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Biomedical subjects

Y Takakura

Publications and source records attributed to Y Takakura.

At least 127 records · Page 7Linked to original sources

Long-term results of Watson-Jones tenodesis of the ankle. Clinical and radiographic findings after ten to eighteen years of follow-up.

Thirty-seven chronically unstable ankles in thirty-six patients were operated on with use of a Watson-Jones tenodesis. Thirty-four ankles (thirty-three patients) were followed for a mean duration of thirteen years and eight months (range, ten to eighteen years) after the operation. There were nine male and twenty-four female patients. The mean age of the patients was thirty-one years (range, fourteen to fifty-seven years) at the time of the operation and forty-four years (range, twenty-eight to seventy years) at the time of the latest follow-up. At the time of the most recent follow-up evaluation, twenty-seven patients (twenty-eight ankles) were examined directly by one of us and twenty-five patients (twenty-six ankles) also were evaluated radiographically. The other six patients were interviewed, with use of a questionnaire, by telephone. Of the thirty-four ankles, nineteen had an excellent result (grade 1), eleven had a good result (grade 2), three had a fair result (grade 3), and one had a poor result (grade 4) according to the rating system of Good et al. The mean score (and standard deviation) on the ankle-hindfoot scale of the American Orthopaedic Foot and Ankle Society for the twenty-eight ankles that were examined directly by one of us was 90 +/- 9.3 points (range, 68 to 100 points). Progression of an exostosis at the edge of the joint was detected in eighteen (69 percent) of the twenty-six ankles that were examined radiographically, but narrowing of the joint space was not seen in any ankle. No relationship was detected between the clinical results and radiographic osteoarthrotic changes or the duration of follow-up. The results did not deteriorate over the long term.

Adolescent↗

Biological effects and cellular uptake of c-myc antisense oligonucleotides and their cationic liposome complexes.

The biological effects and cellular uptake of human c-myc antisense oligonucleotides and their liposome complexes were investigated in vitro using human promonocytic leukemia U937 cells. Antisense phosphorothioate oligonucleotides (S-Oligo) significantly inhibited the growth of U937 cells in a dose-dependent manner. However, no significant effect on cell proliferation was observed with unmodified phosphodiester (P-Oligo) and partially phosphorothioated (PS3-Oligo) oligonucleotides with an antisense sequence and S-Oligo with sense and G-quartet control sequences. In cellular uptake experiments, radiolabeled S-Oligo was taken up by U937 cells more than P-Oligo and PS3-Oligo. Similar results were obtained in mouse peritoneal macrophages used for comparison. Confocal microscopic studies demonstrated a significant distribution of FITC-labeled oligonucleotides on the cell surface and in the cytoplasm in a punctate pattern, but not in the nucleus. When complexed with cationic liposomes, cellular uptake of FITC-labeled P-Oligo or S-Oligo was significantly increased and the fluorescence was located mainly in the nucleus, indicating that the uptake and intracellular pharmacokinetics of both oligonucleotides can be modified by complexation. An inhibitory effect of the complexes was observed at a dose which is ineffective in the case of the oligonucleotides alone. However, this effect was also associated with cytotoxicity of the cationic liposomes, suggesting that optimization of this formulation will be necessary to achieve a more efficient delivery of the oligonucleotides to U937 cells.

Animals↗

Disposition characteristics of glycosylated poly(amino acids) as liver cell-specific drug carrier.

