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.

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.

BOX DNA: a novel regulatory element related to embryonal carcinoma cell differentiation.

BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of mutant polyomavirus (fPyF9) DNA. We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant (PyhrN2) in F9-28 cells, a subclone of mouse F9 embryonal carcinoma (EC) cells expressing the polyomavirus large T antigen. In this study, we demonstrate that BOX DNA enhances transcription from the thymidine kinase (TK) promoter in various EC cells. One or three copies of BOX DNA, linked to the bacterial chloramphenicol acetyltransferase gene under the control of the herpes simplex virus TK promoter, activated promoter activity in F9, P19, and ECA2 cells. Band shift assays using BOX DNA as a probe revealed that specific binding proteins were present in all EC cells examined; the patterns of BOX DNA-protein complexes were the same among them. A mutation introduced within BOX DNA abolished enhancer activity as well as the formation of specific DNA-protein complexes. In non-EC cells, including L and BALB/3T3 cells, the enhancer activity of BOX DNA on the TK promoter was not observed, although binding proteins specific to the sequence exist. In band shift assays, the patterns of the DNA-protein complexes of either L or BALB/3T3 cells were different from those of EC cells. Furthermore, the enhancer activity of BOX DNA decreased upon differentiation induction in all EC cells examined, of different origins and distinct differentiation ability. In parallel with the loss of enhancer activity, the binding proteins specific for BOX DNA decreased in these cells. Moreover, we cloned a genomic DNA of F9, termed BOXF1, containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene. BOXF1, containing a TATA-like motif and the binding elements for Sp1 and Oct in addition to BOX DNA, possessed promoter activity deduced by a BOXF1-chloramphenicol acetyltransferase construct. Deletion analyses of the construct revealed that the transcription of BOXF1 gene is regulated by BOX DNA, preferentially in undifferentiated EC cells versus differentiated cells. Hence, BOX DNA is probably a novel transcriptional element related to EC cell differentiation.

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.

CA54/61 as a marker for epithelial ovarian cancer.

Using a new one-step, double-determinant enzyme immunoassay, we performed quantitative measurements of a mucin-type glycoprotein antigen (CA54/61) that we recently detected in sera of ovarian carcinoma patients. When the cutoff value was set at 12 units/ml, at which a high diagnostic efficiency was demonstrated [or at 20 units/ml (mean + 3 SD of healthy females)], the positive rates of ovarian serous, mucinous, clear cell, and endometrioid carcinomas were 76% (or 63%), 63% (or 55%), 57% (or 52%), and 50% (or 38%), respectively. Even in mucinous cystadenocarcinoma, more than one-half of the cases were positive, indicating the potential utility of the assay in the diagnosis of mucinous tumors. In sera from patients with benign ovarian tumors, only 9% (or 4%) of the cases were positive, indicating the quite high specificity of this test for ovarian carcinomas. To make a comparison between CA54/61 and CA125, we set the cutoff level of CA125 at 110 units/ml, at which value a high diagnostic efficiency was demonstrated [or at 35 units/ml (mean + 3 SD of healthy females)]. When both CA54/61 and CA125 were assessed in sera from 36 patients with mucinous cystadenocarcinoma, the positive rates of CA54/61 and CA125 were 64% (or 56%) and 36% (or 56%), respectively, suggesting that CA54/61 is of clinical value as a new tumor marker for ovarian cancers, including mucinous tumors.

Protein complexes bearing myc-like antigenicity recognize two distinct DNA sequences.

The products of the myc proto-oncogene family have been suggested to carry functions important for cell proliferation, differentiation and neoplasia, but the molecular mechanisms of such biological effects have not yet been clarified. We have previously reported that the c-myc protein or protein(s) complexed with the c-myc protein bind to a specific sequence in the region upstream of the c-myc gene, where there exist an origin of cellular DNA replication (ori) and also a transcriptional enhancer. It was recently reported that the c-myc protein forms complexes with a novel helix-loop-helix leucine zipper protein (Max), and that the Myc-Max complex specifically recognizes a DNA sequence different from the sequence we have defined in the c-myc gene. In this report we examine the nuclear extract prepared from human Raji cells for binding to the two different sequences which have been reported as specific binding sequences for c-myc protein or c-myc protein complexes. The binding to one sequence was inhibited in the presence of excess amount of the other sequence, suggesting that the same protein(s) may be necessary for binding to either of the two sequences. Since binding to both sequences was cancelled by pretreatment of the Raji extract with an anti-c-myc protein antibody, it is suggested that proteins carrying myc-like antigenicity are necessary for efficient binding of the nucleoprotein complexes to the two distinct DNA sequences.

