New class of oxygen carriers improves islet isolation from long-term stored rat pancreata.

OBJECTIVE: Pancreas shipment is frequently associated with prolonged ischemia deteriorating islet graft function. The strategy to prevent ischemic damage utilizing perfluorodecalin (PFD) for human pancreas oxygenation does not seem to improve isolation outcome. The present study investigated the efficiency of perfluorohexyloctane (F6H8), a hyperoxygen carrier characterized by low specific density (1.33 g/cm3) and lipophilic qualities, to facilitate islet isolation from long-term stored rat pancreata. MATERIALS AND METHODS: Prior to islet isolation, pancreata were intraductally flushed in situ with Kyoto solution (KS) and stored for 24 hours in KS, oxygenated PFD, or F6H8. RESULTS: Islet isolation performed after 24-hour storage in KS failed completely. The intrapancreatic pO2 in PFD- and F6H8-incubated pancreata was almost the same. In correspondence, the ATP content and viability of isolated islets were similar as well. In contrast, islet yield and in vitro function were significantly reduced after storage in PFD compared with F6H8. CONCLUSION: This study suggested that islet isolation performed after long-term pancreas preservation can be significantly improved utilizing semifluorinated alkanes as oxygen carriers.

The burst-phase intermediate in the refolding of beta-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy.

The kinetics of the guanidine hydrochloride-induced unfolding and refolding of bovine beta-lactoglobulin, a predominantly beta-sheet protein in the native state, have been studied by stopped-flow circular dichroism and absorption measurements at pH 3.2 and 4.5 degrees C. The refolding reaction was a complex process composed of different kinetic phases, while the unfolding was a single-phase reaction. Most notably, a burst-phase intermediate of refolding, which was formed during the dead time of stopped-flow measurements (approximately 18 ms), showed more intense ellipticity signals in the peptide region below 240 nm than the native state, yielding overshoot behavior in the refolding curves. We have investigated the spectral properties and structural stability of the burst-phase intermediate and also the structural properties in the unfolded state in 4.0 M guanidine hydrochloride of the protein and its disulfide-cleaved derivative. The main conclusions are: (1) the more intense ellipticity of the intermediate in the peptide region arises from formation of non-native alpha-helical structure in the intermediate, apparently suggesting that the folding of beta-lactoglobulin is not represented by a simple sequential mechanism. (2) The burst-phase intermediate has, however, a number of properties in common with the folding intermediates or with the molten globule states of other globular proteins whose folding reactions are known to be represented by the sequential model. These properties include: the presence of the secondary structure without the specific tertiary structure; formation of a hydrophobic core; broad unfolding transition of the intermediate; and rapidity of formation of the intermediate. The burst-phase intermediate of beta-lactoglobulin is thus classified as the same species as the molten globule state. (3) The circular dichroism spectra of beta-lactoglobulin and its disulfide-cleaved derivative in 4.0 M guanidine hydrochloride suggests the presence of the residual beta-structure in the unfolded state and the stabilization of the beta-structure by disulfide bonds. Thus; if this residual beta-structure is part of the native beta-structure and forms a folding initiation site, the folding reaction of beta-lactoglobulin may not necessarily be inconsistent with the sequential model. The non-native alpha-helices in the burst-phase intermediate may be formed in an immature part of the protein molecule because of the local alpha-helical propensity in this part.

Beneficial effect of FR183998, a Na+/H+ exchanger inhibitor, on porcine pancreas allotransplantation retrieved from non-heart-beating donors.

Activation of Na(+)/H(+) exchanger (NHE) may have an important role in the ischemia/reperfusion injury by producing intracellular calcium overload. Recent studies have shown a beneficial effect of an NHE inhibitor on the ischemia/reperfusion injury in the heart. In this study, we examined the effect of FR183998, a potent NHE inhibitor, in porcine pancreas allotransplantation from non-heart-beating Landrace pig donors (NHBDs). The four experimental groups included: untreated with no preservation (group 1; n = 3), treated with no preservation (group 2; n = 5), untreated with preservation (group 3; n = 6), and treated with preservation (group 4; n = 4). The preservation was made in ice-cold University of Wisconsin (UW) solution for 24 hours. The groups treated received 1 mg/kg FR183998 before donor cardiac arrest and 10 mg in the UW solution flush in situ. Serum blood glucose, insulin, and amylase were measured daily. An intravenous glucose tolerance test (IVGTT) was performed on the postoperative day (POD) 7 when pigs were sacrificed for histological examination. Graft survival rates on that day in groups 1,2,3, and 4 were 3 of 3; 5 of 5; 3 of 6; and 4 of 4, respectively. The mean K values of IVGTT in groups 3 and 4 were 0.78 +/- 0.10 and 1.27 +/- 0.16, respectively, which were significantly different (P < .05). Upon histological examination, pancreatic tissue in group 3 showed more severe edema and necrosis than other groups. FR183998 may be considered beneficial for ischemia/reperfusion injury to pancreatic grafts from NHBDs.

