ABSTRACT
UNLABELLED: Regulation of nitric oxide (NO) production is considered essential in mechanical load-related osteogenesis. We examined whether osteoblast endothelial NO synthase (eNOS)-derived NO production was regulated by HSP90. We found that HSP90 is essential for strain-related NO release but appears to be independent of eNOS in cultured osteoblasts. INTRODUCTION: NO is a key regulator of bone mass, and its production by bone cells is regarded as essential in mechanical strain-related osteogenesis. We sought to identify whether bone cell NO production relied upon eNOS, considered to be the predominant NOS isoform in bone, and whether this was regulated by an HSP90-dependent mechanism. METHODS: Using primary rat long bone-derived osteoblasts, the ROS 17/2.8 cell line and primary mouse osteoblasts, derived from wild-type and eNOS-deficient (eNOS(-/-)) mice, we examined by immunoblotting the expression of eNOS using a range of well-characterised antibodies and extraction methods, measured NOS activity by monitoring the conversion of radiolabelled L-arginine to citrulline and examined the production of NO by bone cells subjected to mechanical strain application under various conditions. RESULTS: Our studies have revealed that eNOS protein and activity were both undetectable in osteoblast-like cells, that mechanical strain-induced NO production was retained in bone cells from eNOS-deficient mice, but that this strain-related induction of NO production was, however, dependent upon HSP90. CONCLUSIONS: Together, our studies indicate that HSP90 activity is essential for strain-related NO release by cultured osteoblasts and that this is highly likely to be achieved by an eNOS-independent mechanism.
Subject(s)
Nitric Oxide Synthase Type III/metabolism , Nitric Oxide/biosynthesis , Osteoblasts/metabolism , Animals , Benzoquinones/pharmacology , Cells, Cultured , Female , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/physiology , Humans , Lactams, Macrocyclic/pharmacology , Mechanotransduction, Cellular/drug effects , Mechanotransduction, Cellular/physiology , Mice , Mice, Knockout , Nitric Oxide Synthase Type III/deficiency , Nitric Oxide Synthase Type III/physiology , Osteoblasts/drug effects , Osteoblasts/physiology , Rats , Rats, Sprague-Dawley , Stress, MechanicalABSTRACT
Recent literature contains numerous articles on osteonecrosis of the jaws (ONJ) associated with bisphosphonate treatment (BPT), with most advocating a conservative approach to management. A prospective study was designed to review the surgical management of cases of ONJ that did not respond to conservative management. Forty patients, referred with ONJ that did not respond to conservative management, were treated surgically and followed up for 6 months to 4 years. Four patients were taking i.v. BPT as part of their bone cancer management and 16 were taking oral BPT for osteoporosis. The surgical management of ONJ involved antibiotic therapy, surgical debridement of all necrotic bone and tension-free primary closure. All 40 cases healed uneventfully with no wound breakdown during follow-up. Most of the literature supports the conservative management of ONJ, but the condition leaves the patient debilitated. Many cases do not respond to conservative management and the infection and bone destruction is progressive. The conservative management of ONJ is to be supported, but this prospective study has shown that those cases that do not respond may be managed surgically. It should be recognized that while the results of this paper are encouraging, some cases will be resistant to all treatments.
