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ABSTRACT: Transplantation of microencapsulated islets is proposed as an ideal therapy for the treatment of type 1 diabetes mellitus without immunosuppression. This strategy is based on the principle that foreign cells are protected from the host immune system by an artificial membrane. The aim of this study was to establish an ideal condition of microencapsulation using an air-driven droplet generator and alginate in vitro. The optimal conditions for islet encapsulation were an alginate inflow rate of 10 mL/h, CO2 flow rate of 2.0 L/min in a concentration of 2% alginate. For 2.5% alginate, the alginate inflow rate of 20 mL/h, CO2 flow rate 3.0 L/min was ideal; alginate inflow rate of 40 mL/h, CO2 flow rate of 4.0 L/min showed good microcapsules at 3% alginate. Viability of encapsulated islets was greater than 90%. In terms of insulin secretion, encapsulated islets secreted insulin in response to glucose in static culture medium. However, there was no normal response to low or high glucose challenge with a stimulation index less than 2.0. Microencapsulation of pig islets was successfully performed with air-driven droplet generator and alginate in vitro. Further studies about biocompatibility and glucose control in vivo may provide a useful tool for treatment of patients with diabetes mellitus.
Transplantation Proceedings 11/2008; 40(8):2578-80. · 1.00 Impact Factor
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ABSTRACT: Adult porcine islet xenotransplantation into humans is greatly diminished by the difficulty to isolate islets because of their fragility. The goal of this study was to improve the efficacy of islet yields using endogenous trypsin inhibitor and histidine-tryptophan-ketoglutarate (HTK) perfusate.
We compared two porcine islet isolation protocols: Eurocollins solution for in situ pancreas perfusion without use of an endogenous trypsin inhibitor versus HTK solution including endogenous trypsin inhibitor for pancreas perfusion.
Endogenous trypsin inhibitor and HTK strategies significantly improved total islet yield, recovery, and islet index after purification (P < .05), whereas unpurified islet yield did not increase. An average of 228,000 +/- 95,000 islet equivalents (IEQ) (n = 20) purified islets were obtained in the first group compared with 115,000 +/- 56,000 IEQ (n = 18) in the second group. The average islet index was significantly increased in the first group compared with the second group before and after purification: before: 0.28 versus 0.49 versus after: 0.25 versus 0.4 (P < .05). At this time, islet purity, viability, and stimulation index did not show a significant difference between groups.
Our study showed that endogenous trypsin inhibitor and HTK strategies significantly improved purified islet isolation efficacy because of reduction of islet fragility.
Transplantation Proceedings 10/2008; 40(8):2585-7. · 1.00 Impact Factor
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ABSTRACT: Mutations in the SLC26A4 gene are responsible for Pendred syndrome and non-syndromic hearing loss (DFNB4). This study analysed non-synonymous SLC26A4 mutations newly identified in East Asians, as well as three common mutations in Caucasians, to characterise their molecular pathogenic mechanisms and to explore the possibility of rescuing their processing defects.
A total of 11 non-synonymous disease associated mutations were generated and their effects on protein processing and on ion transporting activities were examined.
Most of the mutations caused retention of the SLC26A4 gene product (pendrin) in the intracellular region, while wild-type pendrin reached the plasma membrane. Accordingly, these mutations abolished complex glycosylation and Cl(-)/HCO(3)(-) exchange activities of pendrin. However, significant heterogeneity in the processing of mutant pendrin molecules was observed. Each mutant protein exhibited a different cellular localisation, a different degree of N-glycosylation, and a different degree of sensitivity to the treatments that rescue processing defects. For example, H723R-pendrin, the most common mutation in East Asians, was mostly expressed in endoplasmic reticulum (ER), and its defects in protein processing and ion transporting activities were restored considerably by low temperature incubation. On the other hand, L236P-pendrin, the most common mutation in Caucasians, was mainly in the centrosomal region and was temperature insensitive.
These results indicate that the processing of pendrin mutant protein is determined by mutant specific mechanisms, and that a mutant specific method would be required to rescue the conformational defects of each folding mutant.
Journal of Medical Genetics 08/2008; 45(7):411-9. · 6.36 Impact Factor
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ABSTRACT: The alpha-melanocyte-stimulating hormone (alpha-MSH) has been shown to interact with various cells of the immune and inflammatory systems and down-regulate either the production or the action of proinflammatory cytokines. In this study, we investigated the potential of alpha-MSH to prevent pancreatic islet cells from cytotoxic injury by inflammatory cytokines released from peripheral blood mononuclear cells (PBMCs) in rats. Pancreatic islets were cocultured with PBMCs in a transwell system during stimulation by phorbol myristic acid and ionomycin. alpha-MSH (50 nmol/L) was added to PBMCs for 2 hours before coculture. Viability and apoptosis of islets were observed by the 3-(4,5-dimethylthiazole-2-yl)-, 5-diphenyltrazolium bromide assay and flow cytometry. We measured inflammatory cytokines and nitric oxide (NO). Insulin release from islets cocultured with mononuclear cells was checked as the metric of islet function. In comparison to the control group, the viability of islets with alpha-MSH-treated mononuclear cells was increased and apoptosis reduced significantly. Inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-1beta, were significantly reduced among the alpha-MSH-treated group. NO production in the alpha-MSH-treated group was decreased significantly. Insulin secretory function of the islets recovered in conditions of alpha-MSH treatment. This study demonstrated that alpha-MSH protected pancreatic islet cells from PBMC-mediated cytotoxicity and preserved insulin secretory function. This treatment may have the potential to improve graft survival in clinical islet transplantation.
