Thirty-year follow-up of a TMD case treated based on the neuromuscular concept.

AIM: Occlusal therapy is employed to alleviate the symptoms of a temporomandibular disorder (TMD) at times. However, the long-term effect of occlusal therapy in the masticatory system is not well understood. This case study aims to present a 30-year follow-up of a TMD case. METHODOLOGY: The patient developed TMD with intermittent closed lock of the left temporomandibular joint (TMJ). Chief complaints included trismus, pain, and noise of the left TMJ during function. The patient's occlusal disharmony was assessed with use of electronic instruments and corrected based on the neuromuscular concept. A minimum-invasive and reversible approach using adhesive occlusal restorations was used. RESULTS: The jaw movement and masticatory muscle activity assessed at the 7- and 23-year follow-ups revealed that the established occlusion was well adapted, and re-established the patient's functional occlusion system. The patient has been free from TMD symptoms with the corrected occlusion for 30 years. CONCLUSIONS: Occlusal reconstruction based on the neuromuscular concept can be stably integrated into the patient's functional occlusion system.

Quantitative analysis of condensation/decondensation status of pDNA in the nuclear sub-domains by QD-FRET.

Recent studies indicate that controlling the nuclear decondensation and intra-nuclear localization of plasmid DNA (pDNA) would result in an increased transfection efficiency. In the present study, we established a technology for imaging the nuclear condensation/decondensation status of pDNA in nuclear subdomains using fluorescence resonance energy transfer (FRET) between quantum dot (QD)-labeled pDNA as donor, and rhodamine-labeled polycations as acceptor. The FRET-occurring pDNA/polycation particle was encapsulated in a nuclear delivery system; a tetra-lamellar multifunctional envelope-type nano device (T-MEND), designed to overcome the endosomal membrane and nuclear membrane via step-wise fusion. Nuclear subdomains (i.e. heterochromatin and euchromatin) were distinguished by Hoechst33342 staining. Thereafter, Z-series of confocal images were captured by confocal laser scanning microscopy. pDNA in condensation/decondensation status in heterochromatin or euchromatin were quantified based on the pixel area of the signals derived from the QD and rhodamine. The results obtained indicate that modulation of the supra-molecular structure of polyrotaxane (DMAE-ss-PRX), a condenser that is cleaved in a reductive environment, conferred euchromatin-preferred decondensation. This represents the first demonstration of the successful control of condensation/decondensation in specific nuclear sub-domain via the use of an artificial DNA condenser.

Construction and analysis of EST libraries of the trans-polyisoprene producing plant, Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.

Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes.

The ABCD1 protein, one of the four ATP-binding cassette (ABC) proteins in subfamily D, is located on the peroxisomal membrane and is involved in the transport of very long chain fatty acid (VLCFA)-CoA into peroxisomes. Its mutation causes X-linked adrenoleukodystophy (X-ALD): an inborn error of peroxisomal ß-oxidation of VLCFA. Whether ABCD1 transports VLCFA-CoA as a CoA ester or free fatty acid is controversial. Recently, Comatose (CTS), a plant homologue of human ABCD1, has been shown to possess acyl-CoA thioesterase (ACOT) activity, and it is suggested that this activity is required for transport of acyl-CoA into peroxisomes. However, the precise transport mechanism is unknown. Here, we expressed human His-tagged ABCD1 in methylotrophic yeast, and characterized its ACOT activity and transport mechanism. The expressed ABCD1 possessed both ATPase and ACOT activities. The ACOT activity of ABCD1 was inhibited by p-chloromercuribenzoic acid (pCMB), a cysteine-reactive compound. Furthermore, we performed a transport assay with ABCD1-containing liposomes using 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled acyl-CoA as the substrate. The results showed that the fatty acid produced from VLCFA-CoA by ABCD1 is transported into liposomes and that ACOT activity is essential during this transport process. We propose a detailed mechanism of VLCFA-CoA transport by ABCD1.

