Integrins: utility as cell type- and stage-specific markers for hepatocellular carcinoma and cholangiocarcinoma.
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Biomarkers, Tumor↗
Biomedical subjects
H Sakai
Publications and source records attributed to H Sakai.
Expression and localization of ferritin mRNA in ameloblasts of rat incisor.
At the maturation stage, the ameloblasts of the rat incisor incorporate iron, supplied through the bloodstream, and deposit it into the surface layer of the enamel. In this unique iron transport system, ferritin functions as a transient iron reservoir in the cells. Here the expression of ferritin mRNA and its localization in the rat enamel organ was examined. Among various tissues, the enamel organ showed the highest expression for both ferritin H- and L-chain mRNA, as quantified by reverse transcription-polymerase chain reaction. In situ hybridization using digoxigenin-labelled cRNA probes for each chain demonstrated that both ferritin H- and L-chain mRNA were abundantly expressed in presecretory and secretory ameloblasts. The intensity of the positive hybridization signal gradually decreased toward the incisal direction. Differing from the mRNA localization, ferritin protein was immunologically undetectable in presecretory or secretory ameloblasts but was found in ameloblasts at the maturation stage, into which iron is known to be incorporated from the bloodstream. Thus, the expression of ferritin mRNA precedes the protein expression in the developmental stages of rat incisor ameloblasts, and the translation of ferritin and its half-life are probably controlled by the iron entry, as has been reported for other cell types.
Ameloblasts↗
Subtalar instability following lateral ligament injuries of the ankle.
A stress radiograph of the subtalar joint was taken using Telos equipment, while the X-ray beam was directed onto the posterior subtalar joint at angles of 30 degrees latero-medially and 40 degrees caudocranially. From this radiograph the subtalar tilt angle was measured as an index of stability of the subtalar joint. Intra-and interobserver evaluations of measurement of this angle were performed on 20 unstable ankles. The errors at a 95 per cent confidence level were 1.9 degrees for intraobserver A, 1.4 degrees for intraobserver B and 2 degrees in interobservation. Stability of the subtalar joint was evaluated on 46 lateral ligament injuries of the ankle (23 acute injuries and 23 chronic injuries) and 80 normal ankles. The subtalar tilt angle was 9.7 degrees +/ 3.2 degrees in acute injuries 10.3 degrees #/ 2.9 degrees in chronic injuries, and 5/2 degrees +/ 2/6 degrees in normal ankles. There were significant differences between the acute or chronic injury and the normal ankles. These results suggested that stability of the subtalar joint was disturbed following acute and chronic lateral ligament injuries of the ankle.
Adolescent↗
Neuropathological study of gazelle herpesvirus 1 (equine herpesvirus 9) infection in Thomson's gazelles (Gazella thomsoni).
Gazelle herpesvirus (GHV-1), correctly designated as equine herpesvirus 9, is a new type of equine herpesvirus immunologically related to equine herpesvirus 1 (EHV-1). As a sequel to a virological study, the neuropathology of encephalitis caused by GHV-1 in Thomson's gazelles (Gazella thomsoni) was examined. Seven gazelles died with or without neurological symptoms between early September and mid-October in 1993. No gross abnormalities were observed at necropsy, but all animals had non-suppurative encephalitis, characterized by necrosis and degeneration of neurons, glial reactions and perivascular cuffing in the cerebrum. Five cases showed intranuclear inclusion bodies, with the appearance of herpesvirus in the degenerating neurons. Immunohistochemically, all seven animals showed a positive reaction to EHV-1 antigen in neurons in the necrotic areas of the cortex. The clinical course and morphological features of GHV-1 encephalitis were distinct from those of EHV-1-induced encephalitis in the horse, which is characterized by vasculitis, thrombosis, ischaemia, and lack of intranuclear inclusions in neurons.
Animals↗
Inhibition of integrin signaling with Arg-Gly-Asp motifs in rat hepatic stellate cells.
