Evaluation of the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems.

Glycyrrhiza uralensis roots used in this study were produced using novel cultivation systems, including artificial hydroponics and artificial hydroponic-field hybrid cultivation. The equivalency between G. uralensis root extracts produced by hydroponics and/or hybrid cultivation and a commercial Glycyrrhiza crude drug were evaluated for both safety and efficacy, and there were no significant differences in terms of mutagenicity on the Ames tests. The levels of cadmium and mercury in both hydroponic roots and crude drugs were less than the limit of quantitation. Arsenic levels were lower in all hydroponic roots than in the crude drug, whereas mean lead levels in the crude drug were not significantly different from those in the hydroponically cultivated G. uralensis roots. Both hydroponic and hybrid-cultivated root extracts showed antiallergic activities against contact hypersensitivity that were similar to those of the crude drug extracts. These study results suggest that hydroponic and hybrid-cultivated roots are equivalent in safety and efficacy to those of commercial crude drugs. Further studies are necessary before the roots are applicable as replacements for the currently available commercial crude drugs produced from wild plant resources.

Electrical detection of biomolecular interactions with metal-insulator-semiconductor diodes.

We report the label-free detection of DNA hybridization using a metal-insulator-semiconductor (MIS) diode or capacitor. Upon immobilization of single-stranded DNA on the gold gate of a MIS capacitor, the capacitance versus voltage characteristics show a significant shift in the direction of negative voltages as expected from the immobilization of negative charges on the gate. The hybridization with the complementary strand gives rise to a further significant shift in the same direction as before, which is consistent with the increase of negative charges on the gate brought about by the hybridization. Fluorescence studies indicate that the immobilization and hybridization of DNA can be electrostatically promoted by electric fields externally applied to the MIS capacitors. The MIS diode detection method is applicable to all biomolecular interactions that affect the surface dipole at the interface between the metal gate and the electrolyte and can be extended to other chemical and biochemical systems such as proteins and cells.

A sensitive method for detecting Porphyromonas gingivalis by polymerase chain reaction and its possible clinical application.

BACKGROUND: It is useful for the clinical diagnosis of periodontitis to monitor the colonization of periodontopathic bacteria in periodontal pockets. In this study, we attempted to establish and possibly identify the clinical application of a sensitive method to detect Porphyromonas gingivalis (P.g.), one of the putative periodontopathic bacteria related to chronic periodontitis. METHODS: Genomic DNA extracted from cultured P.g. 381 and clinically isolated subgingival plaque samples were used as a template of polymerase chain reaction (PCR). We designed primers to amplify the genomic DNA coding 40 kDa outer membrane protein (OMP), one of the unique proteins to all strains of P.g. The efficiency and specificity of amplification were evaluated by agarose gel electrophoresis and subsequent Southern hybridization with a digoxygenin-labeled oligonucleotide probe. RESULTS: Fewer than 100 P.g. bacterial cells in the specimen were reproducibly detected by PCR-hybridization assay. This PCR-hybridization assay was at least 100 times more sensitive than the conventional indirect immunofluorescence assay (IIF). Furthermore, the imaging analysis showed that there is a linear correlation between the strength of the signal and the cell number of P.g. from which the template DNA was extracted semiquantitatively. It is noteworthy that the PCR assay could also be applied to detect P.g. from clinical plaque samples and that it was approximately 100 times more sensitive than a conventional IIF assay. CONCLUSION: The PCR assay established in this study can be a powerful tool to detect P.g. in periodontal pockets and monitor the colonization and/or recolonization of P.g. at the very early phase.

Evaluation of the electroosmotic medium pump system for preparative disk gel electrophoresis.

This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921-1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the "electroosmotic medium pump system." Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step.

Bactericidal activity of a monoclonal antibody against a recombinant 40-kDa outer membrane protein of Porphyromonas gingivalis.