Excessive portal flow causes graft nonfunction in small size liver transplantation: an experimental study in pigs.

We investigated the effects of portocaval shunt (PCS) on excessive portal flow in producing sinusoidal microcirculatory injury in small-for-size liver transplants in pigs. The posterior segment of a whole liver (25%) was transplanted orthotopically. The pigs were divided two groups: group A, graft with PCS (n = 11), and group B, graft without PCS (n = 11). The PCS was a side-to-side anastomosis of the portal vein and the inferior vena cava. In group A, eight pigs survived for more than 4 days; all pigs except for one died of graft nonfunction within 24 hours in group B. The portal flow after reperfusion decreased in group A, but increased about three times greater in group B than that before the operation (P < .01). In group B, destruction of the sinusoidal lining and bleeding in the periportal areas were observed after reperfusion, findings that were not recognized in group A. These results suggest that graft nonfunction after small-for-size liver transplantation may be attributable to excessive portal flow producing sinusoidal microcirculatory injury.

Alcohol, high blood pressure, and serum gamma-glutamyl transpeptidase level.

The influence of the level of serum gamma-glutamyl transpeptidase, a biological marker of alcohol consumption, on elevations of blood pressure and on the development of hypertension related to increases in alcohol consumption was determined in a cross-sectional study of 1,492 middle-aged male workers and in a subsequent 5-year follow-up study of 1,393 workers. Blood pressure levels, as well as the prevalence and incidence of hypertension, were higher in the subjects with serum gamma-glutamyl transpeptidase levels above 50 units/l than in those with normal levels. These differences were more marked in drinkers who consumed 30 ml or more of alcohol per day. Thus, elevated serum gamma-glutamyl transpeptidase activity may identify drinkers at higher risk for the development of alcohol-related hypertension.

Rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dichroism.

Kinetic refolding reactions of ferricytochrome c and beta-lactoglobulin have been studied by stopped-flow circular dichroism by monitoring rapid ellipticity changes of peptide backbone and side-chain chromophores. In both proteins, a transient intermediate accumulates within the dead time of stopped-flow mixing (18 ms), and the intermediate has an appreciable amount of secondary structure but possesses an unfolded tertiary structure. It is suggested that the rapid formation of a secondary structure framework in protein folding is a common property observed in a variety of globular proteins.

Comparative pharmacodynamic analysis of TAT-59 and tamoxifen in rats bearing DMBA-induced mammary carcinoma.

TAT-59 suppressed the growth of DMBA-induced mammary tumors in rats earlier and more strongly than tamoxifen (TAM). After oral administration of the drugs, DP-TAT-59, one of the main metabolites of TAT-59, was found in 10- to 15-fold higher concentrations in both the tumor and blood compared to 4-OH-TAM, an active metabolite of TAM. In a 3-day antiuterotrophic test, every detected metabolite of TAT-59 showed stronger antiestrogenic activity than did TAM. In a competition assay, the affinity of the metabolites for estrogen receptors ranged from that of estradiol to that of TAM. These results suggest that the superior antiestrogenic activity of TAT-59 compared to TAM was either due to its higher penetration into tumor tissue or to the stronger antiestrogenic activity of its metabolites.

Possible involvement of interleukin-1 in ischemic brain edema formation.

