Benzo[a]pyrene/aryl hydrocarbon receptor signaling inhibits osteoblastic differentiation and collagen synthesis of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Cigarette smoking has detrimental effects on periodontal tissue, and is known to be a risk factor for periodontal disease, including the loss of alveolar bone and ligament tissue. However, the direct effects of cigarette smoking on periodontal tissue remain unclear. Recently, we demonstrated that benzo[a]pyrene (BaP), which is a prototypic member of polycyclic aryl hydrocarbons and forms part of the content of cigarettes, attenuated the expression of extracellular matrix remodeling-related genes in human periodontal ligament (PDL) cells (HPDLCs). Thus, we aimed to examine the effects of BaP on the osteoblastic differentiation and collagen synthesis of HPDLCs. MATERIAL AND METHODS: HPDLCs were obtained from healthy molars of three patients, and quantitative reverse transcription-polymerase chain reaction were performed for gene expression analyses of cytochrome P450 1A1 and 1B1, alkaline phosphatase, bone sialoprotein and aryl hydrocarbon receptor (AhR), a receptor for polycyclic aryl hydrocarbons. We have also analyzed the role of the AhR, using 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), which is an AhR antagonist. RESULTS: The treatment of HPDLCs with BaP reduced mRNA expression of osteogenic genes, alkaline phosphatase activity, mineralization and collagen synthesis. The treatment with CH-223191 subsequently restored the observed suppressive effects of BaP on HPDLCs. CONCLUSIONS: The present results suggest that BaP exerts inhibitory effects on the maintenance of homeostasis in HPDL tissue, such as osteoblastic differentiation and collagen synthesis of HPDLCs, and that this signaling pathway could be suppressed by preventing the transactivity of AhR. Future studies may unveil a role for the inhibition of AhR as a promising therapeutic agent for periodontal disease caused by cigarette smoking.

Mechanical induction of interleukin-11 regulates osteoblastic/cementoblastic differentiation of human periodontal ligament stem/progenitor cells.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is continually exposed to mechanical loading caused by mastication or occlusion. Physiological loading is thus considered a key regulator of PDL tissue homeostasis; however, it remains unclear how this occurs. We recently reported that an appropriate magnitude of mechanical stretch can maintain PDL tissue homeostasis via the renin-angiotensin system. In the present study, we investigated the expression of interleukin-11 (IL-11) in human primary PDL cells (HPDLCs) exposed to stretch loading, the contribution of angiotensin II (Ang II) to this event and the effects of IL-11 on osteoblastic/cementoblastic differentiation of human PDL progenitor cells (cell line 1-17). MATERIAL AND METHODS: Human primary PDL cells, derived from human tissues, with or without antagonists against the Ang II receptors AT1 or AT2, were subjected to cyclical stretch loading with 8% elongation for 1 h. Expression of IL-11 was measured by ELISA in these cultures and by immunohistochemistry in the sectioned maxillae of rats. The osteoblastic/cementoblastic potential of cell line 1-17 was determined using cell proliferation, gene expression and Alizarin Red staining. RESULTS: Positive staining for IL-11 was observed in the PDL of rat maxillae and in cultures of HPDLCs. In HPDLCs exposed to stretch, expression of the IL11 gene and the IL-11 protein were up-regulated, concomitant with an increase in Ang II and via AT2. Recombinant human IL-11 (rhIL-11) stimulated an increase in expression of mRNA for the cementoblast-specific marker, CP-23, and for the osteoblastic markers, osteopontin and bone sialoprotein, and promoted proliferation in cell line 1-17. In addition, rhIL-11 also increased the degree of mineralized nodule formation in cell line 1-17 cultures treated with CaCl2 . CONCLUSION: Mechanical loading appears to control proliferation and osteoblastic/cementoblastic differentiation of human PDL stem/progenitor cells through the regulation of Ang II and AT2 by IL-11.

Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis.