Subject(s)
Bone Density Conservation Agents/adverse effects , Diphosphonates/adverse effects , Jaw Diseases/surgery , Osteonecrosis/surgery , Adult , Aged , Aged, 80 and over , Amoxicillin/therapeutic use , Anti-Bacterial Agents/therapeutic use , Anti-Infective Agents, Local/therapeutic use , Bone Neoplasms/drug therapy , Chlorhexidine/therapeutic use , Clindamycin/therapeutic use , Debridement , Disease Progression , Female , Follow-Up Studies , Humans , Jaw Diseases/chemically induced , Male , Middle Aged , Mouthwashes/therapeutic use , Multiple Myeloma/drug therapy , Osteonecrosis/chemically induced , Osteoporosis/drug therapy , Postoperative Complications , Prospective Studies , Surgical Flaps , Suture Techniques , Treatment OutcomeABSTRACT
Hyperbaric oxygen (HBO) has been used for more than 20 years to assist wound healing in the treatment of the more severe complications associated with the side effects of therapeutic radiation treatment. A prospective study was performed in an irradiated rat model to determine whether HBO is effective in reducing the long-term side effects of therapeutic radiation treatment on normal tissue, when given 1 week after the completion of the radiation treatment. The experimental model was designed to simulate a fractionated course of therapeutic radiation that is commonly used in the treatment of cancer of the mandible. One week following completion of the radiotherapy, the animals underwent a 4-week course of HBO treatment, and two animals from each group were killed at 8-week intervals until the end of the experiment at 36 weeks. Histological sections of tissue clearly showed continued growth of teeth and maintenance of specialized tissues, such as salivary gland and bone, in the treated group compared to the non-treated group. This experimental model demonstrated that HBO is effective in reducing the long-term side effects of therapeutic radiation treatment in normal tissue, when given 1 week after the completion of the radiation treatment.
Subject(s)
Hyperbaric Oxygenation/methods , Mandibular Neoplasms/radiotherapy , Radiation Injuries/therapy , Animals , Biomarkers/analysis , Male , Mandible/growth & development , Models, Theoretical , Prospective Studies , Rats , Rats, Wistar , Salivary Glands/growth & development , Tetracycline/analysis , Tooth/growth & developmentABSTRACT
Prion diseases are caused by misfolding of the cellular prion protein, PrPC. In vitro studies have shown that PrP binds copper via the octarepeat region lying within the unstructured N-terminal segment of the protein, but the significance of copper in PrP metabolism remains unclear. Here, six specific antibodies recognizing different epitope regions of PrP were used to measure the effect of copper on the conformation of the molecule at the cell surface. Binding of an antibody, E149, to an epitope within the octarepeat domain of PrP is halved in the presence of copper, whereas binding of antibodies recognizing epitope motifs C-terminal to residue 90 of PrP remain relatively unaltered under equivalent conditions. These experiments strongly suggest that copper induces localized conformational change within the N-terminal portion of cell-surface PrPC.
Subject(s)
Copper/pharmacology , PrPC Proteins/chemistry , PrPC Proteins/drug effects , Protein Conformation/drug effects , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , CHO Cells , Cricetinae , Epitope Mapping , Epitopes , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Molecular Sequence Data , PrPC Proteins/metabolismABSTRACT
The characteristics of the immunity induced by viral antigens or conferred by antiviral antibody via different routes of administration were evaluated comparatively. C57BL/6 mice were immunized via intranasal, intradermal or enteric routes with a live recombinant vaccinia virus expressing the respiratory syncytial virus (RSV) F glycoprotein (F.rVV) or RSV, and then challenged intranasally with RSV. Inhibition of RSV replication was observed in the lungs of all the mice; however, only intranasal immunization hindered virus replication in the nose. Lung inflammation, characterized by infiltration of neutrophils and of mononuclear cells was strongest in the intradermally immunized mice, but was observed in all F.rVV immunized mice to various degrees. Intranasal administration of a potently neutralizing human anti-RSV antibody Fab fragment to infected mice inhibited RSV replication in the nose and, when combined with intraperitoneal administration, protected both the lung and the nose in the absence of deleterious lung pathology. These data suggest that intranasal immunization with F.rVV reduces RSV replication in the respiratory tract, but still induces pathological lung inflammation, even though this is milder than that observed following intradermal immunization. Local neutralizing antibody is indispensable for protection in the nose.