BACKGROUND/AIMS: Activation of hepatic stellate cells plays a key role in liver fibrogenesis. Disruption of normal hepatic stellate cell-matrix interactions may contribute to this process. However, little is known about the molecular events leading from integrin-extracellular matrix interaction to hepatic stellate cell function. Therefore, we investigated the role of integrin signaling in tyrosine phosphorylation of focal adhesion kinase and cytoskeletal assembly in rat hepatic stellate cells using soluble Arg-Gly-Asp containing peptides. METHODS: Hepatic stellate cells were isolated from normal rat livers. Integrin alpha5beta1 expression in hepatic stellate cells was analyzed by immunoprecipitation and immunocytochemistry. The cytoskeletal assembly and tyrosine phosphorylation of focal adhesion kinase were determined by immunocytochemistry and immunoblotting. We also analyzed the effect of Arg-Gly-Asp containing peptides on the expression of smooth muscle alpha actin by immunocytochemistry and immunoblotting. RESULTS: We identified integrin alpha5beta1 in rat hepatic stellate cells. Stress fiber formation and cell shape were different when hepatic stellate cells were plated on various extracellular matrix components. Treatment of hepatic stellate cells with soluble Arg-Gly-Asp peptides diminished the adhesion-induced tyrosine phosphorylation of focal adhesion kinase and inhibited the formation of stress fibers. The peptides also reduced the expression of smooth muscle alpha actin. CONCLUSIONS: Our results demonstrate that adhesion to extracellular matrix induces tyrosine phosphorylation of focal adhesion kinase and promotes actin stress fiber formation and focal adhesion assembly in rat hepatic stellate cells, and that these events are disturbed by soluble Arg-Gly-Asp peptides.
Animals↗
A cross-validation of the European Organization for Research and Treatment of Cancer QLQ-C30 (EORTC QLQ-C30) for Japanese with lung cancer.
The EORTC QLQ-C30 was developed in English-speaking cultures. To determine if this instrument could cross a broad cultural divide and be used in Japan, the cross-cultural validity of its Japanese version was estimated. In evaluating psychometric testing, internal consistency by Cronbach's alpha, item-discrimination by multitrait scaling analysis, and validity analysis with ECOG performance score (PS) and Karnofsky Performance Status Scale (KPS) were performed. The QLQ-C30 (version 1.0) was given to 105 patients with lung cancer. Although the response rate was low in patients with PS 4, the questionnaire was well accepted by patients with PS 0-3. The Japanese QLQ-C30 has a weak scale of role functioning in terms of item discriminative validity. It also has a weak scale of cognitive functioning in items of discriminative validity and internal consistency. However, known-groups comparison showed the expected clinical validity with PS for all the scales except for financial impact, and longitudinally clinical validity with KPS was shown in scales of cognitive functioning, fatigue, and nausea and vomiting. Multitrait scaling analysis showed that the predicted scales constituting quality of life (QOL) in the English-speaking culture were extracted from the Japanese QLQ-C30, and found to be valid in Japan, indicating its possible usefulness as an instrument that is universally applicable across cultures.
Cross-Cultural Comparison↗
Comparison of three treatment regimens of inhaled sodium cromoglycate in the management of adult patients with severe, steroid-dependent asthma.
BACKGROUND: Asthmatic patients whose asthma remains poorly controlled despite treatment with high doses of inhaled corticosteroids and co-administration of oral corticosteroids are a difficult problem in therapeutics. OBJECTIVE: To investigate the relative efficacy of three treatment regimens of inhaled sodium cromoglycate in the treatment of adult, severe, corticosteroid-dependent patients as determined by the reduction in the dose of oral corticosteroids and change in lung function. METHODS: Open, randomized, group comparative trial of 12 weeks duration in asthmatic patients attending a hospital outpatient department. Patients whose asthma is (1) severe according to the classification of the Japanese Society of Allergology, (2) stable, and (3) needing treatment with at least 1600 microg of inhaled beclomethasone dipropionate and 5 mg or greater of oral prednisolone per day. The three treatment regimens of inhaled sodium cromoglycate were group A received sodium cromoglycate powder at a dose of 16 mg/day administered by a metered dose inhaler. Group N received sodium cromoglycate aqueous solution at a dose of 80 mg/day administered by a nebulizer. Group C received sodium cromoglycate aqueous solution (80 mg/day) combined with salbutamol (3 mg/day) administered by a nebulizer. The main outcome measures were a change in the daily dose of oral corticosteroids and in lung function with twice daily measurements of peak expiratory flow (PEF) recorded in the morning (PEF AM) and in the evening (PEF PM). RESULTS: Mean reduction in oral corticosteroid dose/day was group A, 3.68 mg (95% CI 1.35,5.95); group N, 3.59 mg (95% CI 0.73,6.45); and group C, 3.97 mg (95% CI 1.81,6.13). The dosage reductions are all significant but with no differences between the groups. The mean increase in PEF over the last 4 weeks of treatment compared with baseline values was significant in all groups. The increases in group C are significantly greater than those in the other groups. These changes are all significant and the increases in group C are significantly greater than those in the other groups. CONCLUSIONS: Inhaled sodium cromoglycate may be a useful additional treatment in the management of adult patients with severe, oral steroid-dependent asthma. Of the three methods of administration compared in this trial the most useful immediate results were obtained when the drug was administered as an aqueous solution mixed with salbutamol and delivered by a powered nebulizer.
