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Biomedical subjects

A Sano

Publications and source records attributed to A Sano.

At least 37 records · Page 2Linked to original sources

Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions.

We have previously demonstrated that Atelocollagen, used clinically for wound healing, is a reliable safe carrier for gene delivery. To obtain phenotypic changes by gene expression of cDNA, we developed an efficient technique for high-throughput gene transfer and expression screening in mammalian cells in microarrays by precoating a microplate with an Atelocollagen complexed with cDNA to which cells are then seeded. The complexes with a nanoparticle form were efficiently transduced into cells without use of any additional transfection reagent, and they allowed for long-term gene expression without apparent chromosomal integration. The complex spotted onto the well of a microplate was stable for a long period and allowed the cells to transduce and express reporter genes in a dose-dependent manner. We also showed that the present method using Atelocollagen-based gene transfer is applicable to gene medicines such as antisense ODNs and adenovirus vectors. These results suggest that Atelocollagen may be appropriate for general use in high-throughput screening of large sets of gene medicines for functional analyses in mammalian cells.

Adenoviridae↗

Sustained release of human growth hormone (hGH) from collagen film and evaluation of effect on wound healing in db/db mice.

Collagen films containing human growth hormone (hGH) were prepared and the release of hGH from these films and their effect on healing of full-thickness wounds in db/db mice were evaluated. The release profiles of hGH from the collagen films varied with composition and preparation conditions. The film prepared by air-drying of the mixture of hGH and collagen solution released hGH continuously over 3 days both in vitro and in vivo. By application of collagen film containing 3 mg of hGH twice at an interval of 6 days to wounds, area of wounds on day 21 was significantly reduced compared with that of non-treated wounds. Application of hGH alone at the same dose had no significant effect on wound healing. The maximum serum hGH concentration after single administration of the hGH collagen film was lower than that with hGH alone, and hGH persisted in serum over 3 days. These results suggest that hGH collagen film may be a useful topical formulation for the treatment of wounds.

Animals↗

Semi-quantitative RT-PCR analysis of environmental influence on P450scc and PNMT mRNA expression in rat adrenal glands.

Environmental influence on brain function, particularly spatial learning and memory, has been extensively investigated, but little is known about the influence of environmental conditions on the functions of peripheral organs. In the present study, the effects of different housing conditions on the steady-state levels of mRNAs encoding cholesterol side-chain cleavage enzyme (cytochrome P450scc) and phenylethanolamine N-methyltransferase (PNMT) in adrenal glands was examined to investigate the environmental influence on both adrenocortical and adrenomedullary functions. Behavioral changes of the animals housed in different conditions were first examined to assess the relevance of environmental manipulation used. In consistent with previous findings, housing of the animals in enriched conditions resulted in the significant reduction of spontaneous motor activity (locomotor activity and rearing) in comparison with housing in isolated conditions, thus indicating the relevance of housing conditions used in this work for investigating the environmental influence on adrenal function. Then, the effects of these housing conditions on P450scc and PNMT mRNA levels in adrenal glands were examined using semi-quantitative RT-PCR method. In comparison with the isolated group, the enriched group showed significantly higher levels of P450scc mRNA. In contrast, PNMT mRNA levels in the enriched group were significantly lower than those in the isolated group. These results propose the possibility that the environmental conditions may cause differential alterations in adrenocortical and adrenomedullary functions, although their possible association with behavioral changes still remains to be elucidated.

Adrenal Glands↗

The application of biodegradable collagen minipellets as vaccine delivery vehicles in mice and sheep.

Collagen minipellets are injectable delivery vehicles that release antigen and adjuvant over several days in a first-order release profile. In vaccination experiments in mice, secondary antibody responses induced by minipellets formulated with avidin and IL-1beta as adjuvant were equivalent to those induced by a conventional immunization with avidin in alum. When no adjuvant was used, anti-avidin responses induced by minipellets were 10-20-fold higher than those induced by injection of avidin in saline. In sheep, conventional vaccination with avidin in alum induced antibody responses initially exceeding that induced by minipellets formulated with avidin and IL-1beta, while following a secondary vaccination, the minipellet antibody response was equal to or greater than the alum-adjuvanted control groups. Increasing levels of IL-1beta adjuvant resulted in enhanced persistence of the antibody response. When clostridial vaccine antigens were incorporated into the minipellets, total antibody responses induced in sheep were equivalent to those induced by vaccination with the clostridial antigens in alum. Neutralizing antibody titres exceeded those induced by conventional vaccination. No adverse site reactions were observed at the implant site, with immunohistological study showing that the cellular infiltrate was dominated by a transient influx of neutrophils. This is a typical response to delivery of bioactive IL-1beta. The minipellets were completely degraded within 35 days of implantation.