To determine the contribution of interleukin 1 (IL-1) on ischemic brain edema formation, the effect of recombinant human interleukin 1 beta (rhIL-1 beta), or zinc protoporphyrin (ZnPP) as an IL-1 blocker, on brain edema was studied in rats. The animals were subjected to 60 min of ischemia in a middle cerebral artery occlusion model. Immediately after reperfusion, rhIL-1 beta at a dose of 10 ng/2 microliters, or ZnPP at doses of 1 and 10 micrograms/2 microliters were topically applied into lateral cerebroventricle. In rhIL-1 beta-treated rats, ischemic brain edema formation was significantly increased in the dorsal and ventral areas of the caudate putamen 24 h after reperfusion, compared to that of vehicle-treated control rats. Furthermore, in ZnPP-treated rats, brain edema was decreased in both caudate-putamen areas. This suggests that IL-1 plays an important role in pathogenesis for post-ischemic brain edema.

Elevations of serum angiotensin-converting enzyme and gamma-glutamyl transpeptidase activities in hypertensive drinkers.

Serum angiotensin-converting enzyme (ACE) activity was measured in 70 middle-aged male drinkers with or without elevated serum gamma-glutamyl transpeptidase (gamma-GTP) activity and high BP. Serum ACE activity in hypertensive drinkers with elevated serum gamma-GTP was higher than that in drinkers, both normotensive and hypertensive, with normal serum gamma-GTP. Serum ACE levels were strongly correlated with alcohol consumption and serum gamma-GTP levels. Since the induction of ACE by alcohol in hepatic cells has been disputed, the coincident elevation of serum ACE and gamma-GTP activities in hypertensive drinkers may support the contention that the close association between gamma-GTP levels and BP found in drinkers is not mainly related to the induction of gamma-GTP in the hepatic cells, but related to the hepatic cell damage caused by alcohol.

Relationship between serum gamma-glutamyl transpeptidase activity and blood pressure in middle-aged male and female non-drinkers.

Serum gamma-glutamyl transpeptidase (gamma-GTP) activity is a biological parameter for alcohol consumption. In our previous study, however, serum gamma-GTP levels seemed to correlate with blood pressure levels in both middle-aged male non-drinkers and drinkers. Therefore, the associations between gamma-GTP and BP were analysed more minutely in 385 male and 1126 female non-drinkers. The means of systolic (SBP) and diastolic (DBP) pressure in the males were significantly higher than those in the females, despite similar age and body mass index (BMI). The sex difference of BP disappeared after adjustment for serum gamma-GTP levels. The correlations between gamma-GTP and BP and the contributions of three variables, age, BMI, and gamma-GTP, to BP levels found in the non-drinkers in the present study were similar to those observed in the previous study on males including drinkers. These results suggest that common or possibly similar mechanisms which relate to the elevation of serum gamma-GTP activity are involved in the production of high BP both in non-drinking obese persons and in drinkers.

Relationship between serum gamma-glutamyl transpeptidase activity, blood pressure and alcohol consumption.

The associations among alcohol consumption, gamma-glutamyl transpeptidase (gamma-GTP) activity in serum and blood pressure (BP) were analysed in a cross-sectional study of 1,156 healthy male workers 35 to 59 years of age, consisting of 349 non-drinkers, 682 light-daily drinkers (consuming less than 58 ml of alcohol per day) and 125 heavy-daily drinkers consuming more. No marked elevation of BP with high alcohol consumption was seen in the present subjects. On the other hand, a linear elevation of BP together with an increase in serum gamma-GTP activity was found in subjects above 40 years of age. The BP levels corresponding to the three different drinking habits, non-drinkers, light drinkers and heavy drinkers, were similar to each other regarding gamma-GTP levels. The relationship between gamma-GTP and BP was shown, by multiple regression analysis, to be independent of age, obesity and the dose of alcohol consumed. The contribution to the rise in BP of the dose of alcohol consumed, in comparison with that of gamma-GTP, was negligible. Serum gamma-GTP activity is a useful indicator of the susceptibility to the pressor effect of alcohol.

Urinary excretion levels of sodium and potassium in environmental cadmium-exposed subjects.