BACKGROUND: We have cloned the gene for a 40-kDa outer membrane protein (40-kDa OMP) from Porphyromonas gingivalis 381. The recombinant (r)40-kDa OMP has become the subject of considerable interest because of its potential role in the development of a vaccine useful for passive immunization. To develop such a vaccine, it is essential to fully understand the functions of anti-r40-kDa OMP antibody in the host defense against P. gingivalis. To that end, we developed a panel of monoclonal antibodies by immunizing mice with purified r40-kDa OMP. The objective of this study was to determine the bactericidal activity on P. gingivalis by the IgG1 monoclonal antibody Pg-ompA2. METHODS: Bacterial growth measurement, a complement-mediated anti-P. gingivalis assay based on [3H]thymidine uptake, and a 14C-release assay were performed to test the bactericidal activity of Pg-ompA2 to P. gingivalis. RESULTS: In the presence of complement, Pg-ompA2 was lethal to P. gingivalis 381 as well as to the more virulent P. gingivalis strains, including ATCC 53977 and W83. Using component-deficient complement, we determined that Pg-ompA2 killed P. gingivalis by activating both the classical and alternative complement pathways. CONCLUSIONS: Pg-ompA2 has an in vitro complement-mediated bactericidal activity to P. gingivalis. Pg-ompA2 may contribute to the development of a local immunotherapy that can be applied in the gingival crevice of a patient with P. gingivalis-related periodontitis, or be a vaccine candidate.

Antisense oligonucleotide of tissue inhibitor of metalloproteinase-1 induces the plasminogen activator activity in periodontal ligament cells.

BACKGROUND: Matrix metalloproteinases (MMPs), produced by both infiltrating and resident cells of the periodontium, play a role in physiologic and pathologic events. It is recognized that an imbalance between activated MMPs and their endogenous inhibitors leads to pathologic breakdown of the extracellular matrix during periodontitis. Although it is known that pro-MMPs are activated by the plasminogen activator (PA)/plasmin system, and that the activated MMPs are inactivated by tissue inhibitor of metalloproteinases (TIMPs), participation of TIMPs in the PA/plasmin system has not been defined. METHODS: We investigated the effects of the antisense oligonucleotide, consisting of a 21-base sequence from the human TIMP-1 gene including the first ATG initiation codon, on PA/plasmin activities in the cultured medium of periodontal ligament (PDL) fibroblastic cells. Antisense or sense oligonucleotides were directly added into cell-cultured medium, and enzyme activities from the PDL cells were measured. RESULTS: Antisense TIMP-1 oligonucleotide specifically stimulated the PA activity dose-dependently. Other oligonucleotides, sense TIMP-1 or antisense TIMP-2, did not affect PA activity in PDL cells. The PA activity increased by antisense TIMP-1 oligonucleotide was due to an increase of urokinase-type PA (uPA) protein, but not that of tissue-type PA by means of immunoblotting. Furthermore, the stimulation of PA activity in the conditioned medium by adding antisense oligonucleotide for TIMP-1 was not due to the decreasing levels of PA inhibitor-1, an inhibitor of PA. CONCLUSIONS: TIMP-1 controls the synthesis of uPA in the PDL cells. Control of the TIMP-uPA system is important in inflammatory periodontal ligament healing.

Opsonophagocytic effect of antibody against recombinant conserved 40-kDa outer membrane protein of Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis is associated with the initiation and progression of adult periodontitis. The outer membrane proteins of the bacteria are potentially important targets for interaction with host defense systems. A 40-kDa outer membrane protein (40-kDa OMP) is conserved among many strains of P. gingivalis. We have cloned the gene for 40-kDa OMP from P. gingivalis 381 and produced a recombinant protein. For the development of recombinant 40-kDa OMP as a component of a vaccine for passive immunization, the elucidation of the roles of the anti-recombinant 40-kDa OMP antibody in the host defense against P. gingivalis is essential. The objective of this study was to determine the opsonic capacity of the antibody for phagocytosis by neutrophils which play a key role in the immune response to microbial infections. METHODS: To test the opsonic activity of a rabbit polyclonal antibody against r40-kDa OMP (r40-kDa OMP Ab) on human neutrophils to phagocytize P. gingivalis, we constructed a reproducible in vitro model of P. gingivalis-neutrophil interaction using the human promyelocytic cell line HL-60. RESULTS: We demonstrated that r40-kDa OMP Ab in the presence of human complement successfully opsonized [3H]-thymidine-labeled P. gingivalis as a target for phagocytosis by HL-60 cells differentiated with dimethyl sulfoxide. The phagocytized bacteria were then intracellularly killed and lysed, and the radioactive degradation debris egested into the culture medium. CONCLUSIONS: We conclude that antibody against r40-kDa OMP has opsonic activity on human neutrophil function for phagocytosis of P. gingivalis. Subgingival bacteria are coated in vivo with immunoglobulin and complement. When the antibody is specific for crevicular bacteria, immunological interactions can be expected in the crevice. Our observations suggest that the anti-recombinant 40-kDa OMP antibody in concert with the crevicular complement may prevent P. gingivalis colonization r40-kDa OMP may contribute to the development of a local immunotherapy when applied to the crevice of a patient with P. gingivalis-related periodontitis which relates to susceptibility for certain systemic diseases such as diabetes mellitus, cardiovascular disease, and preterm labor.