Potentials of glycosylated poly(amino acids) as carriers of drugs and/or polynucleotides to the liver were studied in mice in detail. Poly-L-glutamic acid (PLGA) and poly-L-lysine (PLL) were selected as carrier backbones and modified with 2-imino-2-methoxyethyl 1-thiogalactoside or mannoside to obtain galactosylated and mannosylated derivatives. After intravenous injection in mice at a dose of 1 mg/kg, Gal-PLGA and Man-PLGA were selectively taken up by the liver parenchymal cells (PC) and liver nonparenchymal cells (NPC), respectively. Moreover, the uptake of Gal-PLGA and Man-PLGA by the liver were significantly inhibited by the presence of Gal-BSA and Man-BSA, respectively. On the other hand, PLL was targeted to the liver without glycosylation. However, Gal-PLL and Man-PLL showed higher accumulation in the liver than unmodified PLL. Although Gal-PLL and Man-PLL showed different distribution between PC and NPC from that of PLL, the effect of the modifications was less pronounced than the cases of PLGA derivatives. In addition, these glycosylated poly(amino acids) investigated, regardless of the type of amino acid or the sugar grafted, were rapidly degraded to be eluted in the low molecular weight fractions in a gel filtration chromatography. These results suggest that glycosylated PLGAs can be useful carriers of low molecular drugs to the liver cells through conjugation, while glycosylated PLLs could be targetable carriers to the cells after the reduction of their cationic charge by complex formation with polynucleotides.

Animals↗

[Drug delivery systems in gene therapy].

The use of nonviral vectors is an attractive in vivo gene delivery strategy that is simpler and lacks some risks inherent in viral systems. Liposomes and receptor-mediated polycation systems are promising carriers for delivery and expression of plasmid DNA encoding genes into the target cells. Many barriers need to be overcome for successful in vivo DNA delivery using these carrier systems. Various factors such as the extent of DNA condensation, particle size of the DNA complex, route of administration, stability against nucleases, target sites, in vivo disposition, binding to cell surface receptor and internalization, intracellular trafficking affect the in vivo gene delivery and expression. This chapter will focus on the current status and perspectives of the plasmid DNA delivery systems for in vivo gene therapy.

DNA↗

Targeted delivery of plasmid DNA to hepatocytes in vivo: optimization of the pharmacokinetics of plasmid DNA/galactosylated poly(L-lysine) complexes by controlling their physicochemical properties.

In vivo receptor-mediated targeting of plasmid DNA to hepatocytes was achieved through optimizing the physicochemical and pharmacokinetic properties of a plasmid DNA/carrier complex. Galactosylated poly(L-lysine) (Gal-PLL) was synthesized using PLL with a molecular weight of 1,800, 13,000 or 29,000 without loss of the cationic charge. Plasmid DNA encoding chloramphenicol acetyltransferase was complexed with each Gal-PLL. A larger amount of PLL1800 is required for the complex formation than with PLL13000 and PLL29000, and increasing the number of galactose units on Gal-PLL resulted in reduced binding to plasmid DNA. The particle size and zeta-potential of the complexes varied depending on the mixing ratio and Gal-PLL used. Then, plasmid DNA/Gal-PLL complexes having diameters of 200 nm or less and a weak negative charge were prepared. After i.v. injection of [32P]plasmid DNA/Gal13-PLL13000 and [32P]plasmid DNA/Gal26-PLL29000, almost 80% of the radioactivity rapidly accumulated in the liver, preferentially in the parenchymal cells. The hepatic uptake clearances (CLliver) were much greater than any of the other tissue uptake clearances. Compared with these complexes, [32P]plasmid DNA/Gal5-PLL1800 and [32P]plasmid DNA/Gal5-PLL13000 had a smaller CLliver, suggesting that both the molecular weight of PLL and the degree of galactose modification determine the hepatic targeting of plasmid DNA. In vitro and in vivo gene expression studies revealed that plasmid DNA/Gal13-PLL13000 and plasmid DNA/Gal26-PLL29000 complexes are superior to plasmid DNA/Gal5-PLL1800 complex for introducing DNA into cells. These results demonstrated that an optimal design of a DNA/carrier complex based on physicochemical properties and a pharmacokinetic analysis of the distribution properties leads to successful in vivo gene delivery.

Animals↗

Intratumoral pharmacokinetics and in vivo gene expression of naked plasmid DNA and its cationic liposome complexes after direct gene transfer.