Subject(s)
Antigens, Viral/administration & dosage , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Viruses/immunology , Administration, Cutaneous , Administration, Intranasal , Animals , Antibodies, Viral/administration & dosage , Antigens, Viral/adverse effects , Antigens, Viral/immunology , Humans , Immunoglobulin Fab Fragments/administration & dosage , Injections, Intraperitoneal , Lung/immunology , Male , Mice , Mice, Inbred C57BL , Viral Fusion Proteins/administration & dosage , Virus ReplicationABSTRACT
In the pathogenesis of preeclampsia, endothelial cell activation or dysfunction is a central theme, and marked dyslipidemia may contribute to endothelial cell dysfunction. The objective of this study was to evaluate the association between preeclampsia and mutations within the lipoprotein lipase (LPL) gene. DNA was extracted from whole blood or cheek swabs of 250 preeclamptic patients, 265 control subjects, and 106 offspring of preeclamptic patients (all white). Control subjects were women who had undergone >/=2 term pregnancies unaffected by preeclampsia. All samples were genotyped for 3 LPL polymorphisms with the use of polymerase chain reaction of known allelic variants. The 3 mutations studied were the following: (1) Asp9Asn substitution in exon 2, (2) T-to-G substitution at position -93 of the proximal promotor region (-93T/G), and (3) Asn291Ser substitution in exon 6. Results were analyzed with an chi(2) contingency table. The prevalences of the Asp9Asn mutation, -93T/G promotor mutation, and Asn291Ser mutation were not significantly different among the preeclamptic patients and control subjects (Asp9Asn: patients, 2.8%; control subjects, 4.0%; -93T/G: patients, 4.5%; control subjects, 5.5%; Asn291Ser: patients, 4.0%; control subject, 3.0%). In addition, there was no difference in the frequency of any of the mutations in the offspring of preeclamptic women compared with that observed in the control population. Between a small group of patients with nulliparous HELLP syndrome (a variant of severe preeclampsia: hemolysis, elevated liver enzyme, low platelets) patients (n=12) and control subjects, there was a significant difference in the prevalence of the Asn291Ser mutation (16.7% versus 3.0%, P=0.01). In this large white population, the Asp9Asn mutation, -93T/G promotor mutation, and Asn291Ser mutation were not associated with an increased risk for preeclampsia. In a small subgroup of patients, the Asn291Ser mutation was associated with an increased risk for nulliparous HELLP syndrome.
Subject(s)
Lipoprotein Lipase/genetics , Point Mutation , Pre-Eclampsia/genetics , Female , Gene Frequency , Genetic Predisposition to Disease , Genotype , HELLP Syndrome/genetics , Humans , Linkage Disequilibrium , PregnancyABSTRACT
The matrix metalloproteinases (MMPs) are enzymes involved in the turnover of the extracellular matrix. Their overexpression in tumors is implicated in the metastatic process and may provide a target for diagnostic tumor imaging by using a radiolabeled inhibitor. MMPs are inhibited by endogenous tissue inhibitors of metalloproteinases (TIMPs). Thus, TIMPs are potential targeting molecules which could be used as vehicles for selective radionuclide delivery by virtue of their binding to MMPs. The aim of this work was to produce a radiopharmaceutical with which to evaluate this potential. The 127 amino acid N-terminal domain of recombinant human TIMP-2 (N-TIMP-2) was conjugated with the bifunctional chelator diethylenetriamine pentaacetic acid (DTPA). Singly modified DTPA-N-TIMP-2 conjugate (identified by electrospray ionization mass spectrometry) was isolated by anion-exchange chromatography. The primary site of DTPA modification on N-TIMP-2 was mapped to lysine-116, which is distant from the site of MMP interaction. The conjugate was radiolabeled with indium-111 to give 111In-DTPA-N-TIMP-2 with a specific activity of at least 4 MBq/microg and a radiochemical yield and purity of >95%, by incubation with 111InCl3, without need for postlabeling purification. The product was sterile, pyrogen-free, and stable in serum over 48 h and retained full inhibitory activity in a fluorimetric binding assay. With these attributes, 111In-DTPA-N-TIMP-2 is a suitable radiopharmaceutical for in vivo biological and clinical investigation of the potential benefits of imaging MMP expression.
