Are food advanced glycation end products toxic in biological systems?

Model food advanced glycation end products (AGEs) were prepared as glycated casein (GC) and glycated soy protein (GS) by the reaction of casein or soy protein with glucose at 50 degrees C, relative humidity 75% for seven days in a powder state. These browned proteins were used as materials for animal experiments. A mixture of 20% glycated proteins (GC:GS = 1:1) diet was fed to streptozotocin (STZ)-diabetic rats for 11 weeks. The results showed that: (1) fructoselysine was observed in the hepatic portal veins, arteries, and femoral veins of rats fed with glycated proteins after 2 h of feeding; (2) blood sugar of glycated protein-fed rats was lower than that of diabetic rats fed with intact protein, while HbA1C in blood and glucose in urine of both groups were similar; (3) lipid peroxidation status in serum, liver, and kidney of both groups was similar; (4) superoxide dismutase (SOD) and glutathione-S-transferase (GST) enzymatic activity in serum and liver of both groups were also similar; (5) there were no differences in degree of cataract formation and concentration of glucose, fructose, sorbitol, and lipid peroxide in the lenses of both groups. From the above results, it can be estimated that food AGEs are not toxic in biological systems, and reactive oxygen species increase in diabetic rats is not caused by glycated proteins but by other pathways.

Transplantation of normal islets into the portal vein of Otsuka Long Evans Tokushima Fatty rats prevents diabetic progression.

To investigate the long-term effects of normal pancreatic islet transplantation on progression of obese type 2 diabetes mellitus (DM), 1500 normal islets (per rat) from Wistar King A rats at 8 weeks of age were transplanted into the liver through the portal vein of Otsuka Long Evans Tokushima Fatty (OLETF) rats, an animal model of obese type 2 DM, at 12 weeks of age. Body weight in the transplanted OLETF (IT) rats 8 and 28 weeks after islet transplantation did not differ from that in the corresponding sham-operated (SO) rats, but was greater than that in lean littermates (LETO rats; P < 0.05 for each group). In the early phase, 8 weeks after transplantation, rats in both IT and SO groups were normoglycemic, but hyperinsulinemic (P < 0.05 for each compared with LETO rats), probably resulting from increased body weight. In the late phase, 28 weeks after transplantation, hyperglycemia in the IT group was greatly attenuated compared with the SO group (P < 0.05), but hyperinsulinemia remained in both the IT and the SO groups compared with that in the LETO group (P < 0.05 for each). Immunohistochemical studies demonstrated that hypertrophic and fibrotic changes in pancreatic islets, together with mesangial proliferation of the glomerular matrix, an indicator for diabetic nephropathy, were attenuated predominantly in the IT group at the late phase after transplantation compared with those in the corresponding phase of the SO group. Islet transplantation into the liver of OLETF rats thus prevented further progression of obese type 2 DM. A possible mechanism is that islet transplantation may prevent development of hyperglycemia by improving abnormal hepatic glucose metabolism and consequently insulin resistance, which may lead to blockade of a vicious cycle between advancing damage to pancreatic islet cells and increased demand for insulin secretion, thus sparing original pancreatic cells from exhaustion induced by increased demand for insulin secretion.

Sphincter of Oddi motility in patients with hepatolithiasis and common bile duct stones.

The purpose of this study was to explore a difference in sphincter of Oddi (SO) motor activity among patients with intrahepatic (I, N = 5), intra- and extrahepatic (IE, N = 15), and common bile duct (CBD, N = 6) stones. Interdigestive motility of the SO and duodenum was studied by pneumohydraulic infusion manometry via the percutaneous route. SO phasic contractions showed a cyclic change in concert with the duodenal migrating motor complex (MMC) in all these patients. There was no significant difference in the cycle length, frequency, or amplitude of the SO phasic waves among the three groups throughout the whole cycle. The SO basal pressure during duodenal phases I and II of the duodenal MMC was significantly lower in patients with the IE type of hepatolithiasis than in those with the I type (P = 0.04), but there was no significant difference during phase III between the two groups. The SO basal pressure during phases I and II of the CBD group was also significantly lower than that of the I group (P = 0.02). The significance became even more prominent (P = 0.001) when a subgroup of patients with a dilated CBD (diameter > 1 cm) was examined. Lower basal pressure in the IE group or CBD group than in the I group suggested that stones in the common duct might injure or irritate the SO and cause SO dysfunction. In the subgroup with dilated CBD, which may have resulted from repeated and severe SO injury, the statistics became more prominent.