Subcellular localization and lysophospholipase/transacylation activities of human group IVC phospholipase A2 (cPLA2gamma).

cPLA2gamma was identified as an ortholog of cPLA2alpha, which is a key enzyme in eicosanoid production. cPLA2gamma was reported to be located in endoplasmic reticulum (ER) and mitochondria and to have lysophospholipase activity beside phospholipase A2 (PLA2) activity. However, subcellular localization, mechanism of membrane binding, regulation and physiological function have not been fully established. In the present study, we examined the subcellular localization and enzymatic properties of cPLA2gamma with C-terminal FLAG-tag. We found that cPLA2gamma was located not only in ER but also mitochondria even in the absence of the prenylation. Purified recombinant cPLA2gamma catalyzed an acyltransferase reaction from one molecule of lysophosphatidylcholine (LPC) to another, forming phosphatidylcholine (PC). LPC or lysophosphatidylethanolamine acted as acyl donor and acceptor, but lysophosphatidylserine, lysophosphatidylinositol and lysophosphatidic acid (LPA) did not. PC and phosphatidylethanolamine (PE) also acted as weak acyl donors. Reaction conditions changed the balance of lysophospholipase and transacylation activities, with addition of LPA/PA, pH>8, and elevated temperature markedly increasing transacylation activity; this suggests that lysophospholipase/transacylation activities of cPLA2gamma may be regulated by various factors. As lysophospholipids are known to accumulate in ischemia heart and to induce arryhthmia, the cPLA2gamma that is abundant in heart may have a protective role through clearance of lysophospholipids by its transacylation activity.

Intranuclear DNA release is a determinant of transfection activity for a non-viral vector: biocleavable polyrotaxane as a supramolecularly dissociative condenser for efficient intranuclear DNA release.

It has been believed that nuclear gene delivery is the most important process for gene expression, and various non-viral vectors are currently being developed with this assumption. However, some of our earlier studies revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the result of the intranuclear disposition of DNA rather than its delivery to the nucleus (Hama S. et al. (2006), Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Ther., 13, 786-794). Here, we report on some direct evidence that demonstrates the importance of the release of intranuclear DNA on transfection activity. The data show that transfection activity can be substantially enhanced by integrating a multifunctional envelope-type nano device (MEND) and a biocleavable polyrotaxane (DMAE-SS-PRX) as an artificial condenser. Our integration system showed significantly higher transfection activity compared to conventional gene delivery system. Moreover, this system provides a strong support for our hypothesis that intranuclear DNA disposition plays a critical role in gene expression for non-viral vectors.

Carrier-Enhanced Anticancer Efficacy of Sunitinib-Loaded Green Tea-Based Micellar Nanocomplex beyond Tumor-Targeted Delivery.

Although a few nanomedicines have been approved for clinical use in cancer treatment, that recognizes improved patient safety through targeted delivery, their improved efficacy over conventional drugs has remained marginal. One of the typical drawbacks of nanocarriers for cancer therapy is a low drug-loading capacity that leads to insufficient efficacy and requires an increase in dosage and/or frequency of administration, which in turn increases carrier toxicity. In contrast, elevating drug-loading would cause the risk of nanocarrier instability, resulting in low efficacy and off-target toxicity. This intractable drug-to-carrier ratio has imposed constraints on the design and development of nanocarriers. However, if the nanocarrier has intrinsic therapeutic effects, the efficacy would be synergistically augmented with less concern for the drug-to-carrier ratio. Sunitinib-loaded micellar nanocomplex (SU-MNC) was formed using poly(ethylene glycol)-conjugated epigallocatechin-3-O-gallate (PEG-EGCG) as such a carrier. SU-MNC specifically inhibited the vascular endothelial growth factor-induced proliferation of endothelial cells, exhibiting minimal cytotoxicity to normal renal cells. SU-MNC showed enhanced anticancer effects and less toxicity than SU administered orally/intravenously on human renal cell carcinoma-xenografted mice, demonstrating more efficient effects on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors. In comparison, a conventional nanocarrier, SU-loaded polymeric micelle (SU-PM) comprised of PEG-b-poly(lactic acid) (PEG-PLA) copolymer, only reduced toxicity with no elevated efficacy, despite comparable drug-loading and tumor-targeting efficiency to SU-MNC. Improved efficacy of SU-MNC was ascribed to the carrier-drug synergies with the high-performance carrier of PEG-EGCG besides tumor-targeted delivery.

Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis.

Hevea brasiliensis Müll. Arg. is a tree that produces natural rubber, an industrially vital isoprenoid polymer. Biosynthesis of natural rubber is known to take place biochemically by a mevalonate (MVA) pathway, but molecular biological characterization of related genes has been insufficient. From H. brasiliensis, we obtained full-length cDNA of genes encoding all of the enzymes that catalyze the six steps of the MVA pathway. Alignment analysis and phylogenetic analysis revealed that in H. brasiliensis there are three acetyl-CoA acetyltransferase genes, two HMG-CoA synthase (HMGS) genes, and four HMG-CoA reductase (HMGR) genes. Gene expression analysis by type of tissue indicated that MVA pathway genes were highly expressed in latex, as compared to other types of tissue and that HMGS and HMGR, which exist in multiple copies, have different expression patterns. Moreover, these MVA pathway genes in H. brasiliensis were found to complement MVA pathway deletion mutations in yeast.

Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis.

Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.

Supramolecular control of polyplex dissociation and cell transfection: efficacy of amino groups and threading cyclodextrins in biocleavable polyrotaxanes.

A novel strategy for gene delivery using biocleavable polyrotaxanes, in which dimethylaminoethyl-modified alpha-cyclodextrins (DMAE-alpha-CDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with benzyloxycarbonyl-L-tyrosine via disulfide linkages (DMAE-SS-PRX), involves the formation of a stable polyion complex (polyplex) against a counter polyanion and the intracellular plasmid DNA (pDNA) release from the polyplex accompanied by the supramolecular dissociation of DMAE-SS-PRXs. In this study, we prepared biocleavable polyrotaxanes with different numbers of threading alpha-CD and amino (DMAE) groups to enhance the transfection activity of DMAE-SS-PRXs. 29DMAE-alpha18-SS-PRX, in which the numbers of alpha-CD molecules and amino groups were 18 and 29 respectively, exhibited a high transfection activity compared with other PRXs. The transfection activity of DMAE-SS-PRXs seems to be related to the efficacy of pDNA release from those polyplexes, which was controlled by the number of alpha-CD and/or amino groups in the polyrotaxane carrier. Most of the DMAE-SS-PRX polyplexes released the pDNA only in the presence of both 10 mM DTT and of the counter-polyanion, as expected, except for 14DMAE-alpha18-SS-PRX, which released pDNA in the absence of dextran sulfate once the DTT had been added to the polyplex solution. The transfection activity of 14DMAE-alpha18-SS-PRX was significantly lower than that of 29DMAE-alpha18-SS-PRX regardless of the above features. Confocal laser scanning microscopic (CLSM) observation suggested that the specific result for 14DMAE-alpha18-SS-PRX might be due to a premature release of pDNA from the most dissociative 14DMAE-alpha18-SS-PRX polyplex in the cytosol. Therefore, transfection activity seems to be related to an appropriate timing of pDNA release.

Determination of the genome sequence of Porphyromonas gingivalis strain ATCC 33277 and genomic comparison with strain W83 revealed extensive genome rearrangements in P. gingivalis.

The gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of chronic periodontitis. Porphyromonas gingivalis strains have been classified into virulent and less-virulent strains by mouse subcutaneous soft tissue abscess model analysis. Here, we present the whole genome sequence of P. gingivalis ATCC 33277, which is classified as a less-virulent strain. We identified 2090 protein-coding sequences (CDSs), 4 RNA operons, and 53 tRNA genes in the ATCC 33277 genome. By genomic comparison with the virulent strain W83, we identified 461 ATCC 33277-specific and 415 W83-specific CDSs. Extensive genomic rearrangements were observed between the two strains: 175 regions in which genomic rearrangements have occurred were identified. Thirty-five of those genomic rearrangements were inversion or translocation and 140 were simple insertion, deletion, or replacement. Both strains contained large numbers of mobile elements, such as insertion sequences, miniature inverted-repeat transposable elements (MITEs), and conjugative transposons, which are frequently associated with genomic rearrangements. These findings indicate that the mobile genetic elements have been deeply involved in the extensive genome rearrangement of P. gingivalis and the occurrence of many of the strain-specific CDSs. We also describe here a very unique feature of MITE400, which we renamed MITEPgRS (MITE of P. gingivalis with Repeating Sequences).

Improved cell viability of linear polyethylenimine through gamma-cyclodextrin inclusion for effective gene delivery.

A polypseudorotaxane consisting of a linear polyethylenimine with Mn of 22,000 (LPEI22k) and gamma-cyclodextrins (gamma-CDs; LPEI22k/gamma-CD) has been examined as a gene carrier. The polyplex formation with luciferase-encoding plasmid DNA (pDNA), intracellular trafficking of polyplex, cytotoxicity, and transfection efficiency were evaluated by various characteristic methods. LPEI22k/gamma-CD formed a pDNA polyplex at higher N/P ratios than LPEI22k; this suggests that the gamma-CD threading sterically interfered with the polyplex formation. In addition, the zeta potentials of the polyplex significantly decreased due to the reduction in charge density of LPEI22k caused by gamma-CD threading. The cellular uptake of pDNA in the LPEI22k/gamma-CD polyplex was enhanced by free gamma-CDs released from the polyplex that might accelerate the cellular uptake through enhanced membrane affinity. LPEI22k/gamma-CD significantly increased cell viability even at high N/P ratios, and the polyplex showed high transfection efficacy. The low cytotoxicity and high gene expression of LPEI22k/gamma-CD are advantageous to polyplex administration in vivo.

Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei.

This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and alpha-cyclodextrins (alpha-CDs), (iii) synthesis of polyrotaxane and (iv) modification of alpha-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23-32 d.

Biocleavable polyrotaxane-plasmid DNA polyplex for enhanced gene delivery.

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic alpha-cyclodextrins (alpha-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of alpha-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between alpha-CDs and PEG. Transfection of the DMAE-SS-PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and the supramolecular dissociation of the polyrotaxane contributed to the enhanced gene delivery.

Quality of life assessment in patients with implant-supported and resin-bonded fixed prosthesis for bounded edentulous spaces.

OBJECTIVES: Dental implants have become increasingly popular in the prosthetic rehabilitation of patients with bounded edentulous spaces. Oral condition-related quality of life (QOL) levels have rarely been assessed in these patients. MATERIAL AND METHODS: Two groups of subjects with bounded edentulous spaces were studied: an implant-supported fixed prosthesis group (11 patients) and a resin-bonded fixed prosthesis group (33 patients). The two groups were well matched in terms of sex, age, missing units and location of missing units. The patients were requested to answer a self-administered QOL questionnaire with two major subscales - oral condition- and general condition-related QOL scores. The test-retest reliability of each question was pre-examined and found acceptable (mean Spearman rank correlation coefficient was 0.55 +/- 0.16). Mean QOL score differences between the two groups were analyzed by the Mann-Whitney U-test. RESULTS: Mean oral condition-related QOL scores of the implant-supported and resin-bonded fixed prosthesis groups were 87.8 +/- 9.5 and 87.1 +/- 12.3% (P = 0.85), and mean general condition-related QOL scores were 73.8 +/- 14.8 and 71.6 +/- 15.2% (P = 0.95), respectively. No significant QOL differences between the two groups were observed in the two subscales. CONCLUSION: In patients with bounded edentulous spaces, multidimensional QOL levels of patients with an implant-supported fixed prosthesis do not exceed those of patients with a resin-bonded fixed prosthesis in a short follow-up period.