Clinical evaluation of the effect of percutaneous transluminal angioplasty and intra-arterial papaverine infusion for the treatment of vasospasm following aneurysmal subarachnoid hemorrhage.

The clinical efficacy of percutaneous transluminal angioplasty and intra-arterial papaverine infusion for treatment of vasospasm following subarachnoid hemorrhage was investigated. Between 1990 and 1993, 84 patients were treated for cerebral vasospasm in National Defense Medical College Hospital. Angioplasty was performed for asymptomatic vasospasm in 18 patients and for symptomatic vasospasm in 12 patients. Intra-arterial papaverine infusion was performed for asymptomatic vasospasm in 10 patients and for symptomatic vasospasm in four patients. The other 40 patients were treated with standard conservative therapy including hypervolemic and hypertensive hemodilution. The outcomes of these patients were analyzed using the Glasgow Outcome Scale. The outcome tended to be better for patients treated with angioplasty, but not for those treated with papaverine infusion, than for those treated conservatively. Recurrence of vasospasm was more frequent after papaverine infusion than after angioplasty. Undesirable complications such as abrupt development of unconsciousness were experienced during papaverine infusion but not during angioplasty. We conclude that percutaneous transluminal angioplasty is superior to intra-arterial papaverine infusion for prevention and treatment of vasospasm following aneurysmal subarachnoid hemorrhage.

Determination and characterization of the hemagglutinin-associated short motifs found in Porphyromonas gingivalis multiple gene products.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterial species implicated as an important pathogen in the development of adult periodontitis. In our studies of P. gingivalis and ways to protect against periodontal disease, we have prepared the monoclonal antibody mAb-Pg-vc and its recombinant antibody, which are capable of inhibiting the hemagglutinating activity of P. gingivalis (Shibata, Y., Kurihara, K., Takiguchi, H., and Abiko, Y. (1998) Infect. Immun. 66, 2207-2212). To clarify the antigenically related hemagglutinating domains, we attempted to determine the minimum motifs responsible for P. gingivalis hemagglutinin. Initially, the 9-kilobase EcoRI fragment encoding the 130-kDa protein was cloned from the P. gingivalis chromosome using mAb-Pg-vc. Western blot analysis of nested deletion clones, the competition experiments using synthetic peptides, and the binding assay of the phage-displayed peptides using the mAb-Pg-vc allowed us to identify the minimum motifs, PVQNLT. Furthermore, the presence of multi-gene family coding for this epitope was confirmed via Southern blot analysis and PCR using the primers complementary to the domain corresponding to this epitope. It is suggested that the hemagglutinin-associated motif may be PVQNLT and that the gene families specifying this motif found in P. gingivalis chromosome encode many hemagglutinin and/or hemagglutinin-related proteases.

Successful surgical removal of a large arteriovenous malformation in a patient with hemophilia: case report.

OBJECTIVE AND IMPORTANCE: This is the first reported case of the successful surgical removal of a large arteriovenous malformation (AVM) in a patient with hemophilia A. CLINICAL PRESENTATION: A 19-year-old male patient was admitted to our department with intracranial hemorrhage. He had previously been diagnosed with hemophilia A and a cerebral AVM. Carotid angiography revealed a large AVM in the right temporal and parietal lobes. The neurological and neuroradiological findings, especially those of single photon emission computed tomography, identified an area of devitalization around the lesion, which was thought to reduce the risk of new deficits resulting from surgical manipulation. INTERVENTION: We resected the AVM in conjunction with supplemental infusions of Factor VIII before, during, and after the operation. A slight cerebral hemorrhage on the 7th postoperative day was observed despite control with Factor VIII, but the patient was discharged without any new deficits. CONCLUSION: We evaluated and managed all problems of a patient with multiple complications and achieved a medical cure.