AIM: To characterize the microbial composition of persistent periapical lesions of root filled teeth using a molecular genetics approach. METHODOLOGY: Apical lesion samples were collected from 12 patients (23-80 years old) who visited the Kyushu University Hospital for apicectomy with persistent periapical lesions associated with root filled teeth. DNA was directly extracted from each sample and the microbial composition was comprehensively analysed using clone library analysis of the 16S rRNA gene. Enterococcus faecalis, Candida albicans and specific fimA genotypes of Porphyromonas gingivalis were confirmed using polymerase chain reaction (PCR) analysis with specific primers. RESULTS: Bacteria were detected in all samples, and the dominant findings were P. gingivalis (19.9%), Fusobacterium nucleatum (11.2%) and Propionibacterium acnes (9%). Bacterial diversity was greater in symptomatic lesions than in asymptomatic ones. In addition, the following bacteria or bacterial combinations were characteristic to symptomatic lesions: Prevotella spp., Treponema spp., Peptostreptococcaceae sp. HOT-113, Olsenella uli, Slackia exigua, Selemonas infelix, P. gingivalis with type IV fimA, and a combination of P. gingivalis, F. nucleatum, and Peptostreptococcaceae sp. HOT-113 and predominance of Streptococcus spp. On the other hand, neither Enterococcus faecalis nor C. albicans were detected in any of the samples. CONCLUSION: Whilst a diverse bacterial species were observed in the persistent apical lesions, some characteristic patterns of bacterial community were found in the symptomatic lesions. The diverse variation of community indicates that bacterial combinations as a community may cause persistent inflammation in periapical tissues rather than specific bacterial species.

Cannabidiol improves lung function and inflammation in mice submitted to LPS-induced acute lung injury.

We have previously shown that the prophylactic treatment with cannabidiol (CBD) reduces inflammation in a model of acute lung injury (ALI). In this work we analyzed the effects of the therapeutic treatment with CBD in mice subjected to the model of lipopolysaccharide (LPS)-induced ALI on pulmonary mechanics and inflammation. CBD (20 and 80 mg/kg) was administered (i.p.) to mice 6 h after LPS-induced lung inflammation. One day (24 h) after the induction of inflammation the assessment of pulmonary mechanics and inflammation were analyzed. The results show that CBD decreased total lung resistance and elastance, leukocyte migration into the lungs, myeloperoxidase activity in the lung tissue, protein concentration and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) in the bronchoalveolar lavage supernatant. Thus, we conclude that CBD administered therapeutically, i.e. during an ongoing inflammatory process, has a potent anti-inflammatory effect and also improves the lung function in mice submitted to LPS-induced ALI. Therefore the present and previous data suggest that in the future cannabidiol might become a useful therapeutic tool for the attenuation and treatment of inflammatory lung diseases.

Overcrowding stress decreases macrophage activity and increases Salmonella Enteritidis invasion in broiler chickens.

Overcrowding stress is a reality in the poultry industry. Chickens exposed to long-term stressful situations present a reduction of welfare and immunosuppression. We designed this experiment to analyse the effects from overcrowding stress of 16 birds/m(2) on performance parameters, serum corticosterone levels, the relative weight of the bursa of Fabricius, plasma IgA and IgG levels, intestinal integrity, macrophage activity and experimental Salmonella Enteritidis invasion. The results of this study indicate that overcrowding stress decreased performance parameters, induced enteritis and decreased macrophage activity and the relative bursa weight in broiler chickens. When the chickens were similarly stressed and infected with Salmonella Enteritidis, there was an increase in feed conversion and a decrease in plasma IgG levels in the stressed and Salmonella-infected birds. We observed moderate enteritis throughout the duodenum of chickens stressed and infected with Salmonella. The overcrowding stress decreased the macrophage phagocytosis intensity and increased Salmonella Enteritidis counts in the livers of birds challenged with the pathogenic bacterium. Overcrowding stress via the hypothalamic-pituitary-adrenal axis that is associated with an increase in corticosterone and enteritis might influence the quality of the intestinal immune barrier and the integrity of the small intestine. This effect allowed pathogenic bacteria to migrate through the intestinal mucosa, resulting in inflammatory infiltration and decreased nutrient absorption. The data strengthen the hypothesis that control of the welfare of chickens and avoidance of stress from overcrowding in poultry production are relevant factors for the maintenance of intestinal integrity, performance and decreased susceptibility to Salmonella infection.

An in vitro evaluation of two resin-based sealers on proliferation and differentiation of human periodontal ligament cells.

