Gene delivery to periodontal tissue using Bubble liposomes and ultrasound.

BACKGROUND AND OBJECTIVE: Periodontitis is the most common inflammatory disease caused by oral biofilm infection. For efficient periodontal treatment, it is important to enhance the outcome of existing regenerative therapies. The physical action of an ultrasound may be able to deliver a therapeutic gene or drugs into the local area of the periodontium being treated for periodontal regeneration. Previously, we developed "Bubble liposomes" as a useful carrier for gene or drug delivery, and reported that delivery efficiency was increased with high-frequency ultrasound in vitro and in vivo. Hence, the aim of the present study was to examine the possibility of delivering genes into gingival tissues using Bubble liposomes and ultrasound. MATERIAL AND METHODS: We attempted to deliver naked plasmid DNA encoding luciferase or enhanced green fluorescent protein (EGFP) into the lower labial gingiva of Wistar rats using Bubble liposomes, with or without ultrasound exposure. Ultrasound parameters were optimized for intensity (0-4.0 W/cm(2) ) and exposure time (0-120 s) to establish the most efficient conditions for exposure. The efficacy and duration of gene expression in the gingiva were investigated using a luciferase assay and fluorescence microscopy. RESULTS: The strongest relative luciferase activity was observed when rats were treated under the following ultrasound conditions: 2.0 W/cm(2) intensity and 30 s of exposure time. Relative luciferase activity, 1 d after gene delivery, was significantly higher in gingiva treated using Bubble liposomes and ultrasound than in gingiva of the other treatment groups. Histological analysis also showed that distinct EGFP-expressing cells were observed in transfected gingiva when rats were treated under optimized conditions. CONCLUSION: From these results, the combination of Bubble liposomes and ultrasound provides an efficient technique for delivering plasmid DNA into the gingiva. This technique can be applied for the delivery of a variety of therapeutic molecules into target tissue, and may serve as a useful treatment strategy for periodontitis.

Paraneoplastic cerebellar degeneration caused by ovarian clear-cell carcinoma.

Paraneoplastic cerebellar degeneration is a paraneoplastic neurological syndrome caused by the remote effect of certain systemic cancers and is characterized by subacute cerebellar symptoms. A 62-year-old woman suffering from unidentified cerebellar symptoms was admitted to our hospital. Paraneoplastic cerebellar degeneration was suspected and ovarian cancer was detected after the systemic examination for malignancy. The symptoms of vertigo and dysarthria were improved a little after surgical operation and treatments of γ-globulin, steroid pulse and tacrolimus hydrate. The cerebellar symptoms of paraneoplastic cerebellar degeneration are often evident prior to detection of malignancy. It is important to perform systemic examination for malignancy in case of unidentified cerebellar symptoms.

Inwardly rectifying potassium channel Kir4.1 is localized at the calyx endings of vestibular afferents.

Inwardly rectifying potassium (Kir) channel Kir4.1 (also called Kcnj10) is expressed in various cells such as satellite glial cells. It is suggested that these cells would absorb excess accumulated K(+) from intercellular space which is surrounded by these cell membranes expressing Kir4.1. In the vestibular system, loss of Kir4.1 results in selective degeneration of type I hair cells despite normal development of type II hair cells. The mechanisms underlying this developmental disorder have been unclear, because it was thought that Kir4.1 is only expressed in glial cells throughout the entire nervous system. Here, we show that Kir4.1 is expressed not only in glial cells but also in neurons of the mouse vestibular system. In the vestibular ganglion, Kir4.1 mRNA is transcribed in both satellite cells and neuronal somata, whereas Kir4.1 protein is expressed only in satellite cells. On the other hand, in the vestibular sensory epithelia, Kir4.1 protein is localized at the calyx endings of vestibular afferents, which surround type I hair cells. Kir4.1 protein expression in the vestibular sensory epithelia is detected beginning after birth, and its localization gradually adopts a calyceal shape until type I hair cells are mature. Kir4.1 localized at the calyx endings may play a role in the K(+)-buffering action of vestibular afferents surrounding type I hair cells.

Injectable growth/differentiation factor-5-recombinant human collagen composite induces endochondral ossification via Sry-related HMG box 9 (Sox9)expression and angiogenesis in murine calvariae.