Identification and cDNA cloning of single-stranded DNA binding proteins that interact with the region upstream of the human c-myc gene.

We have previously reported that a c-myc protein complex binds to the region upstream of the c-myc gene, where exist an origin of cellular DNA replication (ori) and a transcriptional enhancer. Both functions require a 21 bp long sequence, while the c-myc protein complex recognizes a 7 bp consensus therein. It was recently reported that single-stranded DNA binding proteins bound specifically to sequences that play roles in DNA replication or transcription. We examined for proteins binding to the single-stranded DNAs of the 21 bp element (myc(H-P)21). In a band shift assay with HL60 cells nuclear extract, probes of either the plus strand or the minus strand gave rise to specific signals. Mutation introduced within a short consensus (A/TCTA/TA/TT) present in both strands completely abolished binding in either case. Southwestern blotting analysis showed that proteins of molecular weight 105, 80, 50, 45, 40, 39.5 and 14 kDa bound sequence-specifically to either strand and 22 kDa to minus strand to the cognate A/TCTA/TA/TT consensus. These single-stranded DNA binding proteins were named MSSP, c-myc gene single strand binding proteins. We attempted to isolate the cDNAs encoding these proteins by screening a human cDNA library with the plus single-stranded oligonucleotide as a probe. Among several positive clones, we have characterized one, termed MSSP-1. MSSP-1 produced in E. coli as a fusion protein with GST specifically interacted with single-stranded TCTTAT (plus myc(H-P)21) and ACT-ATT (in minus myc(H-P)21), the consensus of which can be referred to as A/TCTA/TA/TT. Sequence analysis of MSSP-1 cDNA revealed that two domains thereof are homologous to the RNA binding motifs common to several ribonucleoproteins. Interestingly, the MSSP-1/GST fusion protein specifically recognized myc(H-P)21 not only in single-stranded but also in double-stranded forms. Binding properties of MSSP-1 imply its functions in DNA replication. Furthermore, when the AT stretch in the SV40 ori core was substituted by TCTTAT, MSSP-1 promoted viral DNA replication depending on the consensus sequences.

c-myc protein complex binds to two sites in human hsp70 promoter region.

We investigated the binding of partially purified (enriched) c-myc protein to the human hsp70 promoter region by band shift and ultraviolet crosslinking assays. In the hsp70 promoter region, two sites were found to be homologous to the c-myc protein complex binding sequence in the c-myc gene. These sites are located at positions -230 and -160 bases relative to the transcription initiation site, overlapping with the region reported for the regulation of hsp70 gene expression by c-myc, and upstream of other regulatory sequences including the heat shock element and the serum responsive element. The results shown here suggest that the c-myc protein complex from human HL-60 cells binds to the two sites of the region directly and sequence specifically. It is therefore suggested that the c-myc protein or protein complex contribute to the regulation of hsp70 gene expression by binding directly to the promoter region.

A major cellular substrate for protein kinases, annexin II, is a DNA-binding protein.

We have screened a human cDNA expression library in lambda gt11 for clones encoding Alu-binding proteins using direct binding of labeled Alu DNA to recombinant phage lysates fixed on a membrane, and isolated a clone 98% identical in sequence to the well-known substrate of protein kinases, annexin II, which was suggested earlier to play a role in transduction of mitogenic signals and DNA replication. A diagnostic property of annexins is their binding to phospholipids in the presence of calcium ions, and we have found that the interaction of proteins of human nuclear extracts with Alu subsequences is suppressed by Ca/phosphatidylserine liposomes, suggesting overlapping of Ca/phospholipid- and DNA-binding domains in annexin II.

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.

Differential effects of divalent cations on specific 3H-GR 65630 binding to 5-HT3 receptors in rat cortical membranes.