AIM: To evaluate the effects of a polymethyl methacrylate resin-based sealer [Superbond sealer (SB)] on the proliferation and osteogenic differentiation of human periodontal ligament cells (HPDLCs) in vitro, compared with a methacrylate resin-based sealer [Epiphany SE sealer (EP)]. METHODOLOGY: Human periodontal ligament cells were obtained from of healthy third molar teeth of two participants with informed consent. To determine the effects of the eluent from set resin sealers on HPDLCs, the 7-day-washed (washed) or non-washed freshly prepared (fresh) set SB or EP discs were prepared. Cells cultured on these discs were evaluated by the WST-1 proliferation assay and scanning electron microscopy (SEM). The osteogenic differentiation of HPDLCs on washed SB discs was then evaluated by gene expression analysis of osteopontin (OPN) and osteocalcin (OCN) by using quantitative RT-PCR. RESULTS: Human periodontal ligament cells exhibited growth on washed SB discs, whereas fresh SB and EP discs and washed EP discs inhibited proliferation of HPDLCs. SEM observation revealed that HPDLCs tightly attached and spread on the surface of washed SB discs, whilst no HPDLCs were observed on the surface of fresh and washed EP discs. Furthermore, HPDLCs significantly upregulated gene expressions of OPN and OCN when cultured on washed SB discs in osteogenic differentiation medium for 2 weeks. CONCLUSIONS: Although Superbond sealer initially exerted cytotoxic effects on HPDLCs, these effects were reduced during washing for 7 days compared to EP, which continued to be cytotoxic even though the specimens were washed for the same period of time. Washed Superbond allowed HPDLCs to differentiate into osteogenic cells.

Cytotoxicity of one-step dentin-bonding agents toward dental pulp and odontoblast-like cells.

The purpose of this study was to compare the cytotoxicity of five one-step dentin-bonding agents on human dental pulp and odontoblast-like cells (MDPC-23). Photopolymerized and unpolymerized samples of these dentin-bonding agents were prepared and incubated with dental pulp or MDPC-23 cells. After 24 or 72 h of incubation, the number of unstained cells with trypan blue was counted. The staining of cells with trypan blue stands for a cytotoxicity. The pulp cell and MDPC-23 cytotoxicity of polymerized sample treatment increased in the order of AQ Bond Plus (AQ)

Expression of growth/differentiation factor 11, a new member of the BMP/TGFbeta superfamily during mouse embryogenesis.

We have cloned and characterized a new member of the bone morphogenetic protein/transforming growth factor beta (BMP/TGFbeta) superfamily, growth differentiation factor 11 (Gdf11), from rat incisor pulp RNA by reverse transcription-polymerase chain reaction using degenerate primers. The mature carboxyl-terminal domain encoded by Gdf11 is most closely related to Gdf8, being 90% identical to the mouse gene. Northern blot analysis revealed Gdf11 is expressed in adult dental pulp and brain. In situ hybridization of sections and whole-mount embryos demonstrated Gdf11 is first strongly expressed in restricted domains at 8.5 days post coitus (dpc) when it is highest in the tail bud. At 10.5 dpc, it is expressed in the branchial arches, limb bud, tail bud and posterior dorsal neural tube. Later, it is expressed in terminally-differentiated odontoblasts, the nasal epithelium, retina and specific regions of the brain.

A novel gene, GliH1, with homology to the Gli zinc finger domain not required for mouse development.

The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1-expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with approximately 90% homology to that of GliH1, indicating there is a family of GliH genes and raising the possibility of overlapping functions during development.

The shaping effects of three nickel-titanium rotary instruments in simulated S-shaped canals.

The purpose of this study was to compare the shaping effects of three nickel-titanium rotary instruments, ProTaper, K3, and RaCe, with emphasis on canal transportation. Simulated canals with an S-shaped curvature in clear resin blocks were prepared with a torque-control, low-speed engine. Canals were prepared using the crown-down technique to the size of #30. Canal aberrations were assessed by comparing the pre- and postinstrumentation images under a stereomicroscope. ProTaper instruments caused greater widening of canals compared to K3 or RaCe. Furthermore, ProTaper files showed a tendency to ledge or zip formation at the end-point of preparation. These canal aberrations may be caused by ProTaper finishing files, which appear to be less flexible than other files of the same tip-size, because of their greater taper-size. These results suggest that nickel-titanium file systems including less tapered, more flexible instruments, like K3 and RaCe should be used in the apical preparation of canals with a complicated curvature.

Implanted bone particles induce osteoclast development in the presence of osteoblast lineage cells.