BACKGROUND AND OBJECTIVE: The types of collagens available today as biomaterials are purified from animal tissues. A major growing concern, however, is their safety, since there are risks of viral and prion contamination and of unknown and potentially zoonotic infectious diseases. The present study aimed to assess, using immunohistochemistry, the effects of recombinant human growth/differentiation factor-5 (rhGDF-5) combined with recombinant human collagen I (rhCI) on bone formation in murine calvariae. MATERIAL AND METHODS: Composite rhGDF-5-rhCI or rhCI alone was injected subcutaneously into murine calvariae. After 3, 7 or 14 days, tissues were examined radiologically, histologically and immunohistochemically. The production of vascular endothelial growth factor (VEGF) by primary osteoblasts, periosteal cells and connective tissue fibroblasts isolated enzymatically from neonatal murine calvariae was also assessed. RESULTS: A protrusion was observed on the calvariae at the site injected with rhGDF-5/rhCI composite. Its mineral density was shown to be different from that of the existing bone by two-dimensional microcomputed tomography. Type II collagen-positive staining was restricted to newly formed tissues. Thus, the newly formed tissues seemed to be bone- and cartilage-like tissues. A number of vessels with positively stained cells for Von Willebrand factor were detected in the newly formed tissues. The rhGDF-5 enhanced VEGF production in cultured connective tissue fibroblasts. Sry-related HMG box 9 (Sox9)-positive cells were detected in the hypertrophic periosteum, and penetrated into the newly formed tissues. CONCLUSIONS: These results suggest that rhCI seems to allow the release of rhGDF-5 and that rhGDF-5-rhCI composite induces endochondral ossification via Sox9 expression and angiogenesis in murine calvariae.

Gene silencing by the tRNA maturase tRNase ZL under the direction of small-guide RNA.

We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3'-processing endoribonuclease (tRNase Z(L)) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase Z(L) in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase Z(L) method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase Z(L) directed by sgRNA not owing to a simple antisense effect. We also show that tRNase Z(L) together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3beta by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.

Simvastatin decreases IL-6 and IL-8 production in epithelial cells.

Many cardiovascular studies have suggested that 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) have anti-inflammatory effects independent of cholesterol lowering. As a chronic inflammatory disease, periodontitis shares some mechanisms with atherosclerosis. Since oral epithelial cells participate importantly in periodontal inflammation, we measured simvastatin effects on interleukin-6 and interleukin-8 production by cultured human epithelial cell line (KB cells) in response to interleukin-1alpha. Simvastatin decreased production, an effect reversed by adding mevalonate or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate. Simvastatin was found to reduce NF-kappaB and AP-1 promoter activity in KB cells. Dominant-negative Rac1 severely inhibited interleukin-1alpha-induced NF-kappaB and AP-1 promoter activity. Our results may indicate an anti-inflammatory effect of simvastatin on human oral epithelial cells, apparently involving Rac1 GTPase inhibition.

Mercaptan-capturing properties of mushrooms.

Mercaptan-capturing properties of 33 kinds of mushrooms were measured. The mushrooms having a high capturing ability toward methyl mercaptan (MeSH) were Agaricus bisporus, A. campestris, Boletus fraternus, B. subvelutipes, Gyrodon lividus, Leccinum scabrum, Suillus grevillei, Morchella esculenta, Russula nigricans, Hypholoma sublateritium, and Lyophyllum sykosporum. These are liable to change their color when injured. The mixture of their acetone powders, which contain polyphenol oxidases, and phenolic compounds such as tyrosine, gamma-L-glutaminyl-4-hydroxybenzene (GHB), DOPA, variegatic acid, grevillin B and C, and pigments, and fluorescent compounds from H. sublateritium also showed high MeSH-capturing properties. 2,5-Bis(methylthio)-DOPA was isolated from the reaction mixture of tyrosine and MeSH with tyrosinase, and the existence of 2- and 5-methylthio-DOPAs was also suggested. Furthermore, acetone powders from fruits and vegetables oxidized the above diphenolic compounds to bind MeSH.

