IL-1alpha stimulates cathepsin K expression in osteoclasts via the tyrosine kinase-NF-kappaB pathway.

Interleukin-1alpha (IL-1alpha) is a powerful activator of osteoclast cells. However, the underlying mechanism for this activation is unknown. In this study, we reveal that IL-1alpha up-regulates the expression of cathepsin K protein, a key protease in bone resorption, by five-fold. Northern blot analysis and promoter analysis show that this induction occurs at the transcriptional level, in a dose-responsive and time-dependent manner. No increase in expression occurs in the presence of either pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of NF-kappaB, or Genistein, a protein tyrosine kinase inhibitor, suggesting that IL-1alpha up-regulation may be via the tyrosine kinase-NF-kappaB pathway to regulate cathepsin K expression. Antisense oligonucleotides to p65, but not the p50 subunit of NF-kappaB, suppress the IL-1alpha-induced expression of cathepsin K. We therefore conclude that IL-1alpha up-regulates cathepsin K gene expression at the transcription level, and this regulation may be via the tyrosine-kinase-NF-kappaB pathway.

Comparison in localization between cystatin C and cathepsin K in osteoclasts and other cells in mouse tibia epiphysis by immunolight and immunoelectron microscopy.

We compared the distribution of a cysteine proteinase inhibitor, cystatin C, with that of cathepsin K in osteoclasts of the mouse tibia by immunolight and immunoelectron microscopy. Light microscopically, strong immunoreactivity for cystatin C was found extracellularly along the resorption lacuna and intracellularly in the organelles of osteoclasts. In serial sections, various patterns of cystatin C and cathepsin K localization were seen, specifically: (1) some resorption lacuna were positive for both cystatin C and cathepsin K; (2) others were positive for either cystatin C or cathepsin K, but not both; and (3) some lacuna were negative for both. In osteoclasts, the localization of cystatin C was similar to that of cathepsin K. Furthermore, cystatin C immunoreactivity was detected in preosteoclasts and osteoblasts, whereas cathepsin K was seen only in preosteoclasts. Electron microscopically, cystatin C immunoreactive products were found in the rough endoplasmic reticulum (ER), Golgi apparatus, vesicles, granules, and vacuoles of osteoclasts. These cystatin C-positive vesicles had fused or were in the process of fusion with the ampullar vacuoles (extracellular spaces) containing cystatin C-positive, fragmented, fibril-like structures. The extracellular cystatin C was deposited on and between the cytoplasmic processes of ruffled borders, and on and between type I collagen fibrils. In the basolateral region of osteoclasts, cystatin C-positive vesicles and granules also fused with vacuoles that contained cystatin C-positive or negative fibril-like structures. These results indicate that osteoclasts not only synthesize and secrete cathepsin K from the ruffled border into the bone resorption lacunae, but also a cysteine proteinase inhibitor, cystatin C. Therefore, it is suggested that cystatin C regulates the degradation of bone matrix by cathepsin K, both extracellularly and intracellularly.

Age-dependent changes in the distribution of BrdU- and TUNEL-positive cells in the murine gingival tissue.

BACKGROUND: Age-dependent morphological and cell kinetic changes of the gingival tissue seem to be related to the occurrence of periodontal disease. The purpose of this study was to investigate the age-dependent changes in the distribution of BrdU- and TUNEL-positive cells in murine gingival tissue. METHODS: Gingival tissue of the lower first molar region of 2-, 3-, 5-, 7-, 10-, 15-, 20-, 30-, 40-, 50-, 60-, 70- and 80-week-old mice was used in this study. BrdU- and TUNEL-positive cells were evaluated at the following 4 sites: 1) gingival epithelium (GE); 2) junctional epithelium (JE); 3) submucosal connective tissue of the gingival epithelium (GECT); and 4) submucosal connective tissue of the junctional epithelium (JECT). RESULTS: No significant differences in the mean number of BrdU-positive cells at each site were demonstrated among the various age groups. No significant change in the mean number of TUNEL-positive cells was demonstrated in either the GE or JE groups among the various age groups. Meanwhile, a significant increase in the TUNEL-positive cells was observed in the GECT of mice 40 weeks or older, and in the JECT of mice 20 weeks or older. CONCLUSIONS: These results indicate that no age-dependent change in the cell proliferation or cell death occurred in the gingival and junctional epithelial layers as well as in the cell proliferation in the submucosal connective tissue. Meanwhile, a significant decrease in the cellular component of the submucosal connective tissue of both gingival and junctional epithelial layers caused by apoptosis occurred with aging. The decreased cellular component in the submucosal connective tissue thus seems to be related to either gingival recession or to the apical migration of the JE with aging. These morphological changes with aging possibly occur in humans and may be related to the susceptibility to periodontal disease in aged individuals.