Administration, Inhalation↗
Study of immunoelectron microscopic localization of cathepsin K in osteoclasts and other bone cells in the mouse femur.
The localization of cathepsin K protein in mouse osteoclasts was examined by immunolight and immunoelectron microscopy using the avidin-biotin-peroxidase complex method with anti-cathepsin K (mouse) antibody. With light microscopy, a strong immunoreaction for cathepsin K was found extracellularly along the bone and cartilage resorption lacunae and detected intracellularly in vesicles, granules, and vacuoles throughout the cytoplasm of multinuclear osteoclasts and chondroclasts attached to the surface of the bone or cartilage. Mononuclear cells, probably preosteoclasts, some distance from the bone also contained a few cathepsin K-positive vesicles and granules. Cathepsin K was sometimes found in the cisternal spaces of the rough endoplasmic reticulum and vesicles of the Golgi apparatus with electron microscopy of the basolateral region of the osteoclasts. Cathepsin K-positive vesicles and granules as lysosomal compartments were present in various stages of fusion with vacuoles as endosomal compartments that contained fragmented cathepsin K-negative fibril-like structures. Some of the vacuoles (endolysosomes), which seemed to be formed by this process of fusion, contained cathepsin K-positive vesicles and fibril-like structures that did not show the regular cross striation of type I collagen fibrils. In the apical region of the osteoclasts, cathepsin K-positive vesicles and pits had already fused with or were in the process of fusing with the ampullar extracellular spaces. There were large deposits of cathepsin K on fragmented fibril-like structures without regular cross striation in the extracellular spaces, as well as on and between the cytoplasmic processes of the ruffled border. There were also extensive deposits of cathepsin K on the type I collagen fibrils with cross striation in the bone resorption lacunae. Osteoblasts and osteocytes were negative for cathepsin K. In the immunocytochemical controls, no immunoreaction was found in the osteoclasts or preosteoclasts, or on the collagen fibrils in the resorption lacunae. The results indicate that cathepsin K is produced in mature osteoclasts attached to the bone and secreted into the bone resorption lacunae. The findings suggest that cathepsin K participates in the extracellular degradation of collagen fibrils in the resorption lacunae and in the subsequent degradation of the fragmented fibrils in the endolysosomes. It is also suggested that cathepsin K degrades the organic cartilage matrix.
Animals↗
The significance of the expression of tumor suppressor gene DCC in human gliomas.
Deleted in colorectal carcinoma (DCC) gene has been as a candidate of tumor suppressor genes, has been identified recently and is thought to relate to the metastatic potential in some cancers. We examined the gene in 60 human gliomas (26 glioblastomas multiforme (GBMs), 16 anaplastic astrocytomas (AAs), 6 low grade astrocytomas (LGAs) of WHO Grade II, and 11 recurrent gliomas) and A172 human GBM cell line by reverse transcription polymerase chain reaction (RT-PCR). Twenty (77%) GBMs, 11 (69%) AAs, and 1 (17%) LGA revealed the reduced or absent DCC expression. Reduced DCC expression was also shown in 10 (91%) recurrent gliomas. Furthermore, in 5 cases with both primary and recurrent GBM, the DCC expressions of all recurrent tumors were lower than those of primary tumors. No significant correlation between DCC expression and Mib-1 labeling index was confirmed. The survival rate of patients without reduced DCC expression was significantly superior to that of patients with reduced DCC expression in overall malignant astrocytic tumors. In GBM and AA separately, DCC expression also tended to correlate with patient's prognosis. These results suggest that reduced DCC expression is an important marker in tumor malignancy and recurrence in astrocytic tumors and that may be a useful prognostic factor in patients with malignant astrocytic tumors.