Effect of aging on functional changes of periodontal tissue cells.

Although the severity of periodontal disease is known to be affected by age, functional changes of periodontal tissue cells during the aging process are not well characterized. It is important to define how cellular aging affects the progression of periodontal diseases associated with the aging process. In vitro aging of human gingival fibroblast (HGF) and periodontal ligament fibroblast (HPLF) cells was prepared by sequential subcultivations (5 to 6 passages as young, 18 to 20 passages as old). GFs were also prepared from gingiva of Down's syndrome patients and 60-week-old rats. Fetal rat calvarial osteoblasts were prepared by sequential digestion with collagenase. HGF and HPLF cells were treated with lipopolysaccharide (LPS) and cyclic tension force, respectively. Amounts of PGE2, interleukin (IL)-1 beta, IL-6, and plasminogen activator (PA) in conditioned media were measured. Total RNA was extracted, and mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). LPS-stimulated PGE2, IL-1 beta, IL-6, and PA production was increased in "old" HGF compared to younger cells. According to RT-PCR analysis, gene expression of COX-2, IL-1 beta, IL-6, and tissue type (t) PA was higher in old cells than in young cells. Cyclic tension force to HPLF also stimulated phenotypic and gene expression of IL-1 beta, PGE2 (COX-2 gene) and tPA. These findings suggest that aging in both HGF and HPLF may be an important factor in the severity of periodontal disease through higher production of inflammatory mediators in response to both LPS and mechanical stress. In addition, oxygen radical-treated fibronectin (FN) as substratum diminished bone nodule formation by osteoblasts when compared with intact FN. This finding suggests that FN plays an important role in Osteoblast activity and that FN damaged by oxygen radicals during the aging process may be related to less bone formation.

Construction of a functional single-chain variable fragment antibody against hemagglutinin from Porphyromonas gingivalis.

Hemagglutinin is a major glycoprotein of Porphyromonas gingivalis vesicles and likely confers the ability to adsorb and penetrate into host tissue cells. To protect this bacterial invasion, murine monoclonal antibody (MAb) Pg-vc, which inhibited the hemagglutinating activity, was prepared by using P. gingivalis vesicles as an antigen. Western blot analysis revealed that when both MAb Pg-vc and anti-HA-Ag2 antibody raised against the P. gingivalis hemagglutinin adhesin (M. Deslauriers and C. Mouton, Infect. Immun. 60:2791-2799, 1992) were allowed to react with protein blots from P. gingivalis vesicles, a superimposable profile was observed. To obtain a recombinant antibody, cDNAs coding for the variable domains of the L and H chains of MAb Pg-vc were cloned by PCR, and a plasmid specifying a single-chain variable fragment (ScFv) was constructed. Following transformation of Escherichia coli cells, a recombinant ScFv protein was successfully expressed. The immunological properties of this protein were identical to those of the parental murine MAb, specifically recognizing the two proteins (43 and 49 kDa) originating from P. gingivalis vesicles. In addition, the ScFv antibody inhibited the P. gingivalis vesicle-associated hemagglutinating activity. The amino acid sequences deduced from nucleotide sequencing experiments confirmed that variable heavy-chain and variable light-chain regions belonged to VH1 and Vkappa12/13 families, respectively. Since the expression system used in this study can readily provide large quantities of single-chain recombinant antibody, it may be a useful in developing a therapeutic agent for passive immunization in humans.

Contribution of IL-1 beta to the enhancement of Campylobacter rectus lipopolysaccharide-stimulated PGE2 production in old gingival fibroblasts in vitro.

Campylobacter rectus is associated with adult periodontitis. We previously reported that C. rectus lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) production in old cells of human gingival fibroblasts (HGFs) is higher than that in young cells. The present study examined whether an enhancement of C. rectus LPS-stimulated interleukin (IL)-1 beta production in old HGFs contributed to the increased production of PGE2. LPS was prepared from C. rectus ATCC33238. HGFs were established from healthy gingiva in three patients, aged 10-12 years. Cellular aging in culture was determined with increasing doubling. The cultured cells were treated with LPS (0.01-10 micrograms/ml), and the amount of IL-1 beta in the medium was measured after a 24 h incubation. The LPS-stimulated IL-1 beta production in each old cell (corresponding to 57-67% of complete life-span) was increased (1.6-2.6 times) compared to that in the young cells (corresponding to 17-20% of the life-span). The IL-1 beta mRNA synthesis in the presence of LPS in the old cells was higher than that in the young cells. The enhancement of LPS-stimulated PGE2 production was inhibited by anti-IL-1 beta antibody and by IL-1 receptor antagonist. These findings suggest that the greater ability of old cells to produce PGE2 in response to C. rectus LPS is due to their greater level of IL-1 beta.