Transplantation Proceedings 07/2007; 39(5):1604-6. · 1.00 Impact Factor
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H-J Park,
S-J Lee,
H-S Jin,
J O Lee,
S-H Go,
H S Jang,
S-K Moon,
S-C Lee,
Y-M Chun,
H-K Lee,
J-Y Choi,
S-C Jung,
A J Griffith, S K Koo
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ABSTRACT: Sensorineural hearing loss associated with enlargement of the vestibular aqueduct (EVA) can be associated with mutations of the SLC26A4 gene. In western populations, less than one-half of the affected individuals with EVA have two mutant SLC26A4 alleles, and EVA is frequently caused by unknown genetic or environmental factors alone or in combination with a single SLC26A4 mutation as part of a complex trait. In this study, we ascertained 26 Korean probands with EVA and performed nucleotide sequence analysis to detect SLC26A4 mutations. All subjects had bilateral EVA, and 20 of 26 were sporadic (simplex) cases. Fourteen different mutations were identified, including nine novel mutations. Five mutations were recurrent and accounted for 80% of all mutant alleles, providing a basis for the design and interpretation of cost-efficient mutation detection algorithms. Two mutant alleles were identified in 21 (81%), one mutant allele was detected in three (11%), and zero mutant allele was detected in two (8%) of 26 probands. The high proportion of Korean probands with two SLC26A4 mutations may reflect a reduced frequency of other genetic or environmental factors causing EVA in comparison to western populations.
Clinical Genetics 03/2005; 67(2):160-5. · 3.13 Impact Factor
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Human Genetics 02/2004; 114(2):222. · 5.07 Impact Factor
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ABSTRACT: The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.
Journal of Biological Chemistry 03/1999; 274(9):5581-7. · 4.77 Impact Factor
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ABSTRACT: Gap junction channels reconstituted in liposomes provide a pathway for the transfer of second messengers. Gap junction channels were formed in the artificial unilamellar liposomes using immunoaffinity-purified connexin 43 gap junction protein from rat brain. Sucrose-permeable and -impermeable liposomes were separated on the basis of sucrose permeability in the iso-osmolar sucrose density gradient. The liposomes permeable to sucrose were also permeable to a communicating dye molecule, Lucifer yellow. In the present study, we examined the transfer of second messengers through the connexin 43 channels reconstituted in liposomes and first report the direct evidence that the gap junction channels are permeable to second messengers including adenosine 3',5'-cyclic phosphate and inositol 1,4,5-trisphosphate.
Biochimica et Biophysica Acta 08/1998; 1372(2):384-8. · 4.66 Impact Factor
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ABSTRACT: Gap junctional communication during the progression of cell cycle from quiescent G0 to S phase was examined in cultured clone 9 rat liver cells. The transfer of scrape-loaded fluorescent dye was suppressed immediately after the stimulation of cell cycle progression in a synchronized cell population. Northern blot analysis showed that the temporal disturbance of gap junctional communication in cells passing from G0 to S phase did not result from transcriptional down-regulation of connexin 43. It was also found that the PKC inhibitor, calphostin C, was able to restore intercellular communication in serum stimulated cells. Data suggest a control mechanism by PKC mediated phosphorylation in the regulation of gap junction function which is vulnerable to cell cycling. The loss of gap junctional communication correlated with the increased phosphorylation of connexin 43 on serine residues in clone 9 cells.
Molecular and Cellular Biochemistry 03/1997; 167(1-2):41-9. · 2.06 Impact Factor
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ABSTRACT: This study was conducted to assess the effects of chemical carcinogens on the gap junction-mediated intercellular communication in cultured mammalian cells. The method of scrape-loading dye transfer of lucifer yellow was adapted as a measure of gap junctional communication. Clone 9 cells derived from rat liver were treated with a model chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the gap junctional communication was assessed by measuring the transfer of scrape-loaded lucifer yellow dye. When cells were treated with the carcinogen at 0.3 mg/ml, the fluorescent dye transfer was inhibited by 90% in 60 min. Other chemical agents, which include direct or indirect carcinogens and antitumor drugs, were also examined for their effects on the gap junctional communication. Direct carcinogens, such as MNNG, hydroxylamine and ethidium bromide, exhibited strong inhibition of intercellular communication, while indirect carcinogens, such as aflatoxin B1 and ethionine, exerted minor effects. Effects of test chemicals on the cell communication through gap junctions were readily quantitated by counting the number of cells stained with the fluorescent dye.
Toxicology 05/1995; 98(1-3):199-206. · 3.68 Impact Factor