The pharmacokinetic properties and gene expression of naked plasmid DNA and its cationic liposome complexes were studied after direct intratumoral injection. Using a Walker 256 tissue-isolated tumor perfusion system, we quantified the recovery of naked plasmid DNA and cationic liposome complexes in the tumor, leakage from the tumor surface, and the venous outflow after intratumoral injection. Approximately 50% of naked plasmid DNA had been eliminated from the tumor 2 h after injection, whereas more than 90% of plasmid DNA was retained in the tumor when it was complexed with cationic liposomes. However, the distribution of these complexes in the tumor was restricted to the tissue surrounding the injection site. Pharmacokinetic analysis of the venous outflow profiles suggested that the rate-limiting process that determines the retention of plasmid DNA in the tumor is transferred from the injection site in the tumor tissue and that complexation with cationic liposomes may retard this process. Furthermore, we examined the gene expression of chloramphenicol acetyltransferase DNA constructs (naked pCMV-CAT) and the corresponding cationic liposome [3-beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol] complexes. A similar level of gene expression was observed in vivo after direct intratumoral injection of naked DNA and its cationic liposome complexes. In both cases, a great variation was observed between tumors, and localization of gene-transduced cells in the tumor tissue was limited to the area in the vicinity of the injection site. Thus, these pharmacokinetic and gene expression studies have demonstrated that cationic liposomes can enhance the retention of injected DNA in the tumor model, whereas cationic liposome complex does not necessarily improve gene expression because of its poor dissemination in this tumor. The present study also suggested that there is a need to control the behavior of the injected naked plasmid DNA and its cationic liposome complexes to ensure better distribution throughout the tumor.

Animals↗

Augmented inhibitory effect of superoxide dismutase on superoxide anion release from macrophages by direct cationization.

Superoxide dismutase (SOD) was modified into cationized form (Cat-SOD) in order to enhance its pharmacological efficacy based on an electrostatic interaction. The inhibitory effect of Cat-SOD on superoxide anion release from inflammatory macrophages and its cellular interaction were studied in vitro. Cat-SOD exhibited an excellent inhibitory effect on superoxide anion release from the macrophages, and this effect surpassed those of native SOD and SOD modified with mannose (Man-SOD) which is taken up via mannose receptor-mediated endocytosis by macrophages. In the presence of colchicine, a microtubule-disruptive agent, the inhibitory effect of Cat-SOD was slightly impaired, whereas the effect of Man-SOD completely disappeared. The intracellular localization of fluorescein isothiocyanate-labeled SOD, Cat-SOD and Man-SOD observed by confocal laser microscopy supported the difference in their abilities to eliminate superoxide anions. The different sensitivities of Cat-SOD and Man-SOD to colchicine were also confirmed by the confocal laser microscopic images, suggesting their distinct intracellular trafficking pathways in the macrophages. In conclusion, Cat-SOD is desirable for its pharmacological activity, which is probably the result of its ability to be delivered to the vicinity of NADPH-oxidase which locates in the cell membrane and generates superoxide anions.

Animals↗

Physicochemical and disposition characteristics of antisense oligonucleotides complexed with glycosylated poly(L-lysine).

The disposition characteristics of a 20 mer antisense phosphodiester oligonucleotide (PO) and its fully phosphorothioated derivative (PS) alone or complexed with glycosylated poly(L-lysine) (galactosylated polylysine, Gal-PLL; mannosylated polylysine, Man-PLL) were studied in mice in relation to their physicochemical characteristics. Good complex formation was obtained at a ratio of 1:0.6, w/w [oligonucleotides (ODNs)/carrier]. The 1:0.6 weight ratio of ODNs/Gal-PLL and ODNs/Man-PLL complexes had zeta potentials of -27 to -31 mV and mean particle size of 100 to 160 nm. After intravenous injection, 35S-labeled ODNs were eliminated rapidly from the circulation; however, their organ disposition characteristics depended on their type. Complex formation with glycosylated PLL increased the hepatic uptake and decreased the urinary clearance of these ODNs to a great extent. These complexes were taken up by both liver parenchymal cells (PC) and nonparenchymal cells (NPC). However, ODNs/Gal-PLL complexes showed a fairly high PC concentration, whereas ODNs/Man-PLL complexes distributed equally to both PC and NPC. The hepatic uptakes of PS/Gal-PLL and PS/Man-PLL complexes were partially inhibited by prior administration of Gal-BSA and Man-BSA, respectively, suggesting their hepatic uptake via the respective receptor-mediated endocytosis. However, uptake by galactose receptors of Kupffer cells, zeta potential, particle size, and Kupffer cell phagocytosis also seem to influence their uptake process. In conclusion, this study illustrates that ODNs can be delivered to hepatocytes and macrophages via galactose and mannose receptors, respectively.