We implanted mineral-containing bone particles (BPs) in rats to investigate the involvement of osteoblast lineage cells in osteoclast development in vivo. BPs were implanted in subcutaneous regions on calvaria or artificial defects of calvaria, with or without adjacent parathyroid glands prepared from other rats. The structural characteristics of multinucleated giant cells (MGCs) induced by the BPs were investigated. The MGCs induced by subcutaneously implanted BPs showed membrane ruffling at the basolateral site, but not at the apical site, regardless of whether parathyroid glands were also implanted. In contrast, the MGCs induced by intraosseously implanted BPs showed the characteristics of osteoclasts, i.e., ruffled borders at the apical sites, clear zone, vacuoles, many mitochondria, and scattered rough endoplasmic reticulum. When BPs and parathyroid glands were implanted in bone defects, the number of MGCs was increased compared to that induced by BPs only. These MGCs showed the typical characteristics of active osteoclasts, including developed ruffled borders and stacks of Golgi succules. The number of osteoclasts was also investigated quantitatively by counting the numbers of MGCs positive and negative for tartrate-resistant acid phosphatase. In intraosseous implantation, MGCs with the characteristics of osteoclasts were observed close to osteoblastic cells characterized by developed rough endoplasmic reticulum. These results indicate that the osteoclasts were not induced solely by the subcutaneously implant BPs, but required osteoblast lineage cells for development.

Trypsinized osteoclast-like multinucleated cells formed in rat bone marrow cultures efficiently form resorption lacunae on dentine.

Rat bone marrow cultures containing 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] formed multinucleated cells (MNCs) that had many characteristics of osteoclasts. These MNCs, which have a tartrate-resistant acid phosphatase (TRAP) activity, could be classified into two morphological types: one type had smooth cellular margins (smooth-margined MNCs) and the other type had irregular spike-like margins (stellate MNCs). When bone marrow cells depleted of authentic osteoclasts were seeded and cultured on dentine slices, only low numbers of resorption lacunae could be detected. However, when preformed MNCs were detached by trypsinization and replated on dentine slices, numerous resorption lacunae were observed by scanning electron microscopy on these slices. Formation of lacunae occurred reproducibly during the five to ten days of culture. We also examined the effect of retinoic acid on TRAP-positive MNC formation in this bone marrow culture system. Although RA inhibited total TRAP-positive MNC formation, it increased the ratio of stellate MNCs to smooth-margined MNC, suggesting that RA may have the ability to regulate the formation of active osteoclasts.

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.

The relationship between water absorption characteristics and the mechanical strength of resin-modified glass-ionomer cements in long-term water storage.

The purpose of this study is to elucidate the water absorption characteristics of resin-modified glass-ionomer cements and to also investigate the relationship between the characteristics and mechanical strength after long-term water storage. The mechanism of water diffusion in these cements is also discussed. Water absorption was measured using a gravimetric analysis for 12 m, while the diffusion coefficient was calculated using Fick's law of diffusion. Water solubility was determined based on the weight of the residue in the immersed water. The compressive and diametral tensile strength were measured at 1, 2, 6, and 12 m. A correlation was observed between the diffusion coefficient and equilibrium water uptake, which thus suggests the water in the cements to diffuse through micro-voids in accordance with the 'Free volumetric theory'. A correlation was seen between the solubility and diffusion coefficient of the cements. The deterioration ratio, defined as the ratio of the strength at 12 m versus that at 1 m, was also calculated. Finally, a negative correlation was observed between the deterioration ratio of the compressive strength and the diffusion coefficients of the cements.

Release profile of antimicrobial agents from alpha-tricalcium phosphate cement.

A new method for treating carious dentine with alpha-tricalcium phosphate (alpha-TCP) dental cement containing antimicrobial agents has been recently introduced. However, the release behavior of antimicrobial agents from this cement has not yet been clarified. The aim of this study is therefore to examine the release profile of the antimicrobial agents from the alpha-TCP cement. Three kinds of antimicrobial agents (metronidazole, cefaclor and ciprofloxacin) were added to two commercially available alpha-TCP cements (new apatite liner type I and type II). The set cements were then immersed in water at 37 degrees C and the released antimicrobial agents and Ca ion were determined at regular intervals for three months. In addition, scanning electron microscopic observations were conducted before and after immersion for three months. The release profile of the cements containing antimicrobial agents varied depending on the types of antimicrobial agents. The incorporation of antimicrobial agents affected the setting reaction of the cements. The release behavior of the drugs also varied depending on the types of the cements. The differences in the release profile between type I and type II cements reflected the structures and compositions of their matrices.

Biocompatibility of tetracalcium phosphate cement when used as a bone substitute.

We evaluated the biocompatibility of tetracalcium phosphate (4CP) cement, made of 4CP powder and 40 wt% copolymer of polyacrylic acid/itaconic acid and 10 wt% citric acid solution. Light and electron microscopic characteristics were studied 3, 10 and 30 d after implantation. Neither inflammation nor foreign-body giant cell reaction was observed in the tissue adjacent to the implanted material. After 30 d, this material was surrounded with newly formed bone. Ultrastructural examination showed that osteogenesis occurred directly on the surface of the material. These findings suggest that this 4CP cement is biocompatible and possesses osteoconductive properties.