Pain associated with phase III of the duodenal migrating motor complex in patients with postcholecystectomy biliary dyskinesia.

BACKGROUND: Correlation between various gastrointestinal events and particular aspects of the migrating motor complex has been reported. This study correlates postcholecystectomy pain to variations in biliary pressure associated with the duodenal motor cycle. METHODS: In 18 patients with postcholecystectomy pain and 10 control subjects, biliary and duodenal pressures were recorded simultaneously with microtransducers. After recording a spontaneous cycle, morphine was administered to induce a premature phase III and spasm of the sphincter of Oddi, and then cerulein was administered to stop the spasm. RESULTS: Transient but significant elevations of biliary pressure occurred at duodenal phase III in both groups, but a greater percentage of the patients developed pain during phase III (89% vs. 20%, p<0.01). Morphine produced premature phase III and biliary pressure elevation, which were accompanied by pain more frequently in the patients than in the control subjects (78% vs. 30%, p<0.05). Biliary pressure dropped after the cerulein injection, relieving the pain in 13 of 14 patients and in 2 of 3 control subjects who had morphine-induced pain. The phase III-related pain was relieved by endoscopic sphincterotomy in 14 of 15 patients. CONCLUSIONS: The cyclic elevation of biliary pressure in coordination with phase III of the duodenal motor cycle may contribute to the development of pain in patients with postcholecystectomy biliary dyskinesia.

Endoscopic retrograde cholangiopancreatography in infants and children.

A single institutional experience with endoscopic retrograde cholangiopancreatography (ERCP) in pediatric patients was reviewed, focusing on the method of anesthesia, choice of an endoscope, indications, and complications. The medical records of 50 ERCPs performed in 42 infants and children (14 male and 28 female) were reviewed retrospectively. The patients' ages ranged from 57 days to 15 years. Forty-four ERCPs were diagnostic and 6 were therapeutic, including incision of choledochocele, and sphincterotomy and extraction of pancreatic stones. All procedures were successful. The most common indication for ERCP was to evaluate congenital biliary dilatation, in 28 patients (67%). Mild cholangitis occurred as a complication in 1 patient, but was alleviated with medication. A conventional duodenoscope could be used in patients older than 10 years. A pediatric duodenoscope was always used in patients under 1 year of age. Either type was chosen individually for those aged 1 to 10 years depending on the purpose, diagnostic or therapeutic. It is noteworthy that ERCP and/or sphincterotomy in a 1-year-old infant and two 2-year-old children were safely performed with the conventional endoscope. General anesthesia was employed in those younger than 9 years and intravenous sedation and local anesthesia in those older than 11 years. For children aged 9 to 11 years, anesthesia was chosen individually. We concluded that ERCP is a relatively easy and safe technique even for infants and children when performed by skilled hands with an appropriate duodenoscope under suitable anesthesia. The minimum age for use of the conventional duodenoscope may be 1 year.

Long-term consequence of endoscopic sphincterotomy for bile duct stones.

