ABSTRACT
The search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identified in vitro to be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPC mRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23-231) as the substrate and PrPSc seeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90-231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPC globular domain. J8 also reduced the fibrillation of mouse rPrP23-231 seeded with in vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPC on the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPC content on the cell surface.
Subject(s)
Chalcones/pharmacology , Drugs, Investigational/pharmacology , Neurons/drug effects , Oxadiazoles/pharmacology , Prion Proteins/antagonists & inhibitors , Animals , Binding Sites , Cell Line, Tumor , Chalcones/chemical synthesis , Cloning, Molecular , Drugs, Investigational/chemical synthesis , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Kinetics , Mice , Molecular Docking Simulation , Neurons/metabolism , Neurons/pathology , Oxadiazoles/chemical synthesis , Prion Proteins/chemistry , Prion Proteins/genetics , Prion Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , ThermodynamicsABSTRACT
The baculovirus-insect cell expression system is widely used to produce recombinant mammalian glycoproteins, but the glycosylated end products are rarely authentic. This is because insect cells are typically unable to produce glycoprotein glycans containing terminal sialic acid residues. In this study, we examined the influence of two mammalian glycosyltransferases on N-glycoprotein sialylation by the baculovirus-insect cell system. This was accomplished by using a novel baculovirus vector designed to express a mammalian alpha2,6-sialyltransferase early in infection and a new insect cell line stably transformed to constitutively express a mammalian beta1,4-galactosyltransferase. Various biochemical assays showed that a foreign glycoprotein was sialylated by this virus-host combination, but not by a control virus-host combination, which lacked the mammalian glycosyltransferase genes. Thus, this study demonstrates that the baculovirus-insect cell expression system can be metabolically engineered for N-glycoprotein sialylation by the addition of two mammalian glycosyltransferase genes.
Subject(s)
N-Acetyllactosamine Synthase/biosynthesis , N-Acetyllactosamine Synthase/genetics , Nucleopolyhedroviruses/genetics , Sialoglycoproteins/biosynthesis , Sialyltransferases/biosynthesis , Sialyltransferases/genetics , Spodoptera/genetics , Spodoptera/metabolism , Animals , Cattle , Cell Line, Transformed , Cloning, Molecular , Genes, Immediate-Early , Genetic Vectors/chemistry , Genetic Vectors/genetics , Genetic Vectors/isolation & purification , Rats , Recombination, Genetic , Sialoglycoproteins/genetics , Spodoptera/cytology , Spodoptera/enzymology , beta-D-Galactoside alpha 2-6-SialyltransferaseABSTRACT
Recombinant mammalian glycoproteins produced by the baculovirus-insect cell expression system usually do not have structurally authentic glycans. One reason for this limitation is the virtual absence in insect cells of certain glycosyltransferases, which are required for the biosynthesis of complex, terminally sialylated glycoproteins by mammalian cells. In this study, we genetically transformed insect cells with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes. This produced a new insect cell line that can express both genes, serve as hosts for baculovirus infection, and produce foreign glycoproteins with terminally sialylated N-glycans.
Subject(s)
Lepidoptera/cytology , N-Acetyllactosamine Synthase/genetics , Sialyltransferases/genetics , Transformation, Genetic , Animals , COS Cells , Chromatography, Affinity , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Glycoproteins/biosynthesis , Glycoproteins/geneticsABSTRACT
The inability to produce eukaryotic glycoproteins with complex N-linked glycans is a major limitation of the baculovirus-insect cell expression system. Recent studies have demonstrated that metabolic engineering can be used to extend the glycoprotein processing capabilities of lepidopteran insect cells. This approach is being used to develop new baculovirus-insect cell expression systems that can produce more authentic recombinant glycoproteins and obtain new information on insect N-glycosylation pathways.
Subject(s)
Baculoviridae/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Animals , Biotechnology , Carbohydrate Sequence , Cell Line , Glycosylation , Insecta , Models, Biological , Molecular Sequence Data , Protein Engineering , Recombinant Proteins/chemistryABSTRACT
We describe a patient with pauciarticular juvenile rheumatoid arthritis with aortic insufficiency who underwent successful aortic valve replacement with a mechanical prosthetic valve.