Adolescent↗
Effects of local administration of osteocalcin on experimental tooth movement.
The purpose of this study was to evaluate the effects of local administration of osteocalcin, a major noncollagenous bone matrix protein, on experimental tooth movement in rats. An orthodontic elastic band was inserted between the upper first and second molars, and the first molar was moved mesially. Purified osteocalcin (0 to 10 micrograms) in 20 microliters of phosphate-buffered saline was injected into the region of the root bifurcation of the first molar daily for 4 days. Tooth movement increased significantly following the injections. Histological studies revealed that the injections markedly stimulated the appearance of osteoclasts on the pressured side of the alveolar bone surface. The results suggest that osteocalcin has an additive effect on the rate of orthodontic tooth movement through the enhancement of osteoclastogenesis on the pressured side.
Alveolar Process↗
Identification of cellular compartments involved in processing of cathepsin E in primary cultures of rat microglia.
Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome-like vacuoles and partly in the vesicles located in the trans-Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46-kDa polypeptide (reduced form) after a 30-min pulse. Most of it was proteolytically processed via a 44-kDa intermediate to a 42-kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse-labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high-mannose oligosaccharides and processed to the active mature enzyme with complex-type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome-like acidic compartments.
Animals↗
Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis.
The presence of nitric oxide (NO) in the kidney has been implicated in the pathogenesis of human glomerulonephritis. However, the exact type of glomerular cells that express NO synthase (NOS) and the NOS isoform involved in the local production of NO has not been identified in the human diseased kidney. We examined the expression of three isoforms of NOS, inducible NOS (iNOS), endothelial NOS (eNOS) and brain NOS (bNOS) in the renal tissue of patients with IgA nephropathy (IgAN, N = 10), lupus nephritis (LN, N = 5), membranous nephropathy (MN, N = 5) and minimal change nephrotic syndrome (MCNS, N = 5). Sections were immunostained and the correlation between the expression of each NOS and the degree of glomerular injury in that section was also examined. Normal portions of surgically resected kidneys served as controls. eNOS was present in glomerular endothelial cells and endothelium of cortical vessels in the control and diseased kidneys. iNOS was localized in mesangial cells, glomerular epithelial cells and infiltrating cells in the diseased glomeruli, whereas immunostaining for iNOS was hardly detected in control kidneys. In addition, the expression pattern of eNOS in each glomerulus was the reverse of that of iNOS. In IgAN and LN, the extent of staining for eNOS correlated negatively with the degree of glomerular injury, while the extent of staining for iNOS correlated positively with the degree of glomerular injury in the same tissues. bNOS was not detected in normal or nephritic glomeruli. Our results indicate the presence of a NO pathway in human diseased kidney, and suggest that NO derived from eNOS and iNOS may be involved in the progression of renal diseases and that NO derived from each NOS may play an important role in different way in human inflamed glomeruli.
Adult↗
Expression of decay accelerating factor mRNA and complement C3 mRNA in human diseased kidney.
BACKGROUND: Decay accelerating factor (DAF), a product of mesangial cells in vitro, is expressed on the surface of cells and is a candidate for the focal suppression of complement activation. It is not clear at present whether the levels of expression of DAF and intrarenal C3 synthesis correlate with the level of tissue injury. METHODS: Immunohistochemistry for DAF and C3 and nonradioactive in situ hybridization with digoxigenin-labeled oligonucleotide probe for DAF and C3 mRNA were performed in 22 tissue samples of kidneys from patients with IgA nephropathy (IgAN), 6 with membranous nephropathy (MN), 6 with lupus nephritis (LN), and five normal kidneys. RESULTS: In the normal kidney, DAF was confined to the juxtaglomerular apparatus and little or no C3 was detected; however, a few glomerular cells were positive for DAF mRNA but no C3 mRNA positive cells were detected. In diseased kidneys, DAF and C3 as well as their mRNAs were detected in mesangial cells, tubular cells and infiltrating cells. Glomerular epithelial cells and Bowman's capsule cells contained little or no DAF and C3 but were positive for their mRNAs. The mean percentages of mesangial cells positive for DAF and C3 mRNAs were 49.3 +/- 11.5% and 50.7 +/- 10.3% in IgAN, and 17.0 +/- 6.3% and 19.4 +/- 9.0% in MN, respectively. The percentage of mesangial cells positive for DAF and C3 mRNAs among intraglomerular cells correlated positively with the degree of mesangial proliferation and glomerular sclerosis in IgAN. In contrast, in LN the percentage of glomerular cells positive for DAF mRNA correlated negatively with the degree of glomerular injury, while the percentage of cells positive for C3 mRNA did not change with the progression of the disease. The ratio of C3 mRNA/DAF mRNA of glomerular cells correlated with the degree of glomerular injury in both IgAN and LN. In the tubulointerstitium, the percentage of cells expressing mRNA, and C3 mRNA/DAF mRNA radio correlated with the degree of tubular atrophy and interstitial broadening in both IgAN and LN. CONCLUSIONS: We conclude that DAF and C3 mRNAs are synthesized in human diseased kidneys, and that a balance between locally synthesized DAF and C3 may be important in the progression of glomerulonephritis.