Subject(s)
Indium Radioisotopes , Matrix Metalloproteinases/analysis , Pentetic Acid/chemistry , Radiopharmaceuticals/chemical synthesis , Tissue Inhibitor of Metalloproteinase-2/chemistry , Drug Design , Drug Stability , Enzyme Inhibitors/chemistry , Humans , Matrix Metalloproteinases/metabolism , Neoplasm Proteins/analysis , Neoplasm Proteins/metabolism , Peptide Mapping , Protein Binding , Radiopharmaceuticals/metabolismABSTRACT
OBJECTIVES: The increasing number of surviving pediatric organ transplant recipients has resulted in a new clinical controversy surrounding the significance of adenotonsillar hypertrophy. The objective of this study is to evaluate adenotonsillar specimens, understand characteristic histopathology, and to examine the frequency and significance of this finding in this population. METHODS: Twenty-one cases of pediatric transplant recipients with adenoidal and/or tonsillar hypertrophy were reviewed retrospectively in a tertiary-care setting. Particular attention was given to the histopathology of their surgical specimens, including any evidence of posttransplantation lymphoproliferative disorders (PTLD). RESULTS: Using morphologic, immunohistochemical, and molecular genetic analyses, 15 (71%) of 21 patients were noted to have Epstein-Barr virus (EBV)-related lymphoid hyperplasia, including 1 case (4.7%) of PTLD. Six (29%) of 21 had evidence of reactive follicular hyperplasia not related to EBV. B-cell and T-cell markers were nearly uniformly positive when tested for, except in the single patient with PTLD, who exhibited polymorphic, polyclonal B-cell morphology. Kappa and lambda light-chain clonality markers were positive in 11 (92%) of 12 patients. CONCLUSIONS: EBV-related lymphoid hyperplasia is frequently associated with adenotonsillar hypertrophy in pediatric organ transplant patients (71% of our cases); 92% of those cases tested exhibit polyclonal B-cell populations. PTLD, an important cause of morbidity and mortality in this population, represented approximately 5% of our cases. The remainder of cases represent follicular hyperplasia unrelated to EBV or lymphoproliferative abnormalities. Characteristic histopathologic findings are presented.
Subject(s)
Adenoids/pathology , Lymphoproliferative Disorders/etiology , Lymphoproliferative Disorders/pathology , Organ Transplantation/adverse effects , Palatine Tonsil/pathology , Adenoids/microbiology , Adolescent , Child , Female , Herpesvirus 4, Human/isolation & purification , Humans , Hyperplasia , Hypertrophy , Immunohistochemistry , Infant , Male , Palatine Tonsil/microbiology , Retrospective StudiesSubject(s)
Bacterial Proteins/chemistry , Mycobacterium bovis/chemistry , Mycobacterium tuberculosis/chemistry , Antigens, Bacterial/chemistry , Bacterial Proteins/immunology , Humans , Mycobacterium bovis/immunology , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/pathogenicity , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, SecondaryABSTRACT
Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.
Subject(s)
Immunoglobulin Fragments/immunology , Prions/immunology , Animals , Antibody Specificity , Biological Assay , Epitopes, B-Lymphocyte/immunology , Escherichia coli , Mice , PrPC Proteins/immunology , Prions/antagonists & inhibitors , Prions/biosynthesis , Recombinant Proteins , Tumor Cells, CulturedABSTRACT
Matrix metalloproteinases are the major agents responsible for the degradation of extracellular matrix and are produced at high levels by transformed and tumour cells, where they participate in the metastatic process by allowing local invasion. They are also more active at sites of new normal growth and angiogenesis. In the early stages of Kaposi sarcoma (KS), in vitro studies have demonstrated that vascular invasion can be inhibited by inhibitors of matrix metalloproteinases. Imaging of visceral and cutaneous KS presents a problem and therefore the potential use of a labelled inhibitor of metalloproteinases, N-TIMP-2, with indium-111 was thought to present a possible imaging tool. The biokinetics, dosimetry and potential for imaging with 111In-DTPA-N-TIMP-2 were assessed in five patients with HIV infection and KS. Between 103.1 and 108.0 MBq of this agent was injected into each patient, and the dynamic uptake over the kidneys was assessed, whole body scans were performed and blood samples were obtained. The clearance from the blood was rapid, with a first component half-time of 16.6+/-3.4 min and a second component half-time of 9.68+/-2.68 h. Two out of five patients experienced minor shivering but one of these patients was generally unwell before the study. The last three patients had no such problems. The tracer distributed predominantly to the kidneys and did not localise in other tissues. No KS lesions were clearly identified. 111In-DTPA-N-TIMP-2 can be successfully prepared and administered to patients safely, with a biodistribution and dosimetry which would allow its use as an imaging tracer. It is unlikely to be of use for imaging KS, but may have a role in other tumours that produce matrix metalloproteinases.