Subject(s)
Aortic Valve Insufficiency/surgery , Arthritis, Juvenile/complications , Heart Valve Prosthesis Implantation , Heart Valve Prosthesis , Aortic Valve/pathology , Aortic Valve/surgery , Aortic Valve Insufficiency/etiology , Arthritis, Juvenile/surgery , Child , Female , Humans , Neovascularization, Pathologic/pathologyABSTRACT
An established lepidopteran insect cell line (Sf9) was cotransfected with expression plasmids encoding neomycin phosphotransferase and bovine beta 1,4-galactosyltransferase. Neomycin-resistant transformants were selected, assayed for beta 1,4-galactosyltransferase activity, and the transformant with the highest level of enzymatic activity was characterized. Southern blots indicated that this transformed Sf9 cell derivative contained multiple copies of the galactosyltransferase-encoding expression plasmid integrated at a single site in its genome. One-step growth curves showed that these cells supported normal levels of baculovirus replication. Baculovirus infection of the transformed cells stimulated beta 1,4-galactosyltransferase activity almost 5-fold by 12 h postinfection. This was followed by a gradual decline in activity, but the infected cells still had about as much activity as uninfected controls as late as 48 h after infection and they were able to produce a beta 1,4-galactosylated virion glycoprotein during infection. Infection of the transformed cells with a conventional recombinant baculovirus expression vector encoding human tissue plasminogen activator also resulted in the production of a galactosylated end-product. These results demonstrate that stable transformation can be used to add a functional mammalian glycosyltransferase to lepidopteran insect cells and extend their N-glycosylation pathway. Furthermore, stably-transformed insect cells can be used as modified hosts for conventional baculovirus expression vectors to produce foreign glycoproteins with "mammalianized" glycans which more closely resemble those produced by higher eucaryotes.
Subject(s)
N-Acetyllactosamine Synthase/metabolism , Tissue Plasminogen Activator/metabolism , Animals , Baculoviridae/physiology , Cattle , Cell Line , Glycosylation , Humans , Mammals , N-Acetyllactosamine Synthase/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Spodoptera , Tissue Plasminogen Activator/biosynthesis , Transfection , Virus ReplicationABSTRACT
Although medication has yet to cure rheumatic disease, over the past two decades many adolescent patients have had improved quality of life with better control of pain, deformity, and weakness. However, noncompliance resulting from risk-taking behavior and concerns about cosmetic side effects and fertility may reduce the effectiveness of any drug regimen. The author reviews commonly used medications and their indications and side effects, and includes a section about the effect of pregnancy on rheumatic disease.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antirheumatic Agents/therapeutic use , Hydroxychloroquine/therapeutic use , Rheumatic Diseases/drug therapy , Adolescent , Adolescent Medicine , Adrenal Cortex Hormones/adverse effects , Adrenal Cortex Hormones/therapeutic use , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Antirheumatic Agents/adverse effects , Female , Humans , Hydroxychloroquine/adverse effects , Male , Methotrexate/adverse effects , Methotrexate/therapeutic use , Pregnancy , Sulfasalazine/adverse effects , Sulfasalazine/therapeutic useABSTRACT
OBJECTIVES: To determine if serum lactate dehydrogenase levels distinguish patients with malignant neoplasm presenting with musculoskeletal complaints from patients with juvenile rheumatoid arthritis who reported similar symptoms. DESIGN: Retrospective case-comparison study. SETTING: Tertiary care, outpatient clinics. PATIENTS: Twelve patients with malignant neoplasms who presented with arthritis or arthralgias and normal complete blood cell counts and blood smears in whom rheumatologic diagnosis was initially made were compared with 24 children with a final diagnosis of juvenile rheumatoid arthritis. The patients with malignant neoplasms all had normal blood counts and elevated sedimentation rates at symptom onset. INTERVENTIONS: None. RESULTS: Serum lactate dehydrogenase levels were significantly higher in the cancer patients at 2.2 times the normal values vs 0.8 times high normal for patients with juvenile rheumatoid arthritis (P =.004, Mann-Whitney U test) No significant differences were observed in white blood cell counts, hemoglobin levels, platelet counts, erythrocyte sedimentation rate, or uric acid or aspartate aminotransferase levels at initial evaluation. CONCLUSIONS: Serum lactate dehydrogenase values may distinguish patients with malignant neoplasms from those with rheumatic disease early in the course of illness when symptoms and other laboratory values are not helpful.