BACKGROUND: There are many reports of early- and intermediate-term results of endoscopic sphincterotomy. However, few data are available on long-term clinical outcome of endoscopic sphincterotomy for removal of common bile duct stones. METHODS: Of 419 patients who underwent endoscopic sphincterotomy, follow-up data were obtained in 410 patients (98%). The period ranged from 1 month to 20 years (average 122 months). RESULTS: Late complications included recurrence of stones (12.3%), acute cholangitis, acute cholecystitis (22% of 32 patients with gallstones, 0% of 88 patients without gallstones), new gallstone formation (6 patients), liver abscess (5 patients), and biliary carcinoma (8 patients). All of the recurrent stones were bilirubinate irrespective of the type of stone at sphincterotomy. Cholangitis and liver abscess occurred in 31% and 11%, respectively, of patients with residual intrahepatic stones but not in patients with complete intrahepatic stone clearance. CONCLUSIONS: Late complications occur in a considerable proportion of patients after endoscopic sphincterotomy for the treatment of common bile duct stones, including stone recurrence, acute cholecystitis (which occurs only in patients with gallstones), liver abscess in patients with residual intrahepatic stones, and biliary carcinoma. The fact that the recurrent stones are invariably of the bilirubinate type, irrespective of the type of stones at initial treatment, suggests that bacterial infestation due to ablation of the sphincter mechanism may have a causative role.

Adenosquamous carcinoma of the pancreas: report of two cases.

Adenosquamous carcinoma of the pancreas is a rare variant of pancreatic exocrine carcinoma. We herein report two patients with this entity. One patient was a 60-yr-old Japanese man complaining of a palpable mass, 5.5 cm in the greatest diameter, in the epigastrium. Serum CA 19-9 was increased (2010 U/ml). Ultrasonography and computed tomography showed a mass in the pancreatic tail with central necrosis and invading the posterior wall of the stomach. Angiography showed an encasement of the splenic artery and complete obstruction of the splenic vein. Distal pancreatectomy, splenectomy, and partial resection of the stomach were done. The patient died of uncontrolled bleeding from the duodenal ulcer four months after operation. The other patient was a 73-yr-old man who presented with jaundice. The CA 19-9 was also elevated (354.8 U/ml). Ultrasonography showed a pancreatic head mass of heterogeneous echogeneity and computed tomography demonstrated a cystic mass with an enhanced rim, indicating necrosis in the tumor center. Angiography showed a hypervascular mass in the head of the pancreas. Pylorus-preserving pancreatoduodenectomy was done, but the patient died of multiple liver metastases 10 months after the operation. From our experience with the two patients, the presence of central necrosis in an infiltrative huge pancreatic tumor seems to be suggestive of the diagnosis of adenosquamous carcinoma of the pancreas.

Organization of the human carboxypeptidase E gene and molecular scanning for mutations in Japanese subjects with NIDDM or obesity.

Insulin is synthesized in the pancreatic beta cell as a larger precursor molecule proinsulin which is converted to insulin and C-peptide by the concerted action of prohormone convertase 2 (PC2), prohormone convertase 3 (PC3) and carboxypeptidase E (CPE). One of the features of non-insulin-dependent diabetes mellitus (NIDDM) is an elevation in the proinsulin level and/or proinsulin/insulin molar ratio suggesting that mutations in these three proinsulin processing enzymes might contribute to the development of NIDDM. The identification of a mutation in the CPE gene of the fat/fat mouse which leads to marked hyperproinsulinaemia and late-onset obesity and diabetes is consistent with a possible role for mutations in CPE in the development of diabetes and obesity in humans. In order to test this hypothesis, we have isolated and characterized the human CPE gene and screened it for mutations in a group of Japanese subjects with NIDDM and obesity. The human CPE gene consists of 9 exons spanning more than 60 kb. Primer extension analysis identified the transcriptional start site at -141 bp from the translational start site. Single strand conformational polymorphism analysis and nucleotide sequencing of the promoter and entire coding region of the CPE gene in 269 Japanese subjects with NIDDM, 28 nondiabetic obese subjects and 104 nonobese and nondiabetic controls revealed three nucleotide changes, a G-to-T substitution at nucleotide -53, a G-to-A substitution at nucleotide -144 (relative to start of transcription) in the promoter region and a silent G-to-A substitution in codon 219. None of the nucleotide substitutions were associated with NIDDM or obesity. Thus, genetic variation in the CPE gene does not appear to play a major role in the pathogenesis of NIDDM or obesity in Japanese subjects.

