The functional effects of suppression of hypothermia-induced cell swelling in liver preservation by cold storage

It is known that cellular edema and functional impairment develop during anaerobic cold storage of organs. The extent of both is related to the storage time and the composition of the preservation solution used. We studied hypothermia-induced cell swelling and its effect on liver function after cold storage preservation with either Eurocollins (EC), a number of modified EC solutions in which glucose was replaced by various concentrations of raffinose, or UW solution. After 24 h storage, tissue swelling as determined by total tissue water (TTW) in rat liver tissue slices was most pronounced in slices incubated in Eurocollins, whereas the TTW was only moderately increased in slices stored in modified Eurocollins containing 90 to 120 mM raffinose. In contrast, slices incubated in UW solution had a TTW equal to normal rat liver tissue. Furthermore, intact rabbit livers preserved with Eurocollins had an increase in the whole organ weight, while there was no weight change after preservation with the modified solution containing 120 mM raffinose (M120). In contrast, a pronounced weight loss was observed after preservation with UW solution. After cold storage, the livers were reperfused for 2 h at 38 degrees C in an isolated perfusion circuit (IPL) with an acellular perfusate. Bile flow was significantly greater in livers preserved in M120 than in those preserved with the conventional Eurocollins. However, the bile flow in the livers stored in M120 was inferior to that in the livers preserved with UW solution, which in turn was equal to that in control livers. The release of alanine-aspartate-aminotransferase into the perfusate was higher in livers preserved with Eurocollins, with or without modification, than in the livers preserved with UW solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular melibiose and fructose are intermediates in raffinose catabolism during fermentation to ethanol by engineered enteric bacteria.

Contrary to general concepts of bacterial saccharide metabolism, melibiose (25 to 32 g/liter) and fructose (5 to 14 g/liter) accumulated as extracellular intermediates during the catabolism of raffinose (O-alpha-D-galactopyranosyl-1, 6-alpha-D-glucopyranosyl-beta-D-fructofuranoside) (90 g/liter) by ethanologenic recombinants of Escherichia coli B, Klebsiella oxytoca M5A1, and Erwinia chrysanthemi EC16. Both hydrolysis products (melibiose and fructose) were subsequently transported and further metabolized by all three organisms. Raffinose catabolism was initiated by beta-fructosidase; melibiose was subsequently hydrolyzed to galactose and glucose by alpha-galactosidase. Glucose and fructose were completely metabolized by all three organisms, but galactose accumulated in the fermentation broth with EC16(pLOI555) and P2. MM2 (a raffinose-positive E. coli mutant) was the most effective biocatalyst for ethanol production (38 g/liter) from raffinose. All organisms rapidly fermented sucrose (90 g/liter) to ethanol (48 g/liter) at more than 90% of the theoretical yield. During sucrose catabolism, both hydrolysis products (glucose and fructose) were metabolized concurrently by EC16(pLOI555) and P2 without sugar leakage. However, fructose accumulated extracellularly (27 to 28 g/liter) at early stages of fermentation with KO11 and MM2. Sequential utilization of glucose and fructose correlated with a diauxie in base utilization (pH maintenance). The mechanism of sugar escape remains unknown but may involve downhill leakage via permease which transports precursor saccharides or novel sugar export proteins. If sugar escape occurs in nature with wild organisms, it could facilitate the development of complex bacterial communities which are based on the sequence of saccharide catabolism and the hierarchy of sugar utilization.     

Development of a cold storage solution for pancreas preservation

Canine pancreas tissue slices were incubated at 5 degrees C for 24 hr in solutions containing different saccharides (raffinose, sucrose, mannitol, or glucose). At the end of incubation tissue water (TW expressed as kg H2O/kg dry wt) was determined as a measure of tissue edema. Tissue edema was greatest in slices stored in Eurocollins (EC) solution (TW = 4.96 +/- 0.14) which contains glucose for osmotic pressure. The degree of edema was decreased by saccharides in proportion to their molecular mass: mannitol (MW = 180, TW = 3.84 +/- 0.08), sucrose (MW = 348, TW = 3.54 +/- 0.08), and raffinose (MW = 594, TW = 3.30 +/- 0.07). Tissue edema was also greatest in slices incubated in solutions containing the smallest molecular mass anions: Cl- (TW = 4.02 +/- 0.16), gluconate (TW = 3.69 +/- 0.10), and lactobionate (TW = 3.28 +/- 0.13). Cold storage of the intact pancreas in EC solution for 24 hr did not induce as much edema as in slices (TW = 2.88 +/- 0.10). However, on isolated reperfusion at normothermia (37 degrees C) the pancreas became edematous (TW = 3.33 +/- 0.12). Storage of the pancreas in a lactobionate-raffinose solution did not induce edema after 90 min of normothermic reperfusion. The suppression of tissue edema in the pancreas may be essential to obtaining long-term preservation (24-72 hr) of this organ which is currently limited to about 6-8 hr in EC solution. The newly developed lactobionate-raffinose solution appears to control tissue edema in both tissue slices and the intact-flushed out organ.     