Animals↗

A physiological role of interferon (IFN)-beta derived from tumor: tumor growth of a mouse bladder carcinoma line MBT-2 is partially suppressed by autocrine IFN-beta.

Although some tumor cells endogenously produce a wide variety of cytokines, their physiological roles remain to be fully understood. In this study, we found that mouse subcutaneous tumor induced by inoculation of bladder tumor MBT-2 cells into syngeneic mice secreted a significant amount of interferon (IFN), whereas the cells exhibited no IFN production in in vitro cell culture. Typing experiment using IFN-specific neutralizing antibodies showed that the tumor-derived IFN was exclusively beta type. Since the MBT-2 tumor tissues were homogenous and not infiltrated by immune cells, MBT-2 cells themselves were considered to be IFN-beta producers. By intraperitoneal injection of neutralizing anti-IFN-beta antibodies into MBT-2 cell-inoculated mice, the tumor growth was substantially precipitated and survival days of the tumor-bearing mice were shortened. As the in vitro cell growth of MBT-2 cells was dose-dependently inhibited by IFN-beta, it was suggested that apparent immunogenicity of MBT-2 tumor is partially mediated by tumor suppression by autocrine IFN-beta.

Animals↗

Effects of skin metabolism on percutaneous penetration of lipophilic drugs.

Effects of skin metabolism on percutaneous penetration of drugs with high lipophilicity were studied in vitro using rat skin pretreated with and without an esterase inhibitor, diisopropyl phosphorofluoridate [also known as diisopropyl fluorophosphate (DFP)]. Without DFP, about 96% of the total penetrated amount appeared as metabolized p-hydroxybenzoic acid in the receptor fluid after application of butylparaben, whereas about 30% penetrated as intact form after application of propylparaben. On the other hand, metabolized p-hydroxybenzoic acid was not defected in the receptor fluid under pretreatment with DFP. DFP significantly decreased (p < 0.05) the total amount that penetrated after application of butylparaben, but it did not significantly affect that of propylparaben. The results indicate that skin metabolism directly affects total amount that penetrated in the case of highly lipophilic drugs, and it was found that the higher metabolic rate to hydrophilic drugs is, the greater the amount that penetrated the skin would be. Thus, when optimal prodrugs are designed for the purpose of enhancing percutaneous penetration, we propose that the bioconversion rate to parent drugs as well as the lipophilicity of prodrugs becomes an important consideration.

Animals↗

In vivo evaluation of acyclovir prodrug penetration and metabolism through rat skin using a diffusion/bioconversion model.

PURPOSE: In order to evaluate the in vivo penetration of prodrugs which undergo metabolism in skin, we analyzed the in vivo penetration profiles of acyclovir prodrugs based on a two-layer skin diffusion model in consideration of metabolic process. METHODS: Acyclovir prodrugs (e.g., valerate, isovalerate and pivarate) were used as model prodrugs and the amounts excreted in urine were measured after percutaneous application. In vivo penetration profiles were then estimated by employing a deconvolution method and the penetration of acyclovir prodrugs was analyzed using a diffusion model. Subsequently, diffusion, partitioning and metabolic parameters were compared under in vitro and in vivo conditions. RESULTS: Although total penetration amounts at the end of the experiment were similar for the three prodrugs, the ratio of intact prodrug to total penetration amount differed significantly. Moreover, the excretion and absorption profiles were also very different for each prodrug. Enzymatic hydrolysis rate constants calculated under in vivo conditions were considerably larger than those obtained in the skin homogenate and in vitro penetration experiments. CONCLUSIONS: The present skin diffusion/bioconversion model combined with computer analysis enables us to comprehensively account for diffusion, partitioning and metabolism during in vivo percutaneous absorption. Nevertheless, different enzymatic hydrolysis rate constants obtained under both in vivo and in vitro conditions demonstrate the difficulty of obtaining accurate values for in vivo enzymatic activity from related in vitro experiments.