Adult↗
Angiotensin I-converting enzyme gene polymorphism in intracranial saccular aneurysm individuals.
A polymorphism in the angiotensin I-converting enzyme (ACE) gene has been associated with cerebrovascular diseases as a new potent risk factor. The purpose of this study was to investigate an association of the gene polymorphism with intracranial saccural aneurysmal patients. The study population consisted of 83 aneurysmal patients (age range 41-85 years) (the AN group) and 104 matched control subjects (age range 30-81 years) (the Control group). For detection of the ACE gene polymorphism, the standard PCR method was performed by using genomic DNA isolated from peripheral blood leukocytes. The PCR products were a 490-bp in the presence of the insertion (I) and a 190-bp fragment in the absence of the insertion (D). The ACE gene polymorphism was classified into three genotypes: I/I genotype (a 490-bp band); D/D genotype (a 190-bp band); or I/D genotype (both a 490-bp and a 190-bp band). The number of subjects with I/I, I/D, and D/D genotypes was 38, 40, and 5 in the AN group and 43, 45, and 16 in the Control group, respectively. The frequency of the D/D genotype in the AN group was significantly lower (5/83 = 0.06) than that in the Control group (16/104 = 0.15) (chi 2 = 4.06; p = 0.044). There was no significant difference between the genotype sof hypertensive patients and normotensive patients in the AN group. Thus, this present study suggests that genetic heterogeneity of the ACE gene may be correlated with the etiology of intracranial aneurysms.
Adult↗
Phosphorothioate hammerhead ribozymes targeting a conserved sequence in the V3 loop region inhibit HIV-1 entry.
Seven different phosphorothioate DNA-RNA chimeric hammerhead ribozymes (RzV3-nT, n = 1-7) targeted against the V3 loop region of HIV-1 were synthesized. Two of these, RzV3-1T and RzV3-3T, efficiently cleaved transcribed envelope RNA of HXB2 in vitro. The target sequence of RzV3-1T belongs to a conserved region and is completely identical in the HIV-1 HXB2, NL432, and ADA strains. Furthermore, RzV3-1T cleaved the envelope RNA of HIV-1 SF162 with a single base substitution in the distal site. U87 cells expressing CD4 and coreceptors were used as target cells for infections with the SF162 and NL432 strains. Replication of both the NL432 and SF162 strains in RzV3-1T-treated cells was significantly lower than that in control cultures. Envelope gene product formation was measured quantitatively with a single-cycle infection assay using pseudovirus generated from cotransfection with one vector containing a luciferase reporter gene and one vector containing the envelope gene of HXB2, SF162, or ADA. Production of pseudovirus in RzV3-1T-treated cells led to a marked (93% or 87%) inhibition of envelope-mediated entry of resultant HXB2-derived or ADA-derived pseudotype virions, respectively, and a moderate (44%) inhibition was seen for SF162-derived pseudotype virions. Thus, an efficient, stable ribozyme against a functionally important region of HIV-1 was identified by evaluating its activities in vitro and in vivo. This ribozyme may be useful for control of HIV-1 infection.
Animals↗
An elevation of stem cell factor in patients with hyperthyroid Graves' disease.
