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Biomedical subjects

Y Gu

Publications and source records attributed to Y Gu.

396 records · Page 22Linked to original sources
PubMed1985

The effects of a myasthenic serum on the acetylcholine receptors of C2 myotubes. II. Functional inactivation of the receptor.

We have investigated the effect of antibodies from a myasthenic serum on the physiological properties of acetylcholine receptors (AChRs) in myotubes of a mouse muscle cell line, C2. The antibodies in this serum blocked the binding of 125I-alpha-bungarotoxin to the myotubes to an extent of about 50%. The antibodies also inhibited the increase in 22Na influx caused by carbamylcholine (CARB). At a concentration of antibody that blocked about 50% of toxin binding, greater than 80% of the AChR-mediated 22Na influx was blocked. The apparent KD for CARB, estimated from the dose-response curve for 22Na influx, was unaffected. The effect of the antibodies was further examined by patch-clamp recording. In greater than 30% of the patches from antibody-treated cells, no channel activity in response to acetylcholine was seen; in contrast, every patch from control cells showed activity. The channels that were seen after antibody treatment were indistinguishable from those seen in normal cells, both in their single-channel conductance and in the kinetic constants used to describe channel opening and closing. We conclude that the antibodies in this serum inhibit the functional response of AChRs in C2 myotubes to acetylcholine and do so by inactivating individual receptors.

Animals↗
PubMed1985

The effects of a myasthenic serum on the acetylcholine receptors of C2 myotubes. I. Immunological distinction between the two toxin-binding sites of the receptor.

We have examined the effect of a serum from a patient with myasthenia gravis on the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptors (AChRs) of a mouse muscle cell line, C2. After a 2-hr incubation, antibodies in the serum reduced toxin binding to C2 myotubes to a maximal extent of approximately 50%. The degradation of surface AChRs could account for the loss of only 5% of sites during the incubation; the remainder, therefore, must have been lost by blockage of binding. To investigate whether the antibodies blocked specifically one of the two toxin-binding sites that each AChR possesses, we used an analysis based on that of Sine, S. W., and P. Taylor, [1981) J. Biol. Chem. 255: 10144-10156). Although the two sites could not be distinguished by their rates of binding of alpha-BuTx, d-tubocurarine (dTC) inhibition of the initial rate of toxin binding revealed that the sites had affinities for dTC that differed by approximately 30-fold. Incubation with the myasthenic antibodies reduced the number of high affinity dTC sites, without affecting those of low affinity. We conclude that the two toxin-binding sites of the AChR are immunologically distinct.

Animals↗
PubMedDate not supplied

Protease chymotrypsin mediates the endothelial expression of P- and E-selectin, but not ICAM and VCAM, induced by placental trophoblasts from pre-eclamptic pregnancies.