Sedimentable amyloplasts in starch sheath cells of woody stems of Japanese cherry.

We examined whether sedimentable amyloplasts act as statolith in the perception of gravity in woody stems using the elongated internodes of Japanese cherry (Prunus jamasakura Sieb. ex Koidz.). In the internode of the seedlings grown on earth, amyloplasts were found sedimented at the distal end of each cell of the endodermal starch sheath tissue. In the internode grown on three-dimensional (3-D) clinostat, amyloplasts were dispersed throughout the cell matrix in the endodermal starch sheath tissue. After changing the positions of the internode from vertical to horizontal, re-sedimentation of amyloplasts toward the direction of gravity was completed in 1h, whereas the bending of the internode was observed after 12 days. We propose that sedimentable amyloplasts in the endodermal starch sheath cells may play a role in gravity perception leading to secondary xylem formation in the secondary thickening growth and eccentric growth in gravi-bending of tree stems.

p21Cip-1/SDI-1/WAF-1 gene is involved in chondrogenic differentiation of ATDC5 cells in vitro.

Development of skeletal cartilage is characterized with coupling growth arrest and cell differentiation. Here, to understand the cyclin-dependent kinase inhibitors involved in the progression of chondrogenic differentiation, we examined changes in the expression levels of cyclin-dependent kinase inhibitor members using mouse ATDC5 prechondrocytes as a widely used in vitro model of cartilage differentiation. Up-regulation of p21 and p27 mRNA was observed following a decrease in growth rate of prechondrocytes, and both transcripts subsequently accumulated during chondrogenic differentiation; p15, p18, and p19 mRNA, in contrast, did not change during differentiation. Only the up-regulation of p21 mRNA during differentiation was prevented by the continuous treatment of early chondrogenic inhibitor, parathyroid hormone, indicating a close correlation between differentiation and p21 induction in ATDC5 cells. Therefore, to examine the role of p21 during chondrogenesis, we established stable cell lines overexpressing full-length p21 antisense RNA in ATDC5. The reduction of endogenous p21 in these cell lines caused inhibition of early chondrogenic differentiation in ATDC5, indicating that p21 gene plays an important role in this process of the cells in vitro. Furthermore, the level of p21 protein and p21.CDK2 complexes transiently increased during differentiation, but not in undifferentiated cells, leading to a decrease in CDK2-associated kinase. However, differentiation-dependent expressed p21 protein was degraded by a proteasome-dependent pathway. Thus, the progression of chondrogenic differentiation requires down-regulation of CDK2-associated kinase with an increase in p21 protein and subsequent degradation of this protein by a proteasomal pathway.

Radiotherapy for liposarcoma of the vulva.

BACKGROUND: Primary liposarcoma of the vulva is extremely rare. We report a case of liposarcoma of the vulva which was treated with local excision and postoperative radiotherapy. CASE: A 21-year-old woman complained of a painless lump in her labium majus which she first noticed 3 years earlier. An initial diagnosis of lipoma of the vulva was made. The patient was treated by surgical removal. On examination of the surgical specimen, the final pathological diagnosis was well-differentiated liposarcoma and the stalk was not excised completely. Radiotherapy was initiated because of the uncertainty of the patient's prognosis. Eighteen months after radiotherapy, no evidence of local recurrence, metastasis, and late complication has been seen. CONCLUSION: This case is suggestive of the contribution of postoperative radiotherapy, although not conclusive, and continued monitoring is necessary because recurrence or metastasis can occur years later.

Bidirectional effects of hypergravity on the cell growth and differentiated functions of osteoblast-like ROS17/2.8 cells.

A low level of hypergravity (1.5-2.0 G) stimulated the proliferation of ROS17/2.8 cells, whereas it inhibited the differentiated functions of alkaline phosphatase activity and osteocalcin synthesis. These results were just the opposite of our results obtained when the cells were exposed to a high level of hypergravity (40-80 G): inhibition of cell growth and the stimulation of the differentiated functions. The direction of change in the cAMP contents of the cells was also reversed, with a low level of hypergravity causing a decrease in the cAMP content and a high level of hypergravity an increase in it. Therefore, bidirectional effects of hypergravity on the growth and differentiated functions exist in ROS17/2.8 cells according to the magnitude of the hypergravity.

