and functional analysis of the generated anti-E2 mAbs reveals that two separate antibodies populations can be obtained: those able to form a stable ternary complex with protein and DNA, and those which recognize the DNA-binding surface of the transcription factor, interfering with E2 binding to DNA.
Subject(s)
Adjuvants, Immunologic/chemistry , Antibodies, Monoclonal/metabolism , DNA-Binding Proteins , Oncogene Proteins, Viral/immunology , Transcription Factors/immunology , Animals , Antibodies, Monoclonal/immunology , DNA, Viral/immunology , Epitope Mapping , Mice , Papillomaviridae , Transcription Factors/metabolismABSTRACT
In Campoletis sonorensis Ichnovirus (CsIV), the repeat element genes constitute a gene family of 28 members. In the present work, we document the presence of members of this gene family in two additional ichnoviruses, Hyposoter didymator Ichnovirus (HdIV) and Tranosema rostrale Ichnovirus (TrIV). Two repeat element genes, representing at least one functional gene, were identified in TrIV, whereas HdIV was found to contain at least three such genes. In both HdIV and TrIV, the known repeat element genes are encoded on single genome segments, with hybridization studies suggesting the presence of other, related but as yet uncharacterized genes. The HdIV and TrIV repeat element genes are all transcribed in infected caterpillars, although differences exist among genes in levels and in tissue specificity of expression. A heuristic tree was generated indicating that the repeat element genes are more similar within a species of wasp than between species, with TrIV genes being more closely related to the CsIV than to the HdIV genes. These results suggest that the most significant duplication, divergence, and expansion of the repeat element genes occurred after speciation. The finding that repeat element genes form an interspecific family within the genus Ichnovirus supports the view that the proteins they encode play an important role in ichnovirus biology.
Subject(s)
Genes, Viral , Polydnaviridae/genetics , Repetitive Sequences, Nucleic Acid , Wasps/virology , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , Gene Expression Regulation, Viral , Molecular Sequence Data , Phylogeny , Polydnaviridae/classification , Polymorphism, Genetic , Transcription, GeneticABSTRACT
The anti-CD4 mAb 13B8.2, directed against the CDR3-like loop of the D1 domain of CD4, inhibits signal transduction pathways leading to both T cell activation and HIV replication. VH9/DSP2/JH2 and Vkappa12-13/Jkappa2 rearrangements, corresponding to genes encoding the heavy and light chain variable regions of the 13B8.2 mAb, were inserted into baculovirus cassettes upstream from pre-installed human Fdgamma1 and Ckappa genes, respectively. After expression in insect cells, a complete correctly-processed Fab was secreted into the culture medium; it was protein-G immunopurified with a yield of 5 mg/L. The chimeric Fab 13B8.2 showed anti-CD4 binding activity with an affinity value of 3.3 nM and recognized the same region on the CDR3-like loop as the parental mAb. The mouse-human Fab inhibited IL2 secretion following antigen presentation and displayed a strong capacity to prevent HIV-1 promoter activation. Taken together, these results indicate that the chimeric Fab retained a major part of the parental 13B8.2 mAb properties and suggest that it might be a valuable therapeutic tool.
Subject(s)
Antibodies, Monoclonal/immunology , CD4 Antigens/immunology , HIV Antigens/immunology , HIV-1/genetics , HIV-1/immunology , Immunoglobulin Fab Fragments/immunology , Promoter Regions, Genetic , Recombinant Fusion Proteins/immunology , Virus Activation/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Baculoviridae/genetics , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Humans , Immunoglobulin Fab Fragments/genetics , Mice , Molecular Sequence Data , Recombinant Fusion Proteins/geneticsABSTRACT
It has been previously shown that the HIV-1 envelope glycoprotein 120 (gp120) activates cell signaling by CXCR4, independently of CD4. The present study examines the involvement of different intracellular signaling pathways and their physiopathologic consequences following the CD4-independent interaction between CXCR4 or CCR5 and gp120 in different cell types: primary T cells, CD4(-)/CXCR4(+)/CCR5(+) T cells, or glioma cells. These interactions were compared with those obtained with natural ligands, stromal cell-derived factor 1 alpha (SDF-1alpha) (CXCL12) and macrophage inflammatory protein 1 beta (MIP-1beta) (CCL4) of their respective coreceptors. Thus, both p38 and SAPK/Jun N-terminal kinase mitogen-activated protein kinases (MAPKs) are activated on stimulation of these cells with either T- or M-tropic gp120, as well as with SDF-1alpha or MIP-1beta. In contrast, extracellular signal-related kinase 1 and 2 MAPKs are only activated by MIP-1beta but not by M-tropic gp120. Importantly, T- and M-tropic gp120 are able to induce the secretion of matrix metalloproteinase 9 (MMP-9), an extracellular metalloproteinase present in cerebrospinal fluid of patients with HIV-1 by T cells or glioma cells. Specific inhibition of MAPK p38 activation resulted in a complete abrogation of the induction of the MMP-9 pathogenic factor expression by gp120 or chemokines in both cell types. Because neurodegenerative features in acquired immune deficiency syndrome dementia may involve demyelinization by MMP-9, the specific targeting of p38 could provide a novel means to control HIV-induced cytopathogenic effects and cell homing to viral replication sites. (Blood. 2001;98:541-547)