OBJECTIVES: Soluble endothelial-cell adhesion molecules (ICAM, VCAM and PECAM) are markers of endothelial activation, and are elevated in the maternal circulation during pregnancy and even further increased in pregnancies complicated by pre-eclampsia (PE). To identify possible sources of endothelial activators during pregnancy, we addressed whether factors released from placental trophoblast cells (TCs) activate endothelial cells (ECs) to enhance adhesion molecule expression on ECs. We also examined whether proteases released by placental cells induce the endothelial cell surface molecule expression in PE. METHODS: Confluent ECs were co-cultured with placental TCs derived from normal (n=9) or PE (n=8) pregnancies or with placental conditioned media (CM) derived from PE placental cultures (n=7). ICAM, VCAM, P-selectin and E-selectin were quantified using an enzyme-linked immunosorbent assay (ELISA). The protease inhibitors alpha(2)-macroglobulin (alpha(2)M), thrombin inhibitor (TI) and chymotrypsin inhibitor (CI) were tested in the co-culture system. mRNAs for ICAM, VCAM, P-selectin and E-selectin were determined by RNase protection assay (RPA). NF-kappaB activity in ECs was also determined. RESULTS: (1) ICAM and VCAM expression was significantly increased on ECs co-cultured with both normal-TCs and PE-TCs, compared to control ECs (P<0.01). ICAM and VCAM expression in ECs co-cultured with normal-TCs did not differ from ECs co-cultured with PE-TCs. (2) E-selectin expression was increased on ECs co-cultured with normal-TCs (P<0.05) and further increased in ECs co-cultured with PE-TCs (P<0.01). (3) P-selectin expression was increased on ECs co-cultured with PE-TCs, but not ECs co-cultured with normal-TCs compared to control ECs (P<0.05). (4) alpha(2)M and TI did not alter the ICAM, VCAM, P-selectin and E-selectin expression on ECs induced by PE-CM. (5) CI blocked the upregulation of P-selectin and E-selectin (P<0.05), but not ICAM and VCAM expression, in ECs cultured with PE-CM. (6) Changes in mRNA for ICAM, VCAM, P-selectin and E-selectin paralleled the increases in protein expression on ECs cultured with PE-CM. (7) NF-kappaB activity was also increased in cells challenged with PE-CM. CONCLUSIONS: (1) Factor(s) released from both normal-TCs and PE-TCs promote ICAM and VCAM expression on ECs. (2) Factor(s) released from PE-TCs significantly increase EC P-selectin and E-selectin expression. (3) CI blocks the upregulation of P-selectin and E-selectin on ECs induced by factors released from PE placental cells, suggesting that chymotrypsin is responsible for the increased endothelial expression of P-selectin and E-selectin in pre-eclampsia.

Cell Adhesion Molecules↗
PubMedDate not supplied

Antisense modulation of 5,10-methylenetetrahydrofolate reductase expression produces neural tube defects in mouse embryos.

The role of folate metabolism in producing neural tube defects (NTDs) in humans is unknown. In the current study, antisense oligodeoxyribonucleotide technology was utilized to disrupt normal expression of the gene for 5,10-methylenetetrahydrofolate reductase (MTHFR) in organogenesis-stage mouse embryos. Two different antisense probes were microinjected into the amniotic sac of gestation day (GD) 8 mouse embryos with PBS or scrambled sense oligodeoxyribonucleotides injected into control embryos. Concentration-dependent increases in the frequencies of embryos with NTDs were observed for both antisense sequences. The level of mRNA for MTHFR was decreased in embryos treated with the higher concentration of one antisense sequence, indicating that the sequence is able to decrease gene expression. 5-methyltetrahydrofolate, the product of the MTHFR reaction, was able to decrease the incidence of antisense-induced NTDs, but co-injection with L-methionine did not. These results suggest that reduced expression of MTHFR may play a role in producing NTDs.

Animals↗
PubMedDate not supplied

Modified subcutaneous tissue with neovascularization is useful as the site for pancreatic islet transplantation.

The success rate of subcutaneous transplantation of pancreatic islets has been extremely low. Insufficient oxygen supply to the grafted islets is one possible major obstacle to the preservation of graft function. This study attempted to use basic fibroblast growth factor (bFGF) in subcutaneous transplantation to induce neovascularization and a sufficient blood flow around the space formed for grafted islets in the subcutaneous tissues. A bFGF-releasing device was designed enclosing bFGF in a polyethylene terephthalate mesh bag coated with polyvinylalcohol hydrogel. In the vascularized group (n = 5), two bFGF-releasing devices were implanted bilaterally into the subcutaneous tissue of the back of streptozotocin-induced diabetic Lewis rats. One week after implantation, isolated rat islets (5000) were syngeneically transplanted subcutaneously after the removal of the devices. In the control group (n = 5), no devices were implanted and the same number of rat islets was transplanted directly. One week after the implantation of the devices into the test animals, a thick, well-vascularized capsule was observed in the subcutaneous site. All vascularized recipient rats showed significant decreases in nonfasting blood glucose and maintained normoglycemia for more than 1 month after islet transplantation. However, in the control group, all rats failed to achieve normoglycemia after transplantation. This study provides evidence that the subcutaneous tissue is a promising site for pancreatic islet transplantation, offering convincing advantages in acceptability for diabetic recipients. Establishment of this subcutaneous islet transplantation technique will afford some new perspectives on successful clinical islet transplantation.