We have examined the ability of divalent cations to inhibit 3H-GR 65630 binding to 5-HT3 receptor recognition sites in rat brain cortical membranes. Among the divalent metal cations tested, Cd2+, Zn2+ and Cu2+ inhibited the specific 3H-GR 65630 binding activity to 5-HT3 receptors at a concentration of 0.1-1 mM. The other divalent metal cations tested (i.e. Co2+, Ni2+, Ba2+, Ca2+, Mg2+ and Mn2+) produced no inhibition of the specific 3H-GR 65630 binding. Cd2+, Zn2+ and Cu2+ did not change the Bmax value of the binding activity, but significantly increased the Kd value. It was suggested that these cations inhibited the binding activity by reducing affinity of the 5-HT3 receptor for the antagonist, resulting in apparent inhibition of the binding activity. As to the binding association rate, Cd2+, Cu2+ and Zn2+ were found to have an inhibitory effect. The binding dissociation rate, however, was shown to be decreased by Cu2+ but not by Cd2+ and Zn2+. Furthermore, the Zn(2+)-induced inhibition of 3H-GR 65630 binding was suggested to be antagonized by both concanavalin A and wheatgerm agglutinin. The Cu(2+)-induced inhibition, however, was not influenced by these lectins, indicating that Cu2+ has a different lectin sensitivity for its inhibitory effect. The different mechanism of action between Cu2+ and Zn2+ was suggested in their inhibitory effect on the specific 3H-GR 65630 binding activity.

Serum placental alkaline phosphatase (PLAP) in gynecologic malignancies--with special reference to the combination of PLAP and CA54/61 assay.

Serum placental alkaline phosphatase (PLAP) levels in patients with gynecological tumors were measured by two kinds of enzyme-antigen immunoassay kits provided by Innogenetics (PLAP-I) and Sangtec Medical (PLAP-S), and the combination assays for PLAP with other tumor markers were studied. None of the healthy women studied were PLAP positive, and the positive rate in patients with benign ovarian tumors was less than 6%. The positive rate in patients with ovarian cancers was about 35%, which was higher than the rates for other cancers. It was significantly higher in patients with ovarian serous cystadenocarcinoma (60%). Remarkably high PLAP-I values were observed in patients with dysgerminoma. By the combination assay for PLAP with CA54/61 antigen in ovarian cancers, the diagnostic efficiency increased compared with that for PLAP and CA125. We conclude that PLAP is useful in the diagnosis of ovarian serous cystadenocarcinoma and dysgerminoma and that CA54/61 is the better partner for the combination assay.

[Establishment and characterization of endometrial undifferentiated carcinoma cell line (TMCC-2.U)].

We established new cell line designed TMCC-2.U, which suggested transformation to undifferentiated carcinoma, derived from endometrial clear cell carcinoma cell line (TMCC-2). The monolayer culture cell showed a pavement arrangement and spindle like shape. A rough-endoplasmic reticulum, mitochondria etc. are well developed. But cytoplasmic endocrine granulosa were so poorly, it suggests functional developments are poor. The TMCC-2.U cells were transplanted to nude mice which showed no typical pattern suggested undifferentiated carcinoma. Their chromosome number varied and the mode is 78. Marker chromosome were found frequency. Growth pattern and production of tumor marker are clearly differentiate from TMCC-2. As mensioned above, TMCC-2.U cell line will be very valuable in basic research on mechanism of transformation and effects of patient's serum on hystogenesity.

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.

Type II pneumocytes in pulmonary tumors. Implications for histogenesis.

Bronchioloalveolar carcinomas (n = 45), intravascular bronchiolo-alveolar tumors (n = 8), and sclerosing hemangiomas of the lung (n = 2) were stained for surfactant apoprotein by the immunoperoxidase method. Of the bronchiolo-alveolar carcinomas, 12 were found to be carcinomas of type II pneumocytes and the remaining 33 tumors were designated as nontype II pneumocytic bronchiolo-alveolar carcinomas. Twenty-five of these tumors displayed trapped benign type II pneumocytes within the tumor masses. In the case of intravascular bronchioloalveolar tumors, none of the tumors demonstrated staining of tumor cells for surfactant apoprotein. In the two cases of sclerosing hemangiomas, the type II pneumocytes were exuberant and numerous, and in one case contained intranuclear inclusions of surfactant apoprotein. This article discusses the implication of the presence of benign type II pneumocytes in pulmonary tumors.

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.

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.

[Studies on the excretion of urinary glycylprolyl dipeptidyl-aminopeptidase (GP-DAP) during normal pregnancy].

Several investigators reported that GP-DAP activity, one of dipeptidyl-aminopeptidases, increased in urine of patients with various renal diseases. In this study, we determined urinary GP-DAP and NAG activities of 165 subjects in normal pregnant women. It was clear that urinary GP-DAP was increased less than NAG with the progress of normal pregnancy. In conclusion, it is suggested that urinary GP-DAP may be a better marker than NAG for the discrimination of renal diseases from normal pregnancy.