The urinary excretion levels of sodium (Na) and potassium (K) in cadmium (Cd)-exposed subjects as related with urinary beta2-microglobulin (beta2-MG) and Cd concentrations were investigated. The target population comprised of 3164 inhabitants of Japan who were more than 50 years of age and who lived in cadmium-polluted areas and 294 inhabitants who lived in non-polluted areas of Japan. A significant increase in urinary excretion of K was shown in Cd-exposed subjects although there was no significant difference in urinary excretion of Na between the Cd-exposed and non-exposed subjects. Urinary K concentrations in both sexes were significantly correlated with age, urinary beta2-MG and Cd concentrations by multiple regression analysis. Urinary Na concentration was significantly correlated with age, beta2-MG and Cd in men, but in women it was significantly associated with only the urinary beta2-MG concentration. The present study demonstrated that increased K excretion was a more sensitive effect of cadmium exposure than increased Na excretion.

Down-regulation of Th2 cell-mediated murine peritoneal eosinophilia by antiallergic agents.

Local eosinophilia has been linked to the pathogenesis of the inflammatory aspect of allergic diseases. The present study found that co-injection of D10G4.1 (D10) cells, a murine Th2 clone, with conalbumin (CA) into the peritoneal cavity of AKR/J mice increased the number of peritoneal eosinophils. The accumulation of eosinophils reached a maximum level at 24 to 48 hr and was accompanied by a marked increase in the number of neutrophils and a minor increase in the number of mononuclear cells. D10-induced peritoneal eosinophilia was suppressed by administration of either anti-IL-4 and anti-IL-5 monoclonal antibodies in an additive manner or by cyclosporin A (CsA). Interestingly, suplatast tosilate (IPD-1151T), known to be antiallergic agent capable of suppressing IgE synthesis and chemical mediator release, but not disodium cromoglycate, selectively suppressed eosinophil accumulation. Taken together with the observation that CsA and IPD-1151T suppressed IL-4 and IL-5 production by CA-stimulated D10 cells in vitro, the present results strongly suggest that agents capable of down-regulating Th2 cell cytokine production may attenuate allergic inflammation by impairing the recruitment of eosinophils that is mediated by Th2 cells.

Progress of renal dysfunction in inhabitants environmentally exposed to cadmium.

The reversibility of beta 2-microglobulinuria, glucosuria, and aminoaciduria was examined in 74 inhabitants (32 males and 42 females) over 50 yr of age, who lived in a cadmium-polluted area. The subjects participated in two examinations conducted just after the cessation of cadmium exposure and 5 yr later. All urinary parameters did not show reversible changes. During the 5 yr the geometric mean concentrations of beta 2-microglobulinuria, glucosuria, and aminoaciduria indicated significant increases in excretion. In cases where greater than 1,000 micrograms/g creatinine of beta 2-microglobulinuria was observed (at the time cadmium exposure ended), almost all individuals exposed to cadmium showed deterioration of beta 2-microglobulinuria, whereas in the case of less than 1,000 micrograms/g creatinine of beta 2-microglobulinuria, no significant changes were observed. The present study indicates that cadmium-induced renal dysfunction in individuals environmentally exposed to cadmium is irreversible.

[A familial case of ADPKD with intracranial aneurysms detected by MR angiography].

The management and screening of unruptured asymptomatic intracranial aneurysm (ICA) in patients with ADPKD and those with a family history of ICA remains a subject of considerable controversy. In recent years, it has been revealed that MRA (magnetic resonance angiography) can define the circle of Willis to allow detection of ICA as small as 3-4 mm. We report a case of a 63-year-old man with ADPKD and his family. No definite aneurysm was observed by angiography screening at 46 years of age, when he was referred for hemodialysis. For the past three years, his family history revealed that three relatives were suffering from subarachnoid hemorrhage at the ages of 32, 36 and 39 years, respectively, two of whom had ICA and one had arterio-venous malformation detected by angiography. Whether they had ADPKD was unknown, but two were suggestive of ADPKD. Therefore, our case underwent MRA as screening for ICA, which showed an ICA with a diameter of 5mm in the anterior communicating artery. The ICA was confirmed as being 6 mm in diameter by conventional angiography. His niece and her son, who had ADPKD, also underwent MRA, which showed a suspicious image of a 2 mm ICA in the latter case. These results suggest that prophylactic screening for ICA is important in an ICA clustering family. MRA is useful in screening for ICA and in the follow-up study on the natural course of ICA.

[The pollen survey and dynamic statistics of patients with allergic rhinitis in Hakodate].