Subject(s)
Arthralgia/etiology , L-Lactate Dehydrogenase/blood , Neoplasms/diagnosis , Neoplasms/enzymology , Adolescent , Arthralgia/enzymology , Arthritis, Juvenile/diagnosis , Arthritis, Juvenile/enzymology , Blood Cell Count , Blood Sedimentation , Child , Child, Preschool , Diagnosis, Differential , Discriminant Analysis , Female , Follow-Up Studies , Humans , Male , Neoplasms/complications , Retrospective StudiesABSTRACT
We describe three patients who had infection with human parvovirus B19 in association with new-onset systemic necrotising vasculitis syndromes, two with features of polyarteritis nodosa and one with features of Wegener's granulomatosis. Chronic B19 infection, lasting 5 months to more than 3 years, was shown by enzyme immunoassay for IgG and IgM antibodies to B19 and polymerase chain reaction for B19 DNA in serum and tissue samples. The patients had atypical serological responses to the B19 infection, although none had a recognisable immunodeficiency disorder. Treatment with corticosteroids and cyclophosphamide did not control vasculitis. Intravenous immunoglobulin (IVIG) therapy led to rapid improvement of the systemic vascultis manifestations, clearing of the chronic parvovirus infection, and long-term remission. These observations suggest an aetiological relation between parvovirus B19 infection and systemic necrotising vasculitis in these patients and indicate a potentially curative role for IVIG in such disorders.
Subject(s)
Erythema Infectiosum/complications , Granulomatosis with Polyangiitis/microbiology , Immunoglobulins, Intravenous/therapeutic use , Parvovirus B19, Human/isolation & purification , Polyarteritis Nodosa/microbiology , Adolescent , Base Sequence , Child, Preschool , Chronic Disease , DNA Primers , DNA, Viral/analysis , Erythema Infectiosum/therapy , Female , Granulomatosis with Polyangiitis/immunology , Granulomatosis with Polyangiitis/therapy , Humans , Male , Molecular Sequence Data , Parvovirus B19, Human/genetics , Parvovirus B19, Human/immunology , Polyarteritis Nodosa/immunology , Polyarteritis Nodosa/therapyABSTRACT
The focus of this review is the musculoskeletal side effects of corticosteroids. These untoward effects can be divided into high-dose phenomena (myopathy and aseptic necrosis) and low-dose problems (growth suppression and osteoporosis). For the clinician, the former group may be an uncommon experience whereas the latter group is highly predictable. The low-dose problems are subtle and asymptomatic, and they can occur in spite of alternate day dosing with steroids. Treatments available to prevent or treat these side effects in patients who are unable to discontinue steroids are reviewed.
Subject(s)
Adrenal Cortex Hormones/adverse effects , Musculoskeletal System/drug effects , Female , Growth/drug effects , Humans , Muscular Diseases/chemically induced , Osteoporosis/chemically inducedABSTRACT
In 32 children with pauciarticular juvenile rheumatoid arthritis, we studied residual muscle atrophy and leg length discrepancy after the arthritis had entered remission. Children under the age of 3 years at disease onset had significantly more muscle atrophy and bone overgrowth than children whose disease began after the age of 3 years. These residua have cosmetic and functional significance, and new methods of treatment will be necessary to prevent or reverse the sequelae in a form of arthritis previously thought to have a benign outcome.
Subject(s)
Arthritis, Juvenile/complications , Leg Length Inequality/etiology , Muscular Atrophy/etiology , Age Factors , Child, Preschool , Female , Follow-Up Studies , Humans , Knee Joint/physiopathology , Male , Muscular Atrophy/therapy , Physical Therapy ModalitiesABSTRACT
The autoimmune diseases, juvenile rheumatoid arthritis and systemic lupus erythematosus, are two of the more common acquired chronic diseases of childhood. In this article we will describe features of the diagnosis and treatment of these disorders. Particular emphasis is placed on the complications and how to anticipate them. The management of these diseases must include an appreciation of how chronic illness can affect family function and school relationships. With early attention to these nonmedicinal details, the majority of children can progress through childhood and adolescence with few physical or emotional handicaps.
Subject(s)
Arthritis, Juvenile , Lupus Erythematosus, Systemic , Arthritis, Juvenile/diagnosis , Arthritis, Juvenile/etiology , Arthritis, Juvenile/therapy , Child , Humans , Lupus Erythematosus, Systemic/diagnosis , Lupus Erythematosus, Systemic/etiology , MaleABSTRACT
Skin biopsies from patients with scleroderma and juvenile dermatomyositis (DM) share many histologic features. Characteristics common to both diseases are particularly evident in the dermal microvasculature and include endothelial swelling and concentric thickening of the vascular basement membrane. Biopsies performed on 3 patients with the severe vasculitic form of juvenile DM showed these changes as well as dropout of vessels and linear deposition of collagen. The latter findings, seen late in the course of the disease, are indistinguishable from those of advanced scleroderma. A hypothesis is presented which attempts to relate these histological findings to a common underlying pathophysiologic mechanism.
