Isolation, cloning, and expression of an acid phosphatase containing phosphotyrosyl phosphatase activity from Prevotella intermedia.
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A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an M(r) of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of Morganella morganii, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined. (+info)
Lymphokine-induced production and release of lysosomal enzymes by macrophages.
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MACROPHAGES ARE ASSOCIATED WITH MOST CHRONIC INFLAMMATORY LESIONS, AND THESE CELLS CONTAIN ENZYMES THAT ARE ABLE TO DESTROY CONNECTIVE TISSUE CONSTITUENTS. Normal lymphoid cells responding to a mitogen, phytohemagglutinin-P, release factor(s) that cause a marked increase in the size and enzyme content for mononuclear phagocytes maintained in culture. The stimulated macrophages, which by several criteria remain otherwise viable and healthy, selectively release large quantities of hydrolytic enzymes to the culture medium. (+info)
MHC class II molecules, cathepsins, and La/SSB proteins in lacrimal acinar cell endomembranes.
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Sjogren's syndrome is a chronic autoimmune disease affecting the lacrimal glands and other epithelia. It has been suggested that acinar cells of the lacrimal glands provoke local autoimmune responses, leading to Sjogren's syndrome when they begin expressing major histocompatibility complex (MHC) class II molecules. We used isopycnic centrifugation and phase partitioning to resolve compartments that participate in traffic between the basolateral membranes and the endomembrane system to test the hypothesis that MHC class II molecules enter compartments that contain potential autoantigens, i.e., La/SSB, and enzymes capable of proteolytically processing autoantigen, i.e., cathepsins B and D. A series of compartments identified as secretory vesicle membranes, prelysosomes, and microdomains of the trans-Golgi network involved in traffic to the basolateral membrane, to the secretory vesicles, and to the prelysosomes were all prominent loci of MHC class II molecules, La/SSB, and cathepsins B and D. These observations support the thesis that lacrimal gland acinar cells that have been induced to express MHC class II molecules function as autoantigen processing and presenting cells. (+info)
Adenocarcinoma of the prostate in perspective.
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Adenocarcinoma of the prostate is responsible for one of every nine deaths from cancer in Canada. In this review epidemiologic factors are considered and current staging systems are outlined. The American Urological System is recommended for staging because of its ability to reflect changes in the understanding of the biologic behaviour of this neoplasm. The adoption of a quantitative grading scheme is suggested to complement the information obtained from the staging assessment. The routes of spread of this disease, along with the procedures used to assess metastatic involvement, are described. Immunologic methods for the analysis of prostatic acid phosphatase have been shown to be superior to the enzymatic methods previously used, and the role of the new techniques is discussed. Emphasis is placed on radiotherapy and endocrine therapy for the treatment of this neoplasm, and the concept of withholding endocrine therapy until symptoms appear is discussed. Potential future developments in this field are considered. (+info)
Fetal mouse hearts: a model for studying ischemia.
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A new experimental model for the study of two important aspects of ischemia, namely, oxygen and substrate deprivation, is proposed: the intact, beating fetal mouse heart in organ culture. This model offers long-term stability, ease and reproducibility of preparation, and the ability to manipulate experimental conditions. Hearts deprived of oxygen and glucose ceased beating immediately. After 3-4 hr of deprivation, biochemical and ultrastructural changes consistent with ischemic injury were evident. These include depletion of ATP and glycogen levels, loss of cytoplasmic enzymes, and extensive swelling and disruption of mitochondrial structure. Glucose and insulin partially protected against ATP depletion. Upon resupply of oxygen and glucose , beating resumed immediately, ATP levels rapidly increased to control levels and, consistent with this, mitochondrial structure returned toward normal. During the recovery phase autophagic vacuoles containing damaged mitochondria and myofibrils were seen, indicating that repair mechanisms were activated. Consistent with this, the proportion of lysosomal enzymes that were present in the nonsedimentable fraction of the tissue homogenate increased. We conclude that the cultured fetal mouse heart is a model useful for studying myocardial responses to anoxia and/or substrate deprivation and for assessing interventions designed to limit damage or to stimulate repair after ischemic injury. (+info)
Clinical significance of immunoassays for type-5 tartrate-resistant acid phosphatase.