We have investigated the pollen survey (1994-1998) and dynamic statistics of patients with allergic rhinitis (1999-2000) in Hakodate, which is located southern part of Hokkaido. We have noted the pollen dispersion of Cryptomeria japonica, Cupressaceae, white birch, Gramineae and Artemisia. Especially, a lot of dispersion of Cryptomeria japonica has been noted in April. Concerning the dynamic statistics of patients with allergic rhinitis, we have investigated the 192 patients with allergic rhinitis in Hakodate municipal hospital. There has been a lot of pollinosis in March, April, May and September. Frequency of positive reaction to the specific IgE have been 38.0% of house dust, 16.9% of Artemisia, 13.2% of Gramineae, 10.3% of white birch, 9.0% of Cryptomeria japonica and 6.9% of cat in 379 subjects. In conclusion, we have noted that Cryptomeria japonica and white birch in addition to Gramineae and Artemisia are becoming more important antigen in patients with pollinosis in Hakodate, south part of Hokkaido.

Edaravone, a free radical scavenger, improves the graft viability on liver transplantation from non-heart-beating donors in pigs.

BACKGROUND: Although liver transplantation from non-heart-beating donors (NHBDs) is an effective way to overcome shortage of donors, primary graft nonfunction is often noted in these grafts. We have previously reported that edaravone, a free radical scavenger, has a cytoprotective effect on warm ischemia-reperfusion injury and improves the function of liver grafts from NHBDs in a rat model of ischemia-reperfusion. The purpose of this study was to investigate the effects of edaravone on liver transplantations from NHBDs. METHODS: Pigs were divided into three groups: (1) a heart-beating (HB) group (n = 5), in which liver grafts were retrieved from HB donors; (2) a non-heart-beating (NHB) group (n = 4), in which liver grafts were retrieved under apnea-induced NHB conditions; and (3) an edaravone-treated (ED) group (n = 5), in which liver grafts were retrieved in the same manner as the NHB group and treated with edaravone at the time of perfusion (3 mg/L in University of Wisconsin [UW] solution), cold preservation (1 mg/L in UW solution), and after surgery (1 mg/kg/d). The grafts from all groups were transplanted after 4 hours of cold preservation. RESULTS: In the ED group, the 7-day survival rate was significantly higher than that in the NHB group (80% versus 0%, P = .0042, Kaplan-Meier log-rank test). Furthermore, on histologic examination, the structure of sinusoids in the ED group was well preserved and similar to that in the HB group. CONCLUSIONS: Edaravone may improve the viability of liver grafts from NHBDs.

The impact of ischemic stress on the quality of isolated pancreatic islets.

BACKGROUND: Although the ischemic stress of donated organs has been shown to have strong negative effects on islet recovery, the impact on islet quality remains uncertain. In the present study, therefore, we examined the influence of ischemic stress on the expression of inflammatory mediators among isolated islets. MATERIALS AND METHODS: Islets were isolated from adult porcine pancreata subjected to 16-hour cold ischemia time (CIT) in addition to 40-minute warm ischemia time (WIT). We evaluated the islet yield, islet loss during the first 24 hours in culture, adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio, ATP/DNA ratio, glucose-stimulated respiratory activity, in vivo bioassay, and the expression of inflammatory mediators (tissue factor [TF], [MCP-1], macrophage migration inhibitory factor) on the isolated islets. We also analyzed ATP/DNA ratios of the exocrine tissues during isolation procedures. RESULTS: The islet yield, survival rate during culture, and glucose-stimulated respiratory activity were significantly lower in cases of 16-hour CIT plus 40-minute WIT compared with the control group (P < .0001, .0006, and .002, respectively). In contrast, ADP/ATP ratio as well as TF and MCP-1 expressions on the isolated islets were higher among the ischemic group (P = .005, .16, and .005, respectively). During isolation procedures, the ATP/DNA of the exocrine tissues was extremely lower in the ischemic compared to the control group (P < .0001). Notably, however, both ATP/DNA and ADP/ATP ratio of isolated islets were well preserved even in the ischemic group (P = .45 and .40). DISCUSSION: These data suggest that ischemic stress during the preservation period negatively affects the energy status of exocrine tissues. Destruction of the exocrine tissues, in combination with warm ischemic stress during the isolation procedures, subsequently decreases isolated islet activity, inducing the expression of inflammatory mediators.