Acyclovir↗

Renal disposition of recombinant human interleukin-11 in the isolated perfused rat kidney.

PURPOSE: To clarify the mechanism of the renal clearance of recombinant human interleukin- 11 (rhIL- 1), we investigated the renal disposition characteristics of rhIL-11 in the perfused rat kidney. METHODS: The disposition characteristics of (111)In-labeled rhIL-11 were analyzed using a single-pass indicator dilution technique and statistical moment analysis in the perfused rat kidney under filtering and nonfiltering conditions. RESULTS: Steady-state distribution volume (Vd) calculated from the venous outflow patterns of rhIL-11 at the doses of 0.3 to 10 microg/kidney was between 0.35 and 0.40 ml/g kidney. However, Vd at the highest dose decreased to a value almost identical to that of bovine serum albumin, suggesting that there is a reversible and saturable interaction between the capillary wall and rhIL-11 molecule. In filtering kidney, a remarkable accumulation of rhIL-11 was observed while its urinary excretion was highly restricted at all doses. In nonfiltering kidney, rhIL-11 showed a decreased but still significant renal uptake. Taken together, the marked renal uptake of rhIL-11 may be explained by both efficient tubular reabsorption and significant uptake from the capillary side. These processes were not saturable within the tested dose range. These characteristics of rhIL-11 are likely based on non-specific electrostatic interaction with the tissues due to its cationic charge in the cytokine. CONCLUSIONS: The renal disposition processes of rhIL-11 were clarified at organ level in a quantitative manner. These findings agree well with previous observations in an in vivo disposition study in mice.

Animals↗

Non-polarized secretion of mouse interferon-beta from gene-transferred human intestinal Caco-2 cells.

PURPOSE: The intestinal epithelium is considered to be a feasible target for somatic gene therapy. To this end, Caco-2 cells derived from human colon carcinoma were transfected with a mouse interferon-beta (IFN-beta) expression vector and several stable sublines were established; this hetero-specific cytokine allows unexpected cellular effects to be avoided. Using the highest mouse IFN-beta-producing sublines, the mode of IFN secretion was examined. METHODS: The secretion polarity of mouse IFN-beta in its gene-transduced Caco-2 sublines was studied in a bicameral culture system in which the chambers were separated by microporous filters. RESULTS: Mouse IFN-beta was secreted to the same extent from both apical and basolateral surfaces of the transduced cells regardless of cell aging. CONCLUSIONS: These results suggest that in the intestinal epithelium exogenous gene products such as IFNs can be delivered to both the luminal and blood sides in vivo. Thus, the intestinal epithelium may be suitable for systemic and local delivery of therapeutic proteins by gene transfer.

Animals↗

Radiographic analysis of hallux valgus in women on weightbearing and nonweightbearing.

To investigate changes in the radiographic appearance during weightbearing in hallux valgus and to examine the importance of a weightbearing view, a comparative study was performed of accurate dorsoplantar radiographs of 108 symptomatic hallux valgus feet in 73 female patients and 100 normal feet in 69 females, which were taken weightbearing and nonweightbearing. The hallux valgus angle in the hallux valgus group increased on weightbearing, whereas that in the normal group decreased. The intermetatarsal angle between the first and second metatarsals increased on weightbearing in both groups. The degree of the changes of the angles was closely related with the degree of the hallux valgus deformity. The intermetatarsal angle between the fourth and fifth metatarsals decreased on weightbearing in the both groups. A weightbearing view is necessary to evaluate the structural configuration of hallux valgus, because muscle imbalance around the first metatarsophalangeal joint in hallux valgus causes an increase of a hallux valgus angle on weightbearing.

Adolescent↗

Varus tilt of the tibial plafond as a factor in chronic ligament instability of the ankle.