Effect of powder grinding on hydroxyapatite formation in a polymeric calcium phosphate cement prepared from tetracalcium phosphate and poly(methyl vinyl ether-maleic acid).

The primary aim of this study was to determine if cements based on poly(methyl vinyl ether-maleic acid) (PMVE-Ma) and tetracalcium phosphate resulted in hydroxyapatite formation. In addition, the mechanical strength of this type of polymeric calcium phosphate cement was evaluated. Cements were prepared by mixing, in a powder/liquid mass ratio of 3.0, an aqueous solution of PMVE-Ma (mass fraction = 25%) and tetracalcium phosphate powders ground for various periods of time. The tetracalcium phosphate powders and set cements were characterized by means of X-ray powder diffraction and scanning electron microscopy. Mechanical strengths of the cements were tested 24 h after mixing. Prolonged grinding of tetracalcium phosphate powder decreased particle size and/or crystallite size and increased lattice distortion. This enhanced the reactivity of the tetracalcium phosphate powder and elevated the extent of crosslinking between PMVE-Ma molecules, resulting in improved mechanical strength. Hydroxyapatite formation was detected in the cement prepared with the most finely ground tetracalcium phosphate powder. The conversion of residual tetracalcium phosphate particles to more thermodynamically stable hydroxyapatite crystals will reduce the solubility of the polymeric cement and increase its biocompatibility.

Biochemical properties of the monomeric mutant of human cathepsin E expressed in Chinese hamster ovary cells: comparison with dimeric forms of the natural and recombinant cathepsin E.

Cathepsin E (CE) is the only known aspartic proteinase that exists as a homodimer consisting of two fully catalytically active monomers, which are covalently bound by a disulfide bond between two cysteine residues at the NH2-terminal region (Cys43 in human pro-CE). To understand the physiological significance of the dimer formation, the monomeric mutant of human CE was constructed by site-directed mutagenesis (Cys43 ->Ser43) and expressed in Chinese hamster ovary (CHO) cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The cellular localization of the monomeric protein was compatible with that of the wild-type (dimeric form) of recombinant human CE expressed in the same cells. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannose-type oligosaccharide chain in the cells. In addition to the maximal activation at around pH 3.5, a substantial proportion of the monomeric pro-CE was converted to the mature form by incubation at pH 7 and 37 degrees C for 5 min. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH7. Although catalytic properties of the in vitro-activated monomeric CE appeared to be indistinguishable from those of the dimeric forms of natural and recombinant CE, the monomeric form was more unstable to pH and temperature changes than these dimeric forms. These results indicate that the dimerization of CE is not necessarily required for proper folding to express activity, correct intracellular localization and carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo.

Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D.

Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2,4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km = 10.9 microM(-1) x s(-1) for cathepsin E and 15.6 microM(-1) x s(-1) for cathepsin D). A very acidic pH optimum o was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km = 12.2 microM(-1) x s(-1) for cathepsin E and 16.3 microM(-1) x s(-1) for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.

Expression of TGF-beta superfamily receptors in dental pulp.

Transforming growth factor-beta (TGF-beta) superfamily members and their cell-surface receptors may play inductive and/or regulatory roles in tooth development and repair. It will be important to identify the complete set of TGF-beta superfamily receptors, to examine their temporal and spatial localization during tooth development, and to elucidate the cascade of molecular events of tooth formation induced by the TGF-beta superfamily. In this report, we have cloned the cDNAs encoding potential receptors for TGF-beta superfamily members in rat incisor pulp and bovine adult pulp which are regarded as embryonic and adult pulp, respectively. We analyzed poly (A)+ RNA from rat incisor pulp and bovine adult pulp by reverse-transcriptase/polymerase chain-reaction (RT-PCR), using a degenerate primers corresponding to the most conserved amino acid sequences in the intracellular serine/threonine kinase of type I or type II like kinase-1 (ALK-1), ALK-2, ALK-3 (bone morphogenetic protein receptor type IA, BMPR-IA), ALK-4 (B1), ALK-5, ALK-6 (BMPR-IB), and BMPR-II (BMP type II receptor) was found to be in dental pulp. Northern blot analysis further detected TGF-beta type II receptor (T beta R-II) mRNA transcript in addition to the above-identified receptors. These results provide the first evidence of multiple type I and type II receptors for TGF-beta s, activins, and BMPs expressed in embryonic and adult pulp, implicating diverse function in tooth development and pulp tissue repair.