Nutritional and physiological effects of casein modified by glucose, diacetyl, or hexanal.

Casein was modified by glucose, diacetyl, or hexanal at 50 degrees C, RH 75% for 1, 7, or 11 days. The chemical changes and digestibility in vitro of these nondialyzable caseins were investigated. The effects of these nondialyzable caseins supplemented with lost amino acids, on rats were studied by pair-feeding for 2 months. It was observed that internal organs such as liver, spleen, kidney, stomach, small intestine, cecum, colon and rectum were mostly unchanged. Biochemical values such as hematocrit, cholesterol, triglyceride, GPT, and GOT were also unchanged. However, the quantity of leucocytes was increased and serum glucose was decreased by feeding rats with modified caseins. Significant decrease in weight gain of rats fed with modified casein was observed, and the rate of decrease depended on the degree of modification of casein by carbonyl compounds. From these results, we supported the suggestion that some inhibitory or antinutritional compounds might be formed during the modification of casein by carbonyl compounds.

Analysis of the molecular requirements for T cell recognition and activation by using Ia-containing lipid vesicles and stopped-flow fluorometry.

Using Ia antigen-containing lipid vesicles, we investigated by stopped-flow fluorometry the requirements for helper T cell recognition and activation. When azobenzenearsonate-L-tyrosine (ABA-L-Tyr)-specific, I-Ak-restricted helper T cell hybridomas 2-45-12 were mixed with ABA-L-Tyr and purified I-Ak molecules on lipid vesicles, an increase of intercellular calcium ion concentration ([Ca2+]i) in the T cells were detected within 1-2s. The average increases of [Ca2+]i were not much different when the lipid vesicles were composed of dimyristoylphosphatidylcholine or of egg phosphatidylcholine, but they were dependent on the density of I-Ak molecules on the liposomes. The increase of [Ca2+]i was inhibited in the presence of anti-I-Ak monoclonal antibody 10.2.16, but not by the addition of anti-L3T4 monoclonal antibody GK1.5. However, the addition of anti-L3T4 antibody during the first 3 h of cultivation completely inhibited the T cell activation [interleukin (IL-2) production]. In our experimental system, IL-2 production was observed either when L3T4-positive T cell hybridomas 2-45-12 were stimulated with ABA-L-Tyr and Ia molecules on the vesicles in the presence of phorbol 12-myristate 13-acetate, or when L3T4-negative T cell hybridomas 3H60.12 were incubated with ABA-L-Tyr and Ia molecules on the planar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential sensitivity of anti-IgM-induced and NaF-induced inositol phospholipid metabolism to serine protease inhibitors in BAL17 B lymphoma cells.

A BAL17 B lymphoma cell line bearing mu and delta chains on its surface behaves in a similar manner to normal mature B cells in terms of initial biochemical transmembrane signalling [Mizuguchi, Beaven, Ohara & Paul (1986) J. Immunol. 137, 2162-2167; Mizuguchi, Yong-Yong, Nakabayashi, Huang, Beaven, Chused & Paul (1987) J. Immunol. 139, 1054-1059]. Therefore the effects of protease inhibitors on increases in inositol phospholipid metabolism and intracellular free calcium concentration ([Ca2+]i) were examined. We show that the serine protease inhibitors Tos-Phe-CH2Cl (1-chloro-4-phenyl-3-L-tosylamidobutan-2-one-, TPCK) and Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one; TLCK) inhibit anti-IgM-mediated accumulation of inositol phosphates in a dose-dependent manner. InsP3 production induced by anti-IgM is also inhibited by pretreatment with Tos-Lys-CH2Cl or Tos-Phe-CH2Cl. Tos-Lys-CH2Cl- Tos-Phe-CH2Cl-mediated inhibition is not overcome by high concentrations of anti-IgM. Moreover, anti-IgM-mediated increases in [Ca2+]i are inhibited by pretreatment of the cells with these inhibitors. However, increases in inositol phospholipid metabolism caused by NaF, an activator of guanine-nucleotide-binding proteins (G-proteins), are approx. 10-fold more resistant to Tos-Lys-CH2Cl and Tos-Phe-CH2Cl inhibition compared with anti-IgM-induced changes. Furthermore, NaF-induced increases in [Ca2+]i are not inhibited by Tos-Lys-CH2Cl or Tos-Phe-CH2Cl pretreatment, suggesting that the inhibitors act at a step proximal to phospholipase C activation. The Tos-Lys-CH2Cl or Tos-Phe-CH2Cl treatment does not change the membrane IgM density as measured by flow cytometry, indicating that the active site of the inhibitors is distal to the membrane IgM molecule. These results indicate that serine proteases may be involved in coupling the receptor cross-linkage to G-protein.