The authors performed a radiographic study of 136 patients with acute ligament sprains and 85 patients with chronic lateral ligament instability of the ankle. Varus angulation of the tibial plafond, varus angulation of the line passing both malleolar ends, and varus angulation of the medial malleolus were measured on the AP views, and dorsal angulation of the tibial plafond was measured on the lateral views. The varus angles of the tibial plafond, the line passing both malleolar ends, and the medial malleolus were larger as mean values in patients with chronic ligament instability than in the patients with acute ligament sprain. The varus tilt of the tibial plafond is more often seen in patients with chronic ligament instability of the ankle than in patients with acute ligament sprains.

Adolescent↗

Lengthening of short great toes by callus distraction.

We lengthened seven first metatarsals in four patients with short great toes by callus distraction using an external fixator. Good clinical and cosmetic results were obtained. Bone lengthening is effective in patients with short great toes not only for cosmesis, but also to relieve pain and callosities on the plantar aspect of the second and third metatarsal heads. Excessive lengthening of the first metatarsal resulted in limitation of the range of movement of the metatarsophalangeal joint of the great toe. To prevent this the amount of lengthening should not exceed 40% of the preoperative length of the metatarsal.

Adult↗

Effects of Trp64Arg mutation in the beta 3-adrenergic receptor gene on weight loss, body fat distribution, glycemic control, and insulin resistance in obese type 2 diabetic patients.

OBJECTIVE: To investigate the effects of Trp64Arg mutation in the beta 3-adrenergic receptor gene on weight loss, body fat distribution, glycemic control, and insulin resistance in obese type 2 diabetic patients. RESEARCH DESIGN AND METHODS: We measured body weight, waist-to-hip ratio (WHR), adjusted resting metabolic rate, fasting blood glucose, fasting serum insulin levels, insulin resistance index (fasting glucose x fasting insulin/22.5), and HbA1c levels before and after 12 weeks of obesity treatment in 61 obese women with type 2 diabetes. The MvaI polymorphism of the beta 3-adrenergic receptor gene was determined by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: Of obese type 2 diabetic patients, those with the mutation (n = 24) had a higher WHR (P < 0.001), a lower adjusted metabolic rate, and higher blood glucose levels, serum insulin levels, insulin resistance index (P < 0.001), and HbA1c levels (P = 0.016). Furthermore, patients with the mutation had smaller decreases in body weight, WHR, insulin resistance index, and HbA1c levels after the weight-loss program compared with patients without the mutation (n = 37), even though food intake, exercise, and serum thyroid hormone levels were similar in both groups. CONCLUSIONS: These present findings show that the Trp64Arg allele of the beta 3-adrenergic receptor gene may predict difficulty in losing body weight, lowering WHR, and improving glycemic control and insulin resistance in obese patients with type 2 diabetes.

Adult↗

Beta 3-adrenoreceptor gene polymorphism: a newly identified risk factor for proliferative retinopathy in NIDDM patients.

Proliferative diabetic retinopathy is an important cause of visual impairment. We investigated whether the polymorphism of the beta 3-adrenoreceptor (beta 3-AR) gene, which is associated with insulin resistance and an earlier onset of NIDDM, was associated with proliferative diabetic retinopathy (PDR) in 215 Japanese NIDDM patients with a duration of diabetes of > or = 10 years. The polymorphism of the beta 3-AR gene was determined by polymerase chain reaction-restriction fragment length polymorphism analysis. The Trp64Arg allele of the beta 3-AR gene was significantly more frequent in the NIDDM patients with PDR (P = 0.002), but not in those with non-PDR (P = 0.151), than in NIDDM patients without diabetic retinopathy. Those with the mutation had an earlier onset of diabetes, a longer duration of diabetes, and higher current and maximal BMI values, compared with those without the mutation. Moreover, this mutation was also associated with higher serum triglyceride and decreased HDL-cholesterol levels. When adjustment was made for age, age at diagnosis, duration of diabetes, current BMI, systolic blood pressure, HbA1e, and serum lipids in a multiple regression analysis, a significant association was found between the Trp64Arg allele and diabetic retinopathy (P = 0.039). The Arg/Arg or Arg/Trp genotype was significantly associated with PDR, compared with the Trp/Trp genotype, with an odds ratio of 2.55 (95% CI 1.25-5.16). We concluded that the beta 3-AR gene polymorphism is a newly identified risk factor for PDR.

Aged↗