Subject(s)
HIV Infections/complications , Radiopharmaceuticals , Sarcoma, Kaposi/diagnostic imaging , Adult , Humans , Male , Middle Aged , Pentetic Acid/analogs & derivatives , Radionuclide Imaging , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/pharmacokinetics , Sarcoma, Kaposi/etiology , Tissue Distribution , Tissue Inhibitor of Metalloproteinase-2 , Whole-Body CountingABSTRACT
The tissue inhibitor of metalloproteinases-2 (TIMP-2) is potentially an important inhibitor of all known matrix metalloproteinases (MMPs). However, it has been shown to undergo specific interactions with both MMP-2 (gelatinase A) and MMP-14 (MT1-MMP), and it has been proposed that these three proteins function as a cell surface-based activation cascade for matrix metalloproteinases and as a focus of proteolytic activity. In this study, we have carried out mutagenesis and kinetic analyses to examine the unique interactions between the AB loop of TIMP-2 and MMP-14. The results demonstrate that the major binding contribution of the AB loop is due solely to residue Tyr-36 at the tip of the hairpin. From this work, we propose that TIMP-2 may be engineered to abrogate MMP-14 binding, whereas its binding properties for other MMPs, including MMP-2, are maintained. Mutants of TIMP-2 with more directed specificity may be of use in gene therapeutic approaches to human disease.
Subject(s)
Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Tissue Inhibitor of Metalloproteinase-2/chemistry , Tissue Inhibitor of Metalloproteinase-2/metabolism , Tyrosine , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Crystallography, X-Ray , Humans , Kinetics , Matrix Metalloproteinases, Membrane-Associated , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Protein Denaturation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino AcidABSTRACT
OBJECTIVE: The purpose of this study was to evaluate the association between preeclampsia and 3 relatively common mutations that are important in the development of vascular disease and thrombosis; these are similar to conditions observed in pregnancies complicated by preeclampsia. STUDY DESIGN: Deoxyribonucleic acid was extracted from whole blood or cheek swabs of 281 patients with preeclampsia and 360 control subjects (all white). Control subjects consisted of women who had undergone at least 2 term pregnancies unaffected by preeclampsia. Mutation frequencies among patients with preeclampsia and control subjects were compared by standard chi2 analysis, with P <.05 considered significant. RESULTS: Thirty-three of 281 women with preeclampsia (11.7%) and 22 of 193 women with severe preeclampsia (11.4%) were homozygous for cytosine-to-thymine substitution at nucleotide 677 in the gene for methyltetrahydrofolate reductase (MTHFR), versus 41 of 360 control subjects (11.4%; difference not significant). Forty of 258 women with preeclampsia (15.5%) and 22 of 175 women with severe preeclampsia (12.6%) were heterozygous for the insertion of 68 bases at position 844 in the gene for cystathionine beta-synthase (CBS), versus 58 of 332 control subjects (17.5%). Fifteen of 250 women with preeclampsia (6.0%) and 11 of 169 with severe preeclampsia (6.5%) were heterozygous for the Leiden mutation (glycine-to-alanine substitution at nucleotide 1691) in the gene for factor V (F5), versus 12 of 253 control subjects (4.7%; difference not significant). CONCLUSION: In this white population a missense mutation of MTHFR, an insertion mutation of CBS, and a missense mutation of F5 were not found to be associated with an increased risk for preeclampsia, either independently or in combination.