Adjuvants, Immunologic↗

Role of mitogen-activated protein kinase-mediated cytosolic phospholipase A2 activation in arachidonic acid metabolism in human eosinophils.

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A(2) (PLA(2)) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA(2) activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC(50) = 8.5 nM)- and time-dependent (t(1/2) = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C(4) (LTC(4)) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA(2) (cPLA(2)) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A(2) by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca(2+)-independent phospholipase A(2) inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC(4) release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1-5 min, and declined thereafter. fMLP stimulation also increased cPLA(2) activity in eosinophils, which was inhibited completely by 30 microM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA(2) activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA(2) activation causes AA hydrolysis and LTC(4) secretion. We also find that cPLA(2) activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA(2) isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC(4).

Arachidonic Acid↗

Development of a new drug delivery system for protein drugs using silicone (II).

In order to achieve a zero-order release of protein drugs, we have developed a new drug delivery system using silicone, which is named the covered-rod-type formulation. Preparation of the covered-rod-type formulation was conducted under mild conditions without heat treatment or the use of organic solvents. The covered-rod-type formulation released human serum albumin (HSA) or interferon (IFN) at a constant rate for 30-100 days in vitro without significant initial burst. When the IFN covered-rod-type formulation was implanted in nude mice, the serum IFN concentration was maintained at a constant level during the period of observation, i.e., 28 days. The covered-rod-type formulation enabled precise control of the release of the protein drugs and would be expected to increase the duration of the drug effect and to reduce the frequency of administration and side effects.

Animals↗

Collagen minipellet as a controlled release delivery system for tetanus and diphtheria toxoid.

The use of biodegradable polymer matrices as a single-dose vaccine delivery system was investigated using tetanus toxoid (TT) and diphtheria toxoid (DT). BALB/c mice were immunized with TT or DT in different formulations including individual, in minipellet and aluminum hydroxide (alum), and the antibody responses were monitored for 48 weeks. Antigens entrapped in minipellet elicited higher antibody responses compared to those obtained with individual antigens and antigens adsorbed to alum and the antibody levels remained elevated over 48 weeks. In addition, minipellet formulations induced the same subclasses of antibodies induced by alum formulations. These results raise the possibility to obtain optimal and long-lasting immune responses by a single administration of minipellet formulations.

Adjuvants, Immunologic↗

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of cytosolic phospholipase A2 is essential for human eosinophil adhesion to fibronectin.

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A(2) (cPLA(2)) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 microg/ml FN at 30 min, and decreased after 60-90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA(2) were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA(2) inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA(2) phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA(2) phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA(2) activation is essential for eosinophil adhesion to FN.

Actins↗

The gene encoding a newly discovered protein, chorein, is mutated in chorea-acanthocytosis.

Chorea-acanthocytosis is a neurodegenerative disorder with peripheral red cell acanthocytosis. Linkage of chorea-acanthocytosis to chromosome 9q21 has been found. We refined the locus region and identified a previously unknown, full-length cDNA encoding a presumably structural protein, which we called chorein. We found a deletion in the coding region of the cDNA leading to a frame shift resulting in the production of a truncated protein in both alleles of patients and in single alleles of obligate carriers.

Chorea↗

Cloning of porcine IGF1 receptor cDNA and detection of sequence polymorphisms using RT-PCR.

A cDNA encoding the porcine type 1 insulin-like growth factor receptor (IGF1R) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The sequence of a 4.2-kb product was determined and had an open reading frame, encoding 1367 amino acids with 98.1 and 95.2% sequence similarity to the human and rat IGF1R, respectively. In the comparison of RT-PCR derived IGF1R sequences from 12 unrelated pigs, 12 silent sequence variants were found.

Animals↗

Cerebriform colonies of Paracoccidioides brasiliensis isolated from nine-banded armadillos (Dasypus novemcinctus) at room temperature.

Twelve isolates of Paracoccidioides brasiliensis generated cerebriform colonies at room temperature on potato glucose agar slants (PDA). These isolates contained abundant chlamydospores and yeast-like cells and are a subset of the 65 isolates obtained from nine-banded armadillos (Dasypus novemcinctus). They grew as a yeast form with typical multiple buddings at 37 degrees C on brain heart infusion agar supplemented with 1% glucose. After replating on PDA and culturing at room temperature for 2 months, the mutants appeared as cottonous colonies, which indicated that the morphological characteristics were unstable.

Animals↗

Intraarterial infusion chemotherapy and radiotherapy with or without surgery for patients with locally advanced or recurrent breast cancer.