Animals↗
PubMedDate not supplied

Improved large-scale isolation of breeder porcine islets: possibility of harvesting from nonheart-beating donor.

To establish a large-scale isolation procedure for adult porcine islets usable as a donor source for xenotransplantation and as a model of human islet isolation, we improved several characteristics of the conventional isolation procedure. At a slaughterhouse we first selected a breeder pig over 1.5 years old (and over 200 kg in weight) with warm ischemic time (WIT) of 15 +/- 2 minutes as nonheart-beating donors. Then, we made a special enzymic mixture that consisted of collagenase S-1 (260 U/mg, NittaZelatin, Japan), collagenase P (1.86 U/ml Lyo Boehringer-Mannheim, USA), DNase (Sigma, St. Louis, Mo), Disparse (NittaZelatin, Japan), and protease inhibitor (Sigma). Third, this mixture was injected very gently into the pancreatic duct at the time of pancreatic harvesting. To prevent overdigestion of the pancreas, the mixture was first cooled to less than 10 degrees C. Fourth, during the warm digestion of pancreas, the pancreas with the enzymic mixture was quietly put in a water bath at 37 degrees C without mechanical shaking. Fifth, we purified the islets with a COBE 2991 cell processor by the Dextran 70 gradient method, because Dextran 70 is very cheap and has the same purification effect as the Ficoll gradient. The results of 10 consecutive breeder porcine islet isolations are reported. The total yield of isolations of islets over 50 microm in the longest diameter after staining with Dithizone (DTZ) was 85,900 +/- 19,954 islets, 291,667 +/- 240,452 IEQ (2,900 +/- 2,324 IEQ/g). The purity of the isolated islets was very high: 90.2 +/- 3.8%. Glucose stimulation during in vitro incubation induced significant insulin release from isolated breeder porcine islets. In two of the diabetic rats receiving encapsulated islets grafts using a mesh-reinforced polyvinyl alcohol hydrogel bag (MRPB), a prominent reduction in serum glucose levels (less than 200 mg/dL) persisted for 13 and 19 days, respectively, after intraperitoneal xenotransplantation islets without immunosuppression. In conclusion, we succeeded in a more efficient and less-expensive isolation of a large amount of adult porcine islets from a nonheart-beating donor.

Animals↗
PubMedDate not supplied

Effect of a new immunosuppressive agent, FTY720, on survival of islet allografts.

A newly developed immunosuppressant, FTY720, has a unique mechanism that is quite different from those of conventional immunosuppressants, and is presumed to be mediated through decreases in the number of peripheral lymphocytes, especially helper T cells. This study was performed to ascertain whether this innovative drug could prolong islet allograft survival. The donors were inbred Lewis rats and the recipients were ACI rats rendered hyperglycemic with intravenous streptozotocin. In the study group, FTY720 dissolved in distilled water was orally administered at a dose of 5 mg/kg to the recipient ACI rats 1 day before and on the day of grafting. In the control group, only distilled water was orally administered to the recipient ACI rats on the day before and the day of grafting. Two thousand islets were transplanted into the portal vein of the recipient rats in the study and control groups immediately after isolation. The graft survival time in the study group was significantly longer than that in the control group, indicating that FTY720 retains a potent effect on the prolongation of islet allograft survival. FTY720 could become a useful immunosuppressant for future clinical islet allotransplantation.

Animals↗
PubMedDate not supplied

Novel method for isolation of adult porcine pancreatic islets with two-stage digestion procedure.