A human monoclonal antibody which inhibits the coaggregation activity of Porphyromonas gingivalis.

A B-cell line producing a human monoclonal antibody (HuMAb) against a recombinant 40-kDa outer membrane protein (OMP) of Porphyromonas gingivalis was constructed by in vivo immunization of a severe combined immunodeficiency C.B.-17/Icr mouse, which had been injected with human peripheral blood lymphocytes, with recombinant 40-kDa OMP and subsequent Epstein-Barr virus immortalization of B cells isolated from the spleen of the mouse. This HuMAb inhibited coaggregation between P. gingivalis vesicles and Actinomyces naeslundii cells.

H2O2-derived free radicals treated fibronectin substratum reduces the bone nodule formation of rat calvarial osteoblast.

Fibronectin (FN) is involved in various cellular activities such as adhesion, proliferation and migration as a substratum. Since the metabolic turnover of FN is much slower than other cellular components, it may be affected by the oxygen free radicals produced in the aging process. However, the effect of oxygen free radicals on FN as substratum in bone formation has not been well characterized. The objective of this study was to examine the effect on the bone forming activity of osteoblasts using an oxygen free radical treated FN substratum in vitro (H2O2-Cu2+system). SDS-PAGE, Western blotting and immuno-blotting analysis revealed that FN was degradated and/or modified by H2O2-Cu2+ (.OH) treatment. Bone nodule formation per well was examined for total number, total area and area per nodule, which data were then compared between non-coated and FN-coated, and between FN-coated and .OH treated FN-coated. Bone nodule formation in the FN-coated was significantly greater than in the non-coated. Furthermore, bone nodule formation in .OH treated FN-coated was significantly less than that of FN-coated. These findings suggested that FN plays important roles in osteoblast activity and that FN substratum damaged by the oxygen free radicals produced by the aging process may cause decline of bone nodule formation through inhibition of the proliferation, differentiation and calcification processes.

In vitro cellular aging stimulates interleukin-1 beta production in stretched human periodontal-ligament-derived cells.

Although the severity of periodontal disease is known to be affected by host age, the pathological role of aging in periodontal disease, and especially that attributable to trauma from occlusion, has not been well-characterized. Interleukin (IL)-1 beta is a key mediator involved in periodontal diseases, a potent stimulator of bone resorption. Furthermore, it is produced by human periodontal ligament (PDL) cells in response to mechanical stress. To investigate the age-related changes in the biosynthetic capacity of IL-1 beta in PDL cells, we examined the effects of in vitro cellular aging with mechanical stress on IL-1 beta protein and gene expression by human PDL cells. Human PDL cells (young = 5th or 6th passage; old = 18-20th passage) were cultured on flexible-bottomed culture plates, and the cells were deformed at 6 cycles per min at 2 steps of tension force for 1 to 5 days. We found a two-fold increase in IL-1 beta production by old PDL cells subjected to mechanical tension compared with that by young PDL cells, although the constitutive levels of IL-1 beta were similar in both the young and old PDL cells. This increase was tension-dependent. IL- 1 beta mRNA was also detected in both cell types under basal conditions, and its expression was further enhanced by application of mechanical tension by use of reverse-transcription-polymerase chain-reaction (RT-PCR) and in situ hybridization methods. The increase in signal rate was higher in the old cells than in the young cells. IL-1 beta-converting enzyme mRNA remained unchanged. It is possible that a large amount of IL- 1 beta produced by PDL cells from an aged host in response to mechanical force may be positively related to the acceleration of alveolar bone resorption.

Inhibition of a Porphyromonas gingivalis colonizing factor between Actinomyces viscosus ATCC 19246 by monoclonal antibodies against recombinant 40-kDa outer-membrane protein.