We analyzed response, side effects, and local control rates of a multimodal treatment consisting of intraarterial infusion chemotherapy (IAIC) and radiotherapy with or without surgery for patients with locally advanced or recurred breast cancer. Thirty-three patients, clinically diagnosed as stage IIB in 1, IIIA in 2, IIIB in 12, IV in 18, were treated from 1991 to 1998. Twenty-five were primary and eight were recurrent cases after surgery. IAIC started as initial treatment up to three times maximum. In most cases, doxorubicin 50 mg, cisplatin 50 mg, and mitomycin 10 mg were infused in the subclavian and/or internal mammary artery. After IAIC, patients in primary cases underwent radical mastectomy or breast conservation surgery, after radiotherapy at a total dose of 50 Gy/25 fractions/5 weeks with a boost of 10 Gy. In recurrent cases, a full dose of radiotherapy was delivered. Clinical objective and complete response rates were 78% and 9% after IAIC. Despite a high rate of residual positive margin (67%) or clinically residual carcinoma, local recurrence developed only in 2 patients (6%) and local control rates at 5 years were calculated as 89%. Bone marrow suppression was frequent, and skin vesiculation (15%) and ulceration (9%) were experienced after IAIC. Skin ulcer (6%), brachial plexus neuropathy (3%), and radiation pneumonitis (3%) occurred as late toxicity. IAIC was effective as an induction treatment and radiotherapy played a role of local control for patients with locally advanced or recurrent breast cancer.

Adult↗

Validity of local treatment including intraarterial infusion chemotherapy and radiotherapy for fungating adenocarcinoma of the breast: case report of more than 8-year survival.

A 39-year-old woman came to us complaining of severe anemia (hemoglobin: 2.3 g/dl) and a painful right breast that was entirely occupied by an ulcerative, foul-smelling tumor approximately 20 cm in maximum dimension. The tumor, which was determined to be mucinous adenocarcinoma at biopsy, had invaded the chest wall with multiple lung metastases (T4cN2M1 stage IV). After a blood transfusion, the patient received the following multimodal treatment: concurrent chemoradiotherapy (50 Gy), intraarterial infusion chemotherapy consisting of doxorubicin 50 mg, mitomycin-C 10 mg, and cisplatin 50 mg, and skin graft surgery. After intraarterial infusion chemotherapy, the fungating tumor disappeared. The patient experienced a relapse of right pleural effusion 2 years later and received multimodal treatment. Now, March 30, 2000, the patient is doing well without local recurrence, 8 years after her first admission. The combination of intraarterial infusion chemotherapy and radiotherapy plays a role in successful treatment of extensive local disease of the breast.

Adenocarcinoma, Mucinous↗

Regulation of eosinophil function by phosphatidylinositol-specific PLC and cytosolic PLA(2).

We examined the regulatory role of cytosolic phospholipase A(2) (cPLA(2)) and phosphatidylinositol (PI)-specific phospholipase C (PLC) in the degranulation of human eosinophils and leukotriene (LT) C(4) synthesis. Activation with formyl-Met-Leu-Phe + cytochalasin B (fMLP/B) caused a time-dependent release of eosinophil peroxidase (EPO) and LTC(4), which was inhibited by pertussis toxin. By immunoblotting, eosinophil PLC-beta2 and -gamma2 isoforms were identified, and PLC activation was measured as a function of inositol 1,4,5-trisphosphate concentration. Stimulated release of EPO and intracellular Ca(2+) concentration was inhibited by ET-18-OCH(3), a PI-PLC inhibitor, whereas trifluoromethylketone (TFMK), a cPLA(2) blocker, had no inhibitory effect. Both TFMK and ET-18-OCH(3) attenuated stimulated arachidonate release and LTC(4) secretion, suggesting that activation of both PLC and cPLA(2) is essential for LTC(4) synthesis caused by fMLP/B. The structurally unrelated protein kinase C inhibitors bisindolylmaleimide, Ro-31-8220, and Go-6976 all blocked fMLP/B-induced EPO release but not LTC(4) secretion. 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester, an intracellular Ca(2+) chelator, suppressed both EPO release and LTC(4) secretion. We found that fMLP/B-induced LTC(4) secretion from human eosinophils is regulated by PI-PLC through calcium-mediated activation of cPLA(2). However, cPLA(2) does not regulate eosinophil degranulation.

Arachidonic Acid↗

Reduced lipoprotein lipase activity in postural skeletal muscle during aging.