Immunocytochemical study of cathepsin L and rat salivary cystatin-3 in rat osteoclasts treated with E-64 in vivo.

The localization of cathepsin L and rat salivary cystatin-3 (RSC-3) in rat osteoclasts (rat femoral and alveolar bones) treated with or without E-64 (control) was examined immunocytochemically. In osteoclasts pretreated with E-64, immunoreactivity for cathepsin L was very weak extracellularly compared to that in the control osteoclasts. However, it was strong intracellularly. The localization of RSC-3 was unclear in the control osteoclasts, while in E-64 treated osteoclasts, both the clear zone and ruffled border areas showed a very strong immunoreaction. At the electron-microscopic level, in normal osteoclasts, numerous immunoreaction products for cathepsin L were found extracellularly in the bone matrix under the ruffled border, while few intracellular products were observed. In contrast, in the E-64-treated osteoclasts, only a few immunoreaction products were found extracellularly, while intracellularly cathepsin L was found in numerous endosome-lysosomal vacuoles. In the immunoreaction for RSC-3, the cytoplasm of the ruffled border was positive, and the tips of the RSC-3-positive ruffled border appeared to enter deeply into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in the vacuoles (probably autophagolysosomes). Thus, in E-64-treated osteoclasts, inhibition of the extracellular release of cathepsin L was demonstrated. In addition, intralysosomal accumulation of RSC-3 and deep penetration of the RSC-3-positive ruffled border into the bone matrix were found. These findings suggest that RSC-3 is associated with the inhibition of cathepsin L in both the lysosomes (in the osteoclasts) and bone matrix.

Localization of cathepsins B, D, and L in the rat osteoclast by immuno-light and -electron microscopy.

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and -electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabeled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border.(ABSTRACT TRUNCATED AT 250 WORDS)

Numeric approach to the biomechanical analysis of thermal effects in coated implants.

A two-dimensional, finite element method was used to examine the influence of material selection and suprabony exposure of the implant coating on thermal and mechanical stress distribution. Hydroxyapatite coating reduced the heat conduction to the surrounding tissue because of its low thermal conductivity. However, thermal stress resulted from thermal expansion of the hydroxyapatite and titanium core. This might influence the success of hydroxyapatite-coated implants because the biomechanical properties of ceramics are so poor for tensile and shearing stress. In addition, this tendency becomes more pronounced when the hydroxyapatite coating surface extends beyond the cortical bone.

Photodegradation products of levofloxacin in aqueous solution.

The photodegradation of levofloxacin (DR-3355, CAS 100986-85-4), the S-(-)-isomer of ofloxacin, was investigated. Levofloxacin in aqueous solution was exposed to near ultraviolet light (peak wavelength 352 nm) for 16 h at room temperature. Nine degradation products (P-2-P-10) were isolated from the reaction mixture by preparative high performance liquid chromatography. The structures of these compounds were deduced from their NMR, MS, UV and IR spectra and optical rotations. The elucidated structures showed that all of these degradation products were analogues altered at the N-methylpiperazine moiety of levofloxacin.

Distribution of substance P and calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat temporomandibular joint.