It is particularly difficult to isolate porcine islets (PI). Experience suggests that the success rate of porcine islet isolation (PII) is probably considerably influenced by the distension and digestion of the pancreas. In this study, we divided the digestion procedure into two stages and developed a new enzyme solution to improve both the distension and digestion procedures. As a result, we established a novel and stable method of large-scale adult porcine islet isolation (APII). The harvested pancreata of 2-year-old pigs weighing over 200 kg (n = 18) were distended by introducing our new enzyme solution gently and slowly through the pancreatic ducts. Two-stage digestion (cold, then warm) was then performed by first placing the distended pancreata on ice for 2 h to cause diffusion of the enzyme solution around the islets, and then by incubating the pancreata in a water bath at 37 degrees C for 45 min without shaking. The islets were purified by a COBE 2991 cell processor on dextran T70 discontinuous density gradients. Histological study was performed on porcine pancreata sampled after 0, 15, 30, and 45 min of the second stage, and stained with H&E stain. Next, islet equivalent was calculated. Static incubation study was performed by stimulating the islets with 3.3 and 16.7 mM glucose in Krebs' Ringer bicarbonate buffer (KRBB) solution at 37 degrees C for 1 h, and finally the insulin released was measured. The dilated acinar cells septa around the islets were observed at time 0. Destruction of the acinar cells around the islets by warm digestion was recognized at 15 and 30 min, and destroyed and separated acinar cells present around the islets at 45 min. During the entire course of the warm digestion, the islets remained intact. The number of isolated islets was 291,667 +/- 240,452 IEQ/pancreas (n = 14) and 3,294 +/- 2199 IEQ/g of pancreatic tissue. The purity of recovered porcine islets was over 90%. The concentration of the insulin secreted by 10,000 IEQ islets selected at random was 83.9 +/- 13.4 microU/dish/h in response to 3.3 mM glucose and 104.1 +/- 12.9 microU/dish/h in response to 16.7 mM glucose (n = 20). A success rate of approximately 80% was attained with APII. We demonstrated that this increase in the success rate was due to the improved distension and digestion provided by this method. This two-stage APII method with its new enzyme solution may facilitate the future use of porcine islets in clinical xenotransplantation trials.

Animals↗
PubMedDate not supplied

Effectiveness of acidic oxidative potential water in preventing bacterial infection in islet transplantation.

At a number of points in the current procedures of islet isolation and islet culture after the harvesting of donor pancreata, microorganisms could potentially infect the islet preparation. Furthermore, the use of islets from multiple donors can compound the risks of contamination of individual recipients. Acidic oxidative potential water (also termed electrolyzed strong acid solution, function water, or acqua oxidation water), which was developed in Japan, is a strong acid formed on the anode in the electrolysis of water containing a small amount of sodium chloride. It has these physical properties: pH, from 2.3 to 2.7; oxidative-reduction potential, from 1,000 to 1,100 mV; dissolved chlorine, from 30 to 40 ppm; and dissolved oxygen, from 10 to 30 ppm. Because of these properties, acidic oxidative potential water has strong bactericidal effects on all bacteria including methicillin-resistant Staphylococcus aureus (MRSA), viruses including HIV, HBV, HCV, CMV, and fungi as a result of the action of the active oxygen and active chlorine that it contains. We conducted this study to evaluate the effect of acidic oxidative potential water irrigation on bacterial contamination on the harvesting of porcine pancreata from slaughterhouses for islet xenotransplantation by counting the number of pancreatic surface bacteria using the Dip-slide method, and on the results of islet culture; and to evaluate the direct effect on isolated islets when it is used to prevent bacterial contamination by the static incubation test and by morphological examination. Direct irrigation of the pancreas by acidic oxidative potential water was found to be very effective in preventing bacterial contamination, but direct irrigation of isolated islets slightly decreased their viability and function.

Abattoirs↗
PubMedDate not supplied

Alteration of DNA base excision repair enzymes hMYH and hOGG1 in hydrogen peroxide resistant transformed human breast cells.