Lipoprotein lipase (LPL) is a key enzyme for fatty acid and lipoprotein metabolism in muscle. However, the effect of aging on LPL regulation in skeletal muscle is unknown. We report the effect of aging on LPL regulation in the soleus (red oxidative postural) muscle and the tibialis anterior (white glycolytic non-weight-bearing) muscle in 4- and 24-mo-old Fischer 344 rats and 18- and 31-mo-old Fischer 344 x Brown-Norway F1 (F-344 x BN F1) rats. Total and heparin-releasable LPL (HR-LPL) activities were decreased 38% (P < 0.01) and 52% (P < 0.05), respectively, in the soleus muscle of the older Fischer 344 rats. There was a 32% reduction (P < 0.05) of total LPL protein mass in the soleus muscle with aging. The results were confirmed in another strain. A decrease of total LPL activity (-50%, P < 0.05) was also found in the soleus muscle between 18- and 31-mo-old F-344 x BN F1 rats. LPL mRNA concentration in the soleus muscle was not different between ages. Total LPL protein mass was reduced by 46% (P < 0.05) in the soleus muscle of the 31-mo-old F-344 x BN F1 rats. In the tibialis anterior muscle, neither LPL activity nor mRNA concentration was affected by age in either strain. In conclusion, LPL regulation in a non-weight-bearing muscle was not affected by aging. However, there was a pronounced reduction in LPL activity and LPL protein mass in postural muscle with aging.

Aging↗

Association of the TaqI A polymorphism of the dopamine D(2) receptor gene with predisposition to neuroleptic malignant syndrome.

OBJECTIVE: The pathophysiology of neuroleptic malignant syndrome is mainly explained by a central hypodopaminergic state. The familial occurrence of neuroleptic malignant syndrome suggests the involvement of a genetic mechanism in the predisposition to the syndrome. Therefore, the authors examined the association between the TaqI A polymorphism of the dopamine D(2) receptor gene (DRD(2)), which alters DRD(2) density and function, and the development of neuroleptic malignant syndrome. METHOD: The subjects were 15 psychiatric patients who had developed neuroleptic malignant syndrome (12 patients with schizophrenia and three with major depression) and 138 patients with schizophrenia who had never developed neuroleptic malignant syndrome. The TaqI A genotypes, A1 and A2 alleles, were determined by the polymerase chain reaction method. RESULTS: The frequency of the A1 allele was significantly higher in the patients who had developed neuroleptic malignant syndrome (56.8%) than in the patients who had not (35.1%). The proportion of the A1 carrier was significantly higher in the patients with neuroleptic malignant syndrome (14 [93.3%] of 15 patients) than in those without the syndrome (79 [57.2%] of 138 patients). CONCLUSIONS: These findings suggest that the TaqI A DRD(2) polymorphism is associated with the predisposition to neuroleptic malignant syndrome.

Adult↗

Fluorometric determination of N-nitroso-N-methylurea with nicotinamide and acetophenone.

A fluorometric method for the determination of N-nitroso-N-methylurea (NMU) has been developed. It is based on the N-methylation reaction of nicotinamide with NMU and a subsequent condensation reaction with acetophenone, followed by an acid treatment to form a fluorescent 2,7-naphthyridine derivative. This method enabled the determination of NMU in the range 0.05 - 2 nmol/200 microl with a relative standard deviation of ca. 3%. It was applied to the determination of NMU formed from a precursor N-methylurea (MU) under simulated gastric conditions containing nitrite and thiocyanate ions at pH 3.0 in the presence of fresh orange juice and milk. NMU was extracted by an Extrelut 20 column and then determined. The mean recoveries of NMU added to the simulated gastric juice containing water, orange juice and milk were 86.5, 85.1 and 69.8%, respectively. The amounts of NMU formed from MU were found to decrease to below 25% in the presence of orange juice and milk.

Acetophenones↗

Biomaterials for gene delivery: atelocollagen-mediated controlled release of molecular medicines.

Over the last decade, increasing attention has been paid to the development of systems to deliver drugs for long periods at controlled rates. Some of these systems can deliver drugs continuously for over one year. However, little effort has been given to developing systems for the controlled release of nucleic acids. Recently, a novel gene transfer method which allows prolonged release and expression of plasmid DNA in vivo in normal adult animals was established. In this system, a biocompatible natural polymer such as collagen or its derivatives acts as the carrier for the delivery of DNA vectors. The biomaterial carrying the plasmid DNA was administered into animals and, once introduced, gradually released plasmid DNA in vivo. A single injection of plasmid DNA/biomaterial produced physiologically significant levels of gene-encoding proteins in the local/systemic circulation of animals and resulted in prolonged biological effects. These results suggest that the biomaterials carrying plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Furthermore, the biomaterials can be removed surgically, minimizing the effect of gene products if some unexpected side effects should be observed after application. The application of these systems to expand the bioavailability of molecular medicine, including antisense oligonucleotides and adenovirus vectors, and to aid in stem cell transplantation in the context of DNA-based tissue engineering will be discussed.

Adenoviridae↗