Subject(s)
Cystathionine beta-Synthase/genetics , DNA Transposable Elements , Factor V/genetics , Genetic Predisposition to Disease , Mutation, Missense/physiology , Oxidoreductases Acting on CH-NH Group Donors/genetics , Pre-Eclampsia/genetics , Amino Acid Substitution , Female , Gene Frequency , Heterozygote , Homozygote , Humans , Methylenetetrahydrofolate Reductase (NADPH2) , PregnancyABSTRACT
To investigate the physiological function of syntaxin 4 in the regulation of GLUT4 vesicle trafficking, we used homologous recombination to generate syntaxin 4-knockout mice. Homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, whereas heterozygous knockout mice, Syn4(+/-), had normal viability with no significant impairment in growth, development, or reproduction. However, the Syn4(+/-) mice manifested impaired glucose tolerance with a 50% reduction in whole-body glucose uptake. This defect was attributed to a 50% reduction in skeletal muscle glucose transport determined by 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp procedures. In parallel, insulin-stimulated GLUT4 translocation in skeletal muscle was also significantly reduced in these mice. In contrast, Syn4(+/-) mice displayed normal insulin-stimulated glucose uptake and metabolism in adipose tissue and liver. Together, these data demonstrate that syntaxin 4 plays a critical physiological role in insulin-stimulated glucose uptake in skeletal muscle. Furthermore, reduction in syntaxin 4 protein levels in this tissue can account for the impairment in whole-body insulin-stimulated glucose metabolism in this animal model.
Subject(s)
Glucose/metabolism , Insulin Resistance/genetics , Membrane Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Muscle Proteins , Muscle, Skeletal/physiology , Adipocytes/physiology , Adipose Tissue, Brown , Animals , Biological Transport , Glucose Clamp Technique , Glucose Tolerance Test , Glucose Transporter Type 4 , Glycogen/metabolism , Glycolysis , Heterozygote , Liver/metabolism , Mice , Mice, Knockout , Qa-SNARE ProteinsABSTRACT
A woman presented at 33 weeks gestation with reduced fetal movements and a nonreactive nonstress test. Fetal ultrasound examination revealed a peculiar unilateral arm tremor. At emergency cesarean section, performed for fetal indications, a 1,672-gm male infant was delivered requiring intubation for feeble respiratory effort. After delivery the neonate was transiently hypertonic and later hypotonic. Continuing ventilatory support at minimal settings was necessary. The work-up for aneuploidy, metabolic disorders, and infection was negative. The infant died after being removed from ventilatory support on day 22. Postmortem examination revealed extensive bilateral brain gliosis and mineralization without evidence of inflammation, partial absence of cranial nerve nuclei III-XI, and a total absence of cranial nerve roots VI-XI. Together these finding are compatible with a diagnosis of expanded Möbius syndrome.
Subject(s)
Brain/pathology , Cranial Nerves/pathology , Mobius Syndrome/diagnosis , Adult , Arm , Diagnosis, Differential , Fatal Outcome , Female , Gliosis , Humans , Infant, Newborn , Infant, Premature , Male , Mobius Syndrome/complications , Mobius Syndrome/pathology , Pregnancy , Tremor/etiologyABSTRACT
It is hypothesized that infectious prions are generated as the cellular form of the prion protein (PrP(C)) undergoes pronounced conformational change under the direction of an infectious PrP(Sc) template. Conversion to the infectious conformer is particularly associated with major structural rearrangement in the central portion of the protein (residues 90-120), which has an extended flexible structure in the PrP(C) isoform. Using a panel of recombinant antibodies reactive with different parts of PrP, we show that equivalent major structural rearrangements occur spontaneously in this region of PrP immobilized on a surface. In contrast, regions more towards the termini of the protein remain relatively unaltered. The rearrangements occur even under conditions where individual PrP molecules should not contact one another. The propensity of specific unstructured regions of PrP to spontaneously undergo large and potentially deleterious conformational changes may have important implications for prion biology.