The density and distribution of substance P-like immunoreactive (SP-LI) and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers in rat temporomandibular joint (TMJ) were investigated in whole-mount preparations and frozen sections by immunohistochemistry with the avidin-biotin-peroxidase complex method. Both types of immunoreactive nerves were observed primarily in the joint capsule, the peripheral articular disc, the synovial membrane, and the periosteum. The distribution of CGRP-LI nerves was similar to that of SP-LI nerves. The anterior portion of the joint capsule and disc was most densely innervated, followed by the posterior, lateral, and medial portions. In addition, CGRP-LI nerves were more numerous and more dense in immuno-intensity than SP-LI nerves. In the synovial membrane, many SP- and CGRP-LI nerves terminated in the subsynovial layer, but some branches extended into the superficial synovial lining layer close to the joint cavity. Immunolabeled nerves were prominently located in the disc attachment and peripheral portion of the disc, and occasional nerves were located in the dense collagenous disc band as an actual disc. However, no fibers were detected in the central disc band. Thus, most of the disc was not innervated by any nerves. The present study provides a morphological basis for the possible roles of neuropeptides in endocytosis by synoviocytes, regulation of blood flow in the synovial membrane, nociception mechanisms of the TMJ, and modulation of the inflammatory response in the TMJ.

Synthesis and antiallergy activity of [1,3,4]thiadiazolo[3,2-a]-1,2,3-triazolo[4,5-d]pyrimidin-9(3H)-one derivatives. II. 6-Alkyl- and 6-cycloalkylalkyl derivatives.

A series of 6-alkyl- or 6-(cycloalkylalkyl)-[1,3,4]thiadiazolo[3,2- a]-1,2,3-triazolo[4,5-d]pyrimidin-9(3H)-ones 1b--o was synthesized from the corresponding 1,3,4-thiadiazol-5-amines 3b--o and the antiallergic activities of the products were evaluated. Among the compounds 6-(2-cyclohexylethyl)- [1,3,4]thiadiazolo[3,2-a]-1,2,3-triazolo[4,5-d]pyrimidin-9(3H)-one 1h, whose X-ray crystallographic stereostructure is shown, was found to be a promising new antiallergic agent, which has low toxicity and dual activity as a leukotriene D4 receptor antagonist and as an orally active mast cell stabilizer.

Physico-chemical properties of muroctasin.

N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl-N6-stearoyl-L-lysine (MDP-Lys(L18), muroctasin) was studied to clarify its chemical structure and physico-chemical properties. The chemical structure of MDP-Lys(L18) was confirmed by UV, IR, NMR and MS analyses as well as by elemental analysis. The physico-chemical properties were investigated by thermal analysis, powder X-ray analysis, ionization constant measurement, high-performance liquid and thin-layer chromatography.

Studies on the stability of muroctasin and degradation products built under extreme conditions.

Decomposition of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys(L18), muroctasin) under extreme conditions was investigated. MDP-Lys(L18) in an aqueous solution was heated under reflux for 4 h to give the decomposition products D-1, D-2, D-3 and D-4. Reaction of MDP-Lys(L18) with 0.1N NaOH at room temperature for 5 h gave D-1, and that with 1 N HCl under reflux for 1 h afforded D-4. MDP-Lys(L18) was found to be stable to light and heat. MDP-Lys(L18) in a powder form was stored at room temperature or at 25 degrees C and 75% relative humidity for 3 days to 18 months. Quantitative analysis, thin-layer chromatography, and tests for appearance and color change, etc., were performed in the stored samples. MDP-Lys(L18) was found to be stable, but hygroscopic.

Metabolism of budralazine, a new antihypertensive agent, III.

1. [14C]Budralazine (I) administered orally to normotensive and spontaneously hypertensive rats showed no significant difference in metabolic fate between the two groups. 2. Peak plasma levels of 14C were 3.6 microgram equiv. of I per ml in both normotensive and hypertensive rats. Approx. 5% of total 14C in the plasma consisted of I within 4 h, but thereafter the parent drug was not detected. Approx. 15% of 14C in the plasma consisted of 1-hydrazinophthalazine within 16 h after the administration. 3. 45% and 37% of the dose of 14C was excreted into urine and faeces within 24 h, respectively. 4. A high retention of 14C in the aorta wall was found and the time course of 14C in the aorta wall was compatible with that of the reduction in blood pressure. The radioactive materials retained in the aorta wall were likely to be I or 1-hydrazinophthalazine, probably the latter, following macroautoradiography in hypertensive rats.