Graves' disease is an autoimmune disorder characterized by the presence of antibodies against thyrotropin receptor (TRAb). Stem cell factor (SCF), derived from bone marrow, is known to promote lymphohematopoiesis. To investigate the relation between the alteration in plasma levels of SCF, thyroid hormone status, and TRAb measured by thyrotropin binding inhibition (TBI), 13 untreated, 21 treated, and 4 relapsed hyperthyroid Graves' disease patients, 21 patients with Hashimoto's thyroiditis, 6 patients with subacute thyroiditis, and 11 control subjects were examined. In untreated hyperthyroid Graves' disease patients, serum levels of thyroxine (T4) and triiodothyronine decreased rapidly by methimazole treatment, and TBI decreased progressively, but variably. Simultaneously, the elevated plasma levels of SCF decreased gradually and progressively. The plasma levels of SCF correlated curvilinearly with the serum levels of T4. In 4 patients with relapsed hyperthyroid Graves' disease, TBI was marginally positive in 3 patients and negative in 1, but plasma levels of SCF were elevated significantly in all 4 patients. In patients with subacute thyroiditis and Hashimoto's thyroiditis with or without T4 replacement, plasma levels of SCF did not differ from that of controls. These findings indicate that the elevation of plasma levels of SCF relates to the longstanding thyrotoxic state and that short-term thyrotoxicosis does not significantly affect plasma levels of SCF. It remains to be determined whether the elevation in plasma levels of SCF is induced by excess thyroid hormone, reflecting the hypermetabolic state, or whether the elevation of plasma levels of SCF contributes to stimulation of lymphocytes producing TRAb.
Acute Disease↗
Conversion of neopullulanase-alpha-amylase from Thermoactinomyces vulgaris R-47 into an amylopullulanse-type enzyme.
TVA I, an alpha-amylase from Thermoactinomyces vulgaris R-47, is a versatile enzyme which hydrolyzes the alpha-(1-->4)-glucosidic linkages of pullulan to produce panose, known as neopullulanase activity, and the alpha-(1-->6)-glucosidic linkages of certain oligosaccharides. We modified the Ala-357, Gln-359, and Tyr-360 residues located in region II, one of the four regions conserved in alpha-amylase family enzymes, and deleted 11 consecutive amino acid residues located after the C-terminus of region II of the TVA I sequence by means of site-directed mutagenesis. The action pattern of the mutated enzyme for pullulan was greatly altered and it hydrolyzed mainly the alpha-(1-->6)-glucosidic linkages of pullulan to produce maltotriose, while the action patterns for starch and maltooligosaccharides were almost identical to those of the wild-type enzyme. This means that the mutated TVA I has lost the neopullulanase activity, and thus can be designated as an amylopullulanase-type enzyme. The kcat/Km value of the mutated enzyme for alpha-(1-->6)-glucosidic linkages was virtually unaltered, while that for alpha-(1-->4)-glucosidic linkages was about 100 times smaller than that of the wild-type enzyme.
Amino Acid Sequence↗
Fluorescence microscopic demonstration of cathepsin K activity as the major lysosomal cysteine proteinase in osteoclasts.
The enzyme activity of lysosomal cysteine proteinases in vital rabbit osteoclasts and mouse osteoclast-like cells was visualized with Z-Leu-Arg-4-methoxy-beta-naphthylamide (Z-LR-MNA) as the enzyme substrate. The MNA liberated by proteolysis forms a fluorescent insoluble Schiff-base product in the presence of 5-nitrosalicylaldehyde. Many small fluorescent particles, endproducts of the Z-LR-MNA hydrolysis, were observed in proximity to the bone surface underneath the actively resorbing osteoclasts, as well as in the cytoplasm. The Z-LR-MNA hydrolase activity was markedly diminished by bafilomycin A1 and chloroquine treatment. Moreover, the activity was completely inhibited by cysteine proteinase inhibitors such as leupeptin and E-64d, but not by other classes of proteinase inhibitors. About 60% of the hydrolase activity in mouse osteoclast-like cells was immunoabsorbed by anti-cathepsin K antibody-coupled Sepharose CL-4B beads, and about 10% of the activity was absorbed with the anti-cathepsin L antibody-coupled beads. Thus, the majority of the Z-LR-MNA hydrolase activity in osteoclasts was derived from cathepsin K. In contrast, using the same substrate in the assay, no detectable cathepsin K activity was observed in mouse peritoneal macrophages. The abundant cathepsin K activity in osteoclasts would therefore indicate a significant role of this enzyme in bone matrix degradation.
Amino Acid Sequence↗