Unidirectional inhibition of early signal transduction of helper T cells by cloned suppressor T cells.

Early intercellular events occurring in cloned T helper (Th) cells following interaction with cloned T suppressor (Ts) cells were studied by stopped-flow fluorometry. It was found that the increase of intracellular Ca2+ ([Ca2+]i) in major histocompatibility complex (MHC)-restricted Th clones induced by the stimulation with antigen and antigen-presenting cells (APC) is inhibited by the incubation with antigen-activated Ts clones. Optimal suppression required that the two cells recognize antigen on the same APC, although the restriction element for recognition could be different. There was an absolute requirement for recognition of the same antigen by these two cell types. The inhibitory effect was unidirectional in that Ts clones could inhibit the increase of [Ca2+]i of Th clones but not vice versa. Ts clones could not suppress the [Ca2+]i response of other Ts clones. If Th and Ts clones do not share the same MHC restriction specificity, a longer co-incubation time for activation of Ts is required for the inhibition of the [Ca2+]i response of the Th clone, suggesting the presence of a non-specific suppressive mediator that selectively acts on Th.

T cell-mediated recognition of foreign antigen and the Ia molecule observed by stopped-flow fluorometry.

Th cell-mediated rapid recognition of foreign Ag and the Ia molecule was studied using azobenzenearsonate-L-tyrosine (ABA-L-tyrosine)-specific Th cells (I-Ak restricted), foreign Ag (ABA-L-tyrosine), and APC (H-2k). Initial transmembrane signals in Th cell hybridomas (2-45-12) and in Th cell lines (A24-17 or A33-7) were monitored by stopped-flow fluorometry with fluorescent probes. It was found that Th cells recognized foreign Ag within 1 s at 25 degrees C on the APC (B10.BR spleen cells or L cells into which I-Ak genes were transferred). Recognition of foreign Ag and the Ia molecule was shown to deliver the initial signals to Th cell hybridomas and T cell lines. First, Th cells had membrane fluidity increased and then calcium was transported from the external medium into the T cells. The initial transmembrane signals to Th cell hybridomas were inhibited by the addition of an anti-I-Ak mAb. None of the initial signals were observed in the absence of either specific foreign Ag or APC.

Phorbol myristate acetate inhibits increases in membrane fluidity induced by anti-IgM in B cells.

Anti-IgM or anti-IgD stimulates B cells to induce increases in inositol phospholipid metabolism and intracellular free calcium concentration [( Ca2+]i). Anti-IgM also causes increases in membrane fluidity that occur more promptly than those in [Ca2+]i in resting B cells as well as BAL17 B lymphoma cells. However, other B cell activators such as LPS or PMA did not induce the membrane fluidity changes. Furthermore, sodium fluoride, which is considered to be an activator of the guanine nucleotide-binding protein, caused increases in membrane fluidity as well as increased [Ca2+]i or inositol phospholipid metabolism. Anti-IgM- or sodium fluoride-induced increases in membrane fluidity were inhibited by 20-min pretreatment of cells with PMA, but not by 24-h pretreatment. These results indicate that membrane fluidity changes are closely associated with increased [Ca2+]i after cross-linkage of membrane Ig and are regulated by protein kinase C in B cells.

Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions.

Chimeric genes composed of immunoglobulin (Ig)-derived variable (V) regions and T-cell receptor (TCR)-derived constant (C) regions were constructed. The VL and VH genes showing anti-phosphorylcholine (PC) activity were used in this study. Two pairs of chimeric genes, VL-C beta and VH-C alpha genes, and VL-C alpha and VH-C beta genes, were inserted into an expression vector containing both Ecogpt and neo genes, and transfected into EL4 cells. Cells which express both chimeric receptor molecules were established. The activity of the transformants to the antigen was examined by using stopped-flow fluorometry. An increase in the concentration of cytoplasmic calcium ion was observed after addition of Staphylococcus pneumoniae R36A bacteria grown in the choline-containing medium which express PC molecules, but not after the PC-negative bacteria grown in the ethanolamine-containing medium.

Early transmembrane events in tumor necrosis factor and lymphotoxin-induced cytotoxicity.

We have studied early transmembrane events in tumor necrosis factor (TNF) and lymphotoxin (LT)-induced cytotoxicity by means of stopped-flow fluorometry with 3 different fluorescent probes. Both TNF and LT caused an increase in the membrane fluidity of the target cells (L X P3 cells). After this event, there was a low level of calcium influx from the external medium into the target cells. On the other hand, the release of calcium from intracellular stores in the target cells was negligible. These sequential events, however, were not observed when mutant LT molecules which lacked the lytic activity were used.

A serine protease triggers the initial step of transmembrane signalling in cytotoxic T cells.

We report here by using stopped-flow fluorometry with three different fluorescent probes that a serine protease triggers the initial step of transmembrane signalling in cytotoxic T cells. When cytotoxic T cells (mouse LC7, H-2b anti H-2d) bound to the specific target cells (mouse mastocytoma P815, H-2d), cytotoxic T cells first increased their membrane fluidity, and calcium then was released from intracellular stores. After that, there was a calcium influx from the external medium into the T cells. All of these steps, however, were blocked by serine protease inhibitors (soybean trypsin inhibitor, N alpha-p-tosyl-L-lysine chloromethyl ketone and tosylphenylalanyl chloromethyl ketone). Bovine pancreatic trypsin and chymotrypsin in the external medium mimicked the signalling events which were triggered by the serine protease on the T cell surfaces. From the reaction time (within 1 s) and its specificity, this serine protease in cytotoxic T cells was considered to be different from a protease which works at the killing stage.

Interleukin 2 increases T lymphocyte membrane mobility before the rise in cytosolic calcium concentration.

Using stopped-flow fluorometry with three different fluorescence probes [2-[(1-pyrenyl-butyryl)oxy]stearic acid, chlortetracycline and Quin 2], we have studied initial stage of T lymphocyte activation after interleukin 2 (IL-2) binding to a specific cell-surface receptor. After IL-2 binding to cytotoxic T lymphocyte (IL-2-dependent mouse LC7 and CTLL-2 cells), membrane mobilities of the cells increased first (4.5 +/- 0.3 s-1 for LC7 and 3.8 +/- 0.2 s-1 for CTLL-2), then calcium was released from intracellular stores into the cytoplasm (1.6 +/- 0.1 s-1 for LC7 and 2.1 +/- 0.1 s-1 for CTLL-2), and lastly, calcium was transported from the external medium into the cytoplasm (1.3 +/- 0.1 s-1 for LC7 and 1.5 +/- 0.1 s-1 for CTLL-2). The slowest process, the calcium influx from the external medium, was suppressed in the presence of a calcium channel blocking agent (verapamil). These observations give us a new information to discuss a model in T lymphocyte activation after IL-2 binding to cell-surface receptors.