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BACKGROUND: Tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) is a product of osteoclasts and a biochemical marker of bone resorption rate. However, erythrocytes and platelets contribute to total TRAP activity in serum, reducing the specificity of direct biochemical assays in serum. Osteoclast TRAP is also known as type-5 TRAP and is antigenically unique. Immunoassays are sought to improve the specificity and sensitivity of TRAP as a bone marker. METHODS: We developed two colorimetric microplate assays for type-5 TRAP: an enzyme capture immunoassay to measure antibody-bound enzymatic activity, and a two-site immunoassay to measure bound enzyme protein. Both use the same monoclonal antibody (14G6) to capture type-5 TRAP, which permits determination of specific activity of serum TRAP in health and disease. RESULTS: Both TRAP assays were linear from one-tenth to fivefold the mean value in 18 healthy subjects. In these subjects, the mean (SD) TRAP activity was 3.2 (0.54) U/L for the enzyme capture assay and 37 (13) microg/L for the two-site assay. Mean TRAP activity was not significantly increased in 64 patients with endstage renal disease requiring hemodialysis (HD) or 99 unselected patients with rheumatic diseases. By contrast, TRAP protein was increased in both the HD and rheumatic disease groups. The specific activity of TRAP in the 17 of 64 HD sera that had increased TRAP activity (0.088 U/microg) was similar to that in healthy subjects (0.091 U/microg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/microg) was significantly decreased. CONCLUSIONS: Wide sample distributions for TRAP activity in HD patients and TRAP protein in rheumatic disease patients suggest the presence of subpopulations of HD patients with increased TRAP activity and of rheumatic patients with increased TRAP protein. Each assay for TRAP activity and protein may have its own biological significance and clinical applications in specific groups of patients. (+info)
Gene dosage: evidence for assignment of erythrocyte acid phosphatase locus to chromosome 2.
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A child, trisomic for the distal short arm of chromosome 2 due to a familial 2/18 translocation, has elevated levels of activity of erythrocyte acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), 3.1.3.2] Ferguson-Smith et al. [(1973) Nature New Biol. 243, 271-274] previously had found decreased levels of activity and loss of expression of an erythrocyte acid phosphatase allele in a subject who lacked one of the two homologous regions containing the distal three bands of chromosome 2. They suggested that the locus for erythrocyte acid phosphatase is located on that segment. Our findings provide further evidence for this assignment and also suggest an in vivo gene dosage effect of this autosomal locus, which depends on both the type and number of alleles present. (+info)
Uptake of cationized ferritin by the epithelium of the main excretory duct of the rat submandibular gland.
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Previous studies demonstrated that the main excretory duct (MED) of the rat submandibular gland can internalize exogenous protein in addition to reabsorbing and secreting electrolytes. However, more precise studies have not been conducted. The aim of this study was to elucidate the cell types responsible for endocytosis of an exogenous protein (ferritin) and to follow the movements of the endocytosed protein in the ductal epithelial cells. The MEDs of the right submandibular gland of male Wistar rats were exposed near the glands proper and cationized ferritin solution was injected into each MED through a fine glass cannula. The MEDs were removed at intervals after ferritin injection, fixed and examined by transmission electron microscopy. The epithelium of the MED of the rat submandibular gland was pseudostratified and consisted of light (types I and II), dark, tuft and basal cells. Uptake of ferritin by the light (types I and II) and dark cells occurred frequently. Small vesicles and multivesicular bodies containing ferritin particles were observed in the supra-nuclear and lateral nuclear cytoplasm. Endocytosis of tracers by tuft cells was rare. Some of the small vesicles and the multivesicular bodies were acid phosphatase-positive. By 60 min after treatment, ferritin-containing small vesicles and multivesicular bodies appeared in the basal cytoplasm. Ferritin particles were also observed in basal extracellular spaces. The light (types I and II), dark and tuft cells (latter rarely) participated in endocytosis of exogenous proteins in the epithelium of the MED of the rat submandibular gland. Almost all of the internalized proteins appeared to be processed by the lysosomal system, and some proteins were released into the extracellular spaces. (+info)