Subject(s)
HIV Envelope Protein gp120/pharmacology , Matrix Metalloproteinase 9/drug effects , Mitogen-Activated Protein Kinases/metabolism , Signal Transduction/drug effects , Animals , Chemokine CCL3 , Chemokine CCL4 , Chemokine CXCL12 , Chemokines, CXC/metabolism , Cytopathogenic Effect, Viral/drug effects , Cytopathogenic Effect, Viral/physiology , Enzyme Activation/drug effects , Humans , Ligands , Macrophage Inflammatory Proteins/metabolism , Matrix Metalloproteinase 9/metabolism , Mitogen-Activated Protein Kinase 8 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/physiology , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Tumor Cells, Cultured , p38 Mitogen-Activated Protein KinasesABSTRACT
A chimeric protein containing the catalytic domain of Trypanosoma cruzi trans-sialidase, the transmembrane domain of the major envelope glycoprotein of the baculovirus (gp67), and the signal peptide of ecdysteroid glucosyltransferase of the baculovirus was expressed under the control of the very late promoter p10 in baculovirus-infected lepidopteran cells. The recombinant protein was found to be enzymatically active. Three days after infection, equal amounts of activity were found associated to the plasma membrane and in the infection medium, both forms having the same apparent molecular weight and being N-glycosylated. When exogenous galactosylated acceptors (lactose or asialo-alpha1-acid glycoprotein) were added in the culture medium of cells infected with the recombinant baculovirus in the presence of a sialylated donor, a sialylation could be observed. Therefore, we propose the use of trans-sialidase as a potential tool for sialylation of glycoconjugates in the baculovirus-insect cells system.
Subject(s)
Baculoviridae/genetics , Membrane Glycoproteins/genetics , N-Acetylneuraminic Acid/metabolism , Neuraminidase/genetics , Trypanosoma cruzi/enzymology , Animals , Base Sequence , Blotting, Western , Catalytic Domain , Cell Line , DNA Primers , DNA, Complementary , Flow Cytometry , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Neuraminidase/chemistry , Neuraminidase/metabolism , SpodopteraABSTRACT
The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) the cloning of immunoglobulin (Ig) variable (V) region genes originating from individual IgG anticardiolipin-specific B cells by a single-cell polymerase chain reaction technique. The corresponding V regions were cloned in order to express human recombinant antibodies in insect cells by a baculovirus expression system. The molecular analysis, the fine specificity, and the protein cofactor dependency of the first 5 monoclonal IgG anticardiolipins are reported here. This clonal analysis reveals the extreme heterogeneity of these antibodies, which could account for the difficulties in the previous attempts to define the pathogenic antiphospholipid response. This approach should help to unravel the complex antiphospholipid immune response and the mechanism of the prothrombotic state associated with these antibodies, but it could also shed some light on their possible origins. (Blood. 2001;97:3820-3828)
Subject(s)
Antibodies, Anticardiolipin/blood , Antiphospholipid Syndrome/immunology , Cloning, Molecular/methods , Adult , Antibodies, Anticardiolipin/genetics , Antibodies, Anticardiolipin/metabolism , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Antibody Diversity , Antibody Specificity , B-Lymphocytes/immunology , Cardiolipins/metabolism , Female , Genes, Immunoglobulin , Humans , Immunoglobulin G/immunology , Lupus Coagulation Inhibitor/metabolismABSTRACT
This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa (B. mori) and a 717 aa (S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14 degrees C above physiological growth conditions (42 degrees C).
Subject(s)
Bombyx/genetics , DNA, Complementary/genetics , HSP90 Heat-Shock Proteins/genetics , Spodoptera/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , DNA, Complementary/chemistry , Gene Expression , Molecular Sequence Data , Phylogeny , RNA/genetics , RNA/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
By taking advantage of the extreme stability of a protein-DNA complex, we have obtained two highly specific monoclonal antibodies against a predetermined palindromic DNA sequence corresponding to the binding site of the E2 transcriptional regulator of the human papillomavirus (HPV-16). The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constants in the low and subnanomolar range. This affinity matches that of the natural DNA binding domain and is severalfold higher than the affinity of a homologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These antibodies discriminate effectively among a number of double- and single-stranded synthetic DNAs with factors ranging from 125- to 20,000-fold the dissociation constant of the specific DNA sequence used in the immunogenic protein-DNA complex. Moreover, they are capable of fine specificity tuning, since they both bind less tightly to another HPV-16 E2 binding site, differing in only 1 base pair in a noncontact flexible region. Beyond the relevance of obtaining a specific anti-DNA response, these results provide a first glance at how DNA as an antigen is recognized specifically by an antibody. The accuracy of the spectroscopic method used for the binding analysis suggests that a detailed mechanistic analysis is attainable.