Subject(s)
PrPC Proteins/chemistry , PrPSc Proteins/chemistry , Animals , Antibodies, Monoclonal/immunology , Mice , PrPC Proteins/immunology , PrPSc Proteins/immunology , Protein Conformation , Time FactorsABSTRACT
Experimental evidence indicates Epstein Barr virus (EBV) envelope glycoprotein gp350/220 elicits a potent virus neutralizing response in the infected human host that may play an important role in restricting viral pathogenesis. In this study, we report the molecular cloning in combinatorial phage display vectors, of the IgG1 repertoire of an individual naturally infected with EBV, and describe the recovery and characterization of a monoclonal antibody recognizing gp350/220. A detailed understanding of the human antibody response in EBV infection will identify antibodies of potential use in anti-viral prophylaxis and will advance the production of more effective vaccine candidates.
Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Viral/genetics , Antigens, Viral/immunology , Herpesvirus 4, Human/immunology , Immunoglobulin Fab Fragments/genetics , Viral Matrix Proteins/immunology , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Base Sequence , Humans , Immunoglobulin Fab Fragments/immunology , Molecular Sequence DataABSTRACT
Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrP(Sc)). To explore this postulate, we examined the properties of PrP(Sc) from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrP(Sc) molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrP(Sc) as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have approximately 75-d incubation periods; a concentration of 1.08 M was found for the DY strain with a approximately 170-d incubation period and approximately 1.25 M for the SHa(RML) and 139H isolates with approximately 180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a approximately 320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrP(Sc) conformers encipher the biological properties of prion strains.
Subject(s)
Prions/chemistry , Prions/classification , Protein Conformation , Scrapie/etiology , Animals , Antibodies/genetics , Cricetinae , Endopeptidases , Enzyme-Linked Immunosorbent Assay/methods , Guanidine , Mesocricetus , Protein Denaturation/drug effects , Species SpecificityABSTRACT
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.
Subject(s)
Antibodies, Antinuclear/immunology , B-Lymphocytes/immunology , Brain/immunology , Multiple Sclerosis/immunology , Adult , Antibodies, Antinuclear/blood , Antibodies, Antinuclear/cerebrospinal fluid , Female , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin kappa-Chains/genetics , Immunoglobulin kappa-Chains/immunology , Multiple Sclerosis/blood , Multiple Sclerosis/cerebrospinal fluidABSTRACT
HR325 (2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'(trifluoromethyl)-phenyl]-propenamide) is an immunomodulatory compound through pyrimidine biosynthesis inhibition with antiproliferative properties which was derived from the isoxazol compound A77 1726 [2-cyano-3-cyclopropyl-3-hydroxy-enoic acid (4-trifluoromethylphenyl)-amide]. During studies of the effects on early signal transduction events of this type of compound, it was found that HR325 dose-dependently inhibited adenosine 3',5'-cyclic monophosphate (cAMP) synthesis by Jurkat cells stimulated with prostaglandin E(2), (PGE(2)), cholera toxin (CTX), or forskolin (FKN). The potency of inhibition by HR325 of FKN-stimulated cells (IC(50) 30.4 microM) was approximately 3-fold higher than that of the other agonists (11.6 and 11.7 microM) and was independent of time of preincubation for both PGE(2) and FKN. Interestingly, A77 1726, an analogue of HR325, displayed a markedly different profile of stimulus-dependent potencies. The inhibition of cAMP synthesis by HR325 when stimulated by both PGE(2) and FKN was unaffected by glucose supplementation, in contrast to HR325-inhibited ATP levels, which were restored under such conditions. Further studies revealed that HR325 reduced intracellular ATP levels by uncoupling oxidative phosphorylation, albeit with a 1000-fold lower potency than the antihelmintic drug niclosamide. In addition, glucose supplementation experiments showed that, in contrast to HR325, the niclosamide-mediated reduction of ATP levels was wholly responsible for its inhibition of PGE(2)- and FKN-stimulated cAMP synthesis.