Multipotency of a bone marrow stromal cell line, TBR31-2, established from ts-SV40 T antigen gene transgenic mice.

Bone marrow is believed to contain multipotential stromal stem cells which can differentiate into osteoblasts, chondrocytes, adipocytes, and myoblasts (Prockop, D. J. Science 276, 71-74, 1997). Therefore, characterization and identification of the stem-like cell within the stromal cells are important to understand bone marrow function in relation to the hematopoietic microenvironment, and repair/regeneration of tissue defects. TBR31-2 cell, a bone marrow stromal cell line established from bone marrow of transgenic mice harboring temperature-sensitive (ts) simian virus (SV) 40T-antigen gene for immortality, is induced toward both adipocytic and osteogenic cells under conditions of the inactivation of T-antigen (Okuyama, R., Yanai, N., Obinata, M. Exp. Cell Res. 218, 424-429, 1995). In this work, using a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, mRNA expressions of tissue-specific differentiation markers for adipocyte (lipoprotein lipase), osteoblast (type I collagen and osteocalcin), chondrocyte (type II and X collagen), and muscle cell (desmin) were examined during a long-term culture of the cell. In addition, histochemical studies showed the appearance of adipocytic, osteoblastic, chondrocytic, and muscle cells during this long-term culture. Thus, TBR31-2, which has characteristics of an undifferentiated cell, has the potential to express the multipotential cell lineages. These results indicated that a multipotential progenitor cell including potential to differentiate into a muscle cell and which is situated in the mesenchymal cell lineage was first obtained.

Establishment of permanent cell lines exhibiting vitamin D-dependent expression of beta-galactosidase activity.

The active hormonal form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), has been described as a principal mediator of skeletal homeostasis. Treatment of rat osteosarcoma (ROS)17/2.8, an osteoblast-like cell line, with 1alpha,25(OH)2D3 results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. We isolated permanent cell lines that were established by transfecting ROS 17/2.8 cells with plasmids consisting of the human osteocalcin gene promoter containing the vitamin D responsive element linked to a bacterial beta-galactosidase gene. In one of many cell lines, especially in clone NK-31, 1alpha,25(OH)2D3 strongly stimulated beta-galactosidase activity. Reverse transcription-polymerase chain reaction analysis also showed endogenous osteocalcin gene expression and beta-galactosidase gene expression in clone NK-31 cells, which paralleled the increase in beta-galactosidase activity. Using a synthetic analogue of 1alpha,25(OH)2D3, 24,24-difluoro-1alpha,25-dihydroxyvitamin D3, we found that the levels of this activity and these gene expressions were nearly parallel to those of 1alpha,25(OH)2D3. 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 at high doses (concentration: 10(-7) M) also induced beta-galactosidase activity in clone NK-31. These cell lines, harboring the plasmid-carrying beta-galactosidase gene under the control of the osteocalcin gene promoter, may contribute to studies on the regulation by 1alpha,25(OH)2D3 or to the development of synthetic analogues of 1alpha,25(OH)2D3.

Growth of Prunus tree stems under simulated microgravity conditions.

Stem growth of Prunus trees under simulated microgravity conditions was examined using a three-dimensional clinostat. The stems elongated with bending under such conditions. Stem elongation and leaf expansion were both promoted, whereas the formation of xylem in the secondary thickening growth was inhibited under the simulated microgravity condition. In secondary xylem, sedimentable amyloplasts were observed in the 1g control. The present results suggest that stem elongation and leaf expansion may be inhibited at 1g, while growth direction and secondary xylem formation depend on a gravity stimulus. A space experiment is expected to advance research on thickening growth in trees. Grant Numbers: 07456073, 1D 215.

Growth of pea epicotyl in low magnetic field: implication for space research.