1. Porphyromonas gingivalis, an important pathogen in human periodontal disease, aggregates with Actinomyces viscosus ATCC 19246. 2. Monoclonal antibodies (mAbs) against purified recombinant 40-kDa outer-membrane protein (r40-kDa, OMP) of P. gingivalis 381 inhibited its coaggregation with A. viscosus ATCC 19246 in a dose-dependent manner. 3. Five mAb clones against r40-kDa OMP were selected. The isotype of the five was IgG1. 4. Pg-ompA2 inhibited the coaggregation of several strains of P. gingivalis with A. viscosus ATCC 19246 cells.

Effect of tension-force on plasminogen activator activity from human periodontal ligament cells.

The plasminogen activator (PA)-plasmin proteolytic system has recently received considerable attention because of its participation in a wide variety of biological activities and in pathological conditions involving tissue destruction. Excessive mechanical stress such as occlusal trauma is associated with alveolar bone loss in severe periodontitis. Therefore, mechanical stress may involve degradation of the extracellular matrix by occlusal trauma through activation of the PA-plasmin proteolytic system. We examined the effects of mechanical stress on PA activity, gene expressions of tissue type (t) PA, urokinase type (u) PA and PA inhibitor-1 (PAI-1) in human PDL cells. Human PDL cells were cultured on flexible-bottomed culture plates and placed on a Flexercell Strain Unit. The cells were flexed at 6 cycles (5 s strain, 5 s relaxation) at 9% and 18% elongation for 5 d. Application of tension-force induced significantly higher PA activity in stressed PDL cells than in non-stressed controls, and did so in a time- and magnitude-dependent manner (p < 0.001, ANOVA). Western-blot analysis revealed that the high level of activity was due to tPA and not uPA. Gene expression of tPA mRNA in stressed PDL cells, as examined by RT-PCR, increased on d 5. These findings suggest that tPA may be involved in periodontal metabolism in response to mechanical stress.

Pontine glioma with osteoblastic skeletal metastases in a child.

BACKGROUND: The development of systemic metastases from primary intracranial gliomas is rare. We report here a rare case of pontine glioma with osteoblastic skeletal metastases. CASE: This 12-year-old boy presented with a 4-month history of hoarseness, dysphagia, and a progressively ataxic gait. Cranial computed tomography (CT) and magnetic resonance imaging (MRI) revealed a brain stem tumor that was diagnosed as a low grade glioma by stereotactic biopsy. Twelve months later following chemotherapy and radiotherapy, neurologic examination and neuroradiologic studies disclosed a recurrence of the pontine glioma. Skeletal roentgenograms revealed widespread osteoblastic metastases in the skull, vertebral bodies, pelvis, and long bones. A specimen from the iliac bone demonstrated cells that were immunoreactive glial fibrillary acidic protein (GFAP). DISCUSSION: The mechanism of how glioma cells determine their biologic behavior at bony metastatic sites is not known. Infratentorial gliomas, which occur frequently in young patients and demonstrate active bony metabolism, may stimulate osteoblastic cells, and induce osteoblastic changes.

Effects of in vitro cellular aging on alkaline phosphatase, cathepsin activities and collagen secretion of human periodontal ligament derived cells.

It is believed that the degree of periodontal tissue breakdown and tooth loss increase with age. In periodontal tissues which are gingiva, periodontal ligament (PL), alveolar bone and tooth cementum, the PL which is soft connective tissue, lies between the tooth cementum and alveolar bone, having the primary function of tooth support, and maintaining the homeostasis of supporting tissues, as well as providing the healing process. We therefore investigated the effects of in vitro cellular aging on alkaline phosphatase (ALP), cathepsin activities and collagen secretion from human PL cells obtained from 18-23 year-old patients' teeth. ALP, cathepsin activities and collagen secretion may play important roles in the remodeling and maintaining of periodontal tissues. To investigate the life span of PL cells, the cells were sequentially subcultivated. The maximum population doubling level of the PL cells in the present experiment was 22-25 passages. Investigating some important biological activities of the PL cells at different passage levels (6-7, 30% of life span to 17-20, 75% of life span), ALP activity and collagen secretion were found to have significantly decreased while cathepsin B and L activities significantly increased with cellular aging. Since these biological activities in human PL cells tend to be more catabolic with increase in cellular aging, the increase in periodontal breakdown with age may be partly related to the catabolic changes of the PL cells themselves.