BACKGROUND: Oxidative stress is a major causative agent of carcinogenesis, aging, and a number of diseases. 8-oxoG is the most stable and deleterious lesion of oxidative DNA damage. The 8-oxoG lesions can be eliminated by human repair systems consisting of three enzymes hMTH1, hOGG1, and hMYH homologous to E. coli MutT, MutM, and MutY proteins, respectively. MATERIAL AND METHODS: Human cells (P1, P2, and P3) resistant to H(2)O(2) were derived from the non-tumorigenic human breast cell line MCF10A by sequential treatment of the cells with H(2)O(2). The protein expression levels of DNA repair enzymes were analyzed by Western blotting. The DNA binding and glycosylase activities of hMYH and hOGG1 were measured in the extracts of the H(2)O(2) resistant cells. RESULTS: The H(2)O(2) resistant cells displayed tremendously greater anchorage-independent growth capability and higher expression of the anti-apoptotic protein BCL-2 than the parental cells. H(2)O(2) detoxification ability was elevated in P1 and P2 cells, but not in P3 cells, suggesting P3 cells might employ a different defense mechanism from P1 and P2 cells. In P3 cells, both hOGG1 and hMYH glycosylase activities were reduced but their protein levels increased. Two A/8-oxoG binding complexes were detected with cell extracts: the fast-migrating complex (bottom form) was dominated in MCF10A cells, and was greatly reduced in P3 cells. Interesting, the P3 cells showing the least amount of bottom form had the weakest hMYH glycosylase activity. CONCLUSIONS: Our results demonstrated, for the first time, that alteration of base excision repair pathways is correlated to cell resistance to oxidative stress.

8-Hydroxy-2'-Deoxyguanosine↗
PubMedDate not supplied

Metabolism of cholecystokinin-33 in vivo: effect of L-364,718, a CCK receptor antagonist.

Metabolism of cholecystokinin (CCK) and the effect of L-364,718, a specific CCK-A receptor antagonist, on the metabolism of CCK were examined in dogs. In conscious dogs, 45 min intravenous infusion of synthetic human CCK-33 (100 pmol/Kg/hr) caused an integrated CCK response over 90 min of 675 +/- 51 pmol-90 min/L, and the plasma CCK levels declined promptly with a t1/2 of 2.2 +/- 0.3 min after cessation. Organ extraction of CCK-33 by the kidney, mesenteric organs, and liver was examined in anesthetized dogs. From the gradients of the plasma levels between afferent and efferent vessels for each organ after bolus injection of CCK-33 (50 pmol/Kg), renal extraction ratio was 0.30 +/- 0.04, and mesenteric extraction ratio was 0.19 +/- 0.04. Hepatic extraction was not detected. T1/2 and extraction ratios were not affected by the preinjection of L-364,718 (20 nmol/Kg). The results indicate in dogs that exogenously administered CCK-33 is degraded by the mesenteric organs as well as the kidney but not by the liver, and that receptor-mediated mechanisms are not involved in these degradation pathways of CCK.

Anesthesia↗
PubMedDate not supplied

Insulin release from a bioartificial pancreas using a mesh reinforced polyvinyl alcohol hydrogel tube. An in vitro study.

Islet transplantation with a bioartificial pancreas is a potential alternative to whole pancreas transplantation. The authors constructed a bioartificial pancreas using mesh reinforced polyvinyl alcohol hydrogel tubes (MRPT), in an attempt to clarify the in vitro responsiveness to glucose of islets seeded in the MRPT. When the MRPT were perfused in a small chamber with buffer containing 3.3 mmol or 16.7 mmol glucose, insulin release from the MRPT began to increase at 9 +/- 3 min, reaching a plateau at approximately 40 min after the glucose concentration in the perfusate increased from 3.3 to 16.7 mmol. When MRPT seeded with islets were subjected to static incubation in buffer containing 3.3 mmol or 16.7 mmol glucose, insulin release from the MRPT remained elevated for 3 hr of high glucose stimulation, the amount of secreted insulin depending upon the number of islets seeded. Although pre incubation of semipermeable membranes in culture medium containing fetal bovine serum prior to seeding with islets has recently been reported to improve insulin release, the authors found that such pre treatment of the MRPT did not have a beneficial effect. Their in vitro findings in this study suggest that the bioartificial pancreas using MRPT could be a promising therapeutic approach to human diabetes mellitus.

Animals↗