A magnetic field is an inescapable environmental factor for plants on the earth. However, its impact on plant growth is not well understood. In order to survey how magnetic fields affect plant, Alaska pea seedlings were incubated under low magnetic field (LMF) and also in the normal geo-magnetic environment. Two-day-old etiolated seedlings were incubated in a magnetic shield box and in a control box. Sedimentation of amyloplasts was examined in the epicotyls of seedlings grown under these two conditions. The elongation of epicotyls was promoted by LMF. Elongation was most prominent in the middle part of the epicotyls. Cell elongation and increased osmotic pressure of cell sap were found in the epidermal cells exposed to LMF. When the gravitational environment was 1G, the epicotyls incubated under both LMF and normal geomagnetic field grew straight upward and amyloplasts sedimented similarly. However, under simulated microgravity (clinostat), epicotyl and cell elongation was promoted. Furthermore, the epicotyls bent and amyloplasts were dispersed in the cells in simulated microgravity. The dispersion of amyloplasts may relate to the posture control in epicotyl growth under simulated microgravity generated by 3D clinorotation, since it was not observed under LMF in 1G. Since enhanced elongation of cells was commonly seen both at LMF and in simulated microgravity, all elongation on the 3D-clinostat could result from pseudo-low magnetic field, as a by-product of clinorotation. (i.e., clinostat results could be based on randomization of magnetic field together with randomization of gravity vector.) Our results point to the possible use of space for studies in magnetic biology. With space experiments, the effects of dominant environmental factors, such as gravity on plants, could be neutralized or controlled for to reveal magnetic effects more clearly.

Stimulative effect of high-level hypergravity on differentiated functions of osteoblast-like cells.

The exposure of freshly isolated osteoblasts and osteoblast-like cells to high-level hypergravity caused the inhibition of cell growth, elevation of cAMP content, and the stimulation of differentiated functions such as alkaline phosphatase activity, collagen synthesis, and osteocalcin synthesis. Blockage of elevation of cAMP by SQ22536, an inhibitor of adenylate cyclase, resulted in the inhibition of the hypergravity-stimulated alkaline phosphatase activity, indicating that cAMP is the intracellular mediator of this action of hypergravity. H89, an inhibitor of cAMP-dependent protein kinase (PKA), further inhibited the cell growth that was already inhibited by the hypergravity, and further stimulated the alkaline phosphatase activity that was already stimulated by hypergravity. If cAMP acts through the PKA system, H89 should have blocked the changes in cell function effected by the exposure to hypergravity. Therefore the elevated intracellular cAMP by the exposure of hypergravity caused the changes in cell function by a PKA-independent pathway.

Study of the pharmacokinetics of cefpirome sulphate in the elderly.

OBJECTIVE: To determine the appropriate method of administration of the cephem antibiotic cefpirome sulphate in elderly patients. METHOD: We studied cefpirome's pharmacokinetics in patients with urinary tract infections. Patients received cefpirome sulphate 0.5 g by intravenous drip infusion over 30 mins. RESULTS: Patients with a creatinine clearance rate (Ccr) of 80 ml/min had an AUC of 96.7 microg.h/ml and a T1/2 of 2.36 h, whereas those with Ccr of 40-80 ml/min had an AUC of 172.0 microg.h/ml and a T1/2 of 3.45 h and those with Ccr of < 40 ml/min had an AUC of 152 microg.h/ ml and a T1/2 of 4.86 h. CONCLUSION: These results indicate that decreased kidney function can cause increases in the AUC and T1/2 of cefpirome. Thus in elderly patients and perhaps also in other patients with decreased kidney function, cefpirome should be administered at an initial dose of 0.5 g.

5-HT3 receptor blocking properties of the antiparkinsonian agent, talipexole.

1. Talipexole showed moderate displacement activity of 3H-GR 65630 binding to 5-HT3 receptors in both rat cortical and intestinal membrane fractions with Ki values of 0.35 microM and 0.22 microM, respectively. 2. Bromocriptine failed to displace the binding activity in either experimental system even at a concentration of 10 microM. 3. Both talipexole and tropisetron were found to significantly inhibit 5-HT3 receptor-mediated effects of 5-HT in isolated guinea-pig ileum or atrium; however, the effect of talipexole was weaker than that of tropisetron. 4. Bromocriptine, in contrast, had no antagonistic effects on 5-HT3 receptor-mediated activity in guinea-pig ileum or atrium. 5. It was concluded that talipexole might act as an antagonist on 5-HT3 receptors in both brain and intestinal tissues.