Subject(s)
Dermatomyositis/pathology , Basement Membrane/pathology , Child , Cyclophosphamide/therapeutic use , Dermatomyositis/drug therapy , Dermatomyositis/etiology , Endothelium/pathology , Female , Humans , Male , Microcirculation/pathology , Prednisone/therapeutic use , Scleroderma, Localized/pathologyABSTRACT
Sixty steroid-treated patients with asthma were evaluated for the presence of muscle weakness by use of both manual muscle testing and the Cybex II isokinetic dynamometer. The patients were compared to age and sex-matched sedentary control subjects. Forty-eight percent of the patients (12/25) taking greater than or equal to 40 mg per day of prednisone had hip flexor strength greater than or equal to 2 SD below the mean of age and sex-matched control subjects by Cybex testing (CT). Sixty-four percent of the patients (16/25) taking greater than or equal to 40 mg per day of prednisone were found on manual muscle testing to have hip flexor weakness. Only one patient taking less than 30 mg per day of prednisone was found to have muscle weakness. Biochemical parameters, including CPK, aldolase, SGOT, LDH, and LDH isoenzymes were measured to assess the degree of steroid-induced muscle damage. They neither correlated with the degree of hip flexor weakness as measured by CT, nor did they discriminate between patients receiving small doses and large doses of steroids. Changes in urinary excretion of creatine did not help to confirm the diagnosis of steroid myopathy. Although CT provides an objective means of assessing muscle strength in these patients, at this time no definitive chemical test is available for the diagnosis of steroid myopathy.
Subject(s)
Asthma/drug therapy , Muscular Diseases/diagnosis , Prednisone/adverse effects , Adult , Creatine/urine , Creatinine/urine , Female , Humans , Male , Middle Aged , Muscles/drug effects , Muscular Diseases/chemically induced , Prednisone/therapeutic useSubject(s)
Infant, Premature, Diseases/pathology , Lupus Erythematosus, Systemic/pathology , Antibodies/immunology , Antibodies, Antinuclear/immunology , Blood Platelets/immunology , Humans , Infant, Newborn , Lupus Erythematosus, Systemic/complications , Male , Nephritis/etiology , Prednisone/therapeutic use , Thrombocytopenia/drug therapyABSTRACT
As the presenting complaint in 7 per cent of pediatrician visits, pain in the limbs is a common problem in childhood. It is important that the diagnosis be made expeditiously. The authors review the possible organic cause of limb pain, as well as limb pain from conversion reactions and from growing pains, giving special attention to the differential diagnosis so that appropriate treatment for the pain can be initiated.
Subject(s)
Extremities , Pain/etiology , Arthritis, Infectious/complications , Arthritis, Juvenile/complications , Bone Diseases/complications , Bone Neoplasms/complications , Cartilage Diseases/complications , Child , Dermatomyositis/complications , Endocrine System Diseases/complications , Female , Growth , Hematologic Diseases/complications , Humans , Leukemia/complications , Lupus Erythematosus, Systemic/complications , Male , Muscular Diseases/complications , Myofascial Pain Syndromes/complications , Nutrition Disorders/complications , Osteomyelitis/complications , Pain/psychology , Phobic Disorders/complications , Psychophysiologic Disorders/complications , Rheumatic Fever/complications , Spinal Diseases/complications , Wounds and Injuries/complicationsABSTRACT
A reproducible high-pressure liquid chromatography assay of calmodulin, a multifunctional calcium-dependent modulating protein, was developed for cartilage and bone by using cyclic nucleotide phosphodiesterase activity as the basis for standard curve determination. Calmodulin activity was then measured in rabbits that were made osteopenic by prednisolone injection in an effort to characterize in vivo cellular events. A significant rise in bone calmodulin levels was noted when this data was correlated with osteocyte and osteoblast content by quantitative histomorphometry. This suggests that calmodulin mediates steroid effects on the collagen matrix as well as on calcium homeostasis. Diminished calmodulin levels in weight-bearing cartilage of steroid-treated animals has as yet unresolved significance. Further characterization of calmodulin activity appears warranted in the study of osteopenic states at the subcellular level.