Nonstructural carbohydrates in dormant and afterripened wild oat caryopses

Nonstructural carbohydrates were determined in both embryo and endosperm of dormant (nongerminating) and afterripened (germinating) intact caryopses of wild oat ( Avena fatua L.). No changes in endosperm starch or soluble sugar were observed at the onset of germination (18 h). No changes in glucose, fructose, sucrose or starch within dormant or afterripened embryos correlated with onset of visual germination. In afterripened embryos, depletion of raffinose (18 h), stachyose (18 h) and galactose (24 h) was correlated with germination. In contrast, raffinose-family oligosaccharide levels in dormant embryos remained constant for 7 days following imbibition. Germination of isolated dormant embryos on 88 m M galactose-containing media was accompanied by decreased endogenous levels of raffinose and stachyose. Isolated embryos from dormant caryopses incorporated ¹⁴C from ¹⁴C-fructose into both raffinose and stachyose during 24 h of imbibition. In contrast, no ¹⁴C incorporation into stachyose was observed in embryos from afterripened caryopses. No ¹⁴C incorporation into raffinose was observed at 18 and 24 h. When in vitro activities of α galactosidase were measured, no temporal differences between dormant or afterripened caryopses were detected in either embryo or endosperm tissue. Although the mechanism associated with differences in utilization of raffinose and stachyose is yet unidentified, alterations in raffinose-family oligosaccharide metabolism in the embryo appear to be a unique prerequisite for afterripening-induced germination.     

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 degrees C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9 h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 degrees C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.     

Effects of method of preservation on functions of livers from fed and fasted rabbits

Livers from fed, fasted (48 h) and glucose-fed rabbits were preserved for 24 and 48 h by either simple cold storage (CS) or continuous machine perfusion (MP) with the University of Wisconsin preservation solutions. After preservation liver functions were measured by isolated perfusion of the liver (at 37 degrees C) for 2 h. Fasting caused an 85% reduction in the concentration of glycogen in the liver but no change in ATP or glutathione. Glucose feeding suppressed the loss of glycogen (39% loss). After 24 h preservation by CS livers from fed or fasted animals were similar including bile production (6.2 +/- 0.5 and 5.6 +/- 0.4 ml/2 h, 100 g, respectively), hepatocellular injury (LDH release = 965 +/- 100 and 1049 +/- 284 U/liter), and concentrations of ATP (1.17 +/- 0.15 and 1.18 +/- 0.04 mumol/g, glutathione (1.94 +/- 0.51 and 2.35 +/- 0.26 mumol/g, respectively), and K:Na ratio (6.7 +/- 1.0 and 7.7 +/- 0.5, respectively). After 48 h CS livers from fed animals were superior to livers from fasted animals including significantly more bile production (5.0 +/- 0.9 vs 2.0 +/- 0.3 ml/2 h, 100 g), less LDH release (1123 +/- 98 vs 3701 +/- 562 U/liter), higher concentration of ATP (0.50 +/- 0.16 vs 0.33 +/- 0.07 mumol/g) and glutathione (0.93 +/- 0.14 vs 0.30 +/- 0.13 mumol/g), and a larger K:Na ratio (7.4 vs 1.5). Livers from fed animals were also better preserved than livers from fasted animals when the method was machine perfusion. The decrease in liver functions in livers from fasted animals preserved for 48 h by CS or MP was prevented by feeding glucose. Glucose feeding increased bile formation after 48 h CS preservation from 2.0 +/- 0.3 (fasted) to 6.9 +/- 1.2 ml/2 h, 100 g; LDH release was reduced from 3701 +/- 562 (fasted) to 1450 +/- 154 U/liter; ATP was increased from 0.33 +/- 0.07 (fasted) to 1.63 +/- 0.18 mumol/g; glutathione was increased from 0.30 +/- 0.01 (fasted) to 2.17 +/- 0.30 mumol g; and K:Na ratio was increased from 1.5 +/- 0.9 to 5.3 +/- 1.0. This study shows that the nutritional status of the donor can affect the quality of liver preservation. The improvement in preservation by feeding rabbits only glucose suggests that glycogen is an important metabolite for successful liver preservation. Glycogen may be a source for ATP synthesis during the early period of reperfusion of preserved livers.     

Contents of soluble carbohydrates in yellow lupin seeds maturated at various temperatures

The objective of this paper was to compare the levels of soluble sugars in seeds of yellow lupin cv. Juno matured at different temperatures. The temperature regimes applied were 1). 26 °C for 24 h (high temperature), 2). 24 °C for 12 h and 19 °C for the next 12 h (optimum temperature regime), 3). 26 °C for 16 h and 4 °C for the next 8 h (high-low temperatures). Six soluble carbohydrates (d-galactose, myo-inositol, sucrose, raffinose, stachyose and verbascose) were quantified. Seeds maturing at constant temperature 26 °C accumulated more raffinose (by 100 %) than seeds maturing at optimum temperature regime. Seeds maturing at high temperature accumulated less stachyose and verbascose than those maturing at optimum temperature conditions, the differences being 45 and 24 %, respectively. In seeds maturing at high-low temperature the level of raffinose decreased while the level of stachyose and verbascose increased, compared to those maturing at optimum conditions. The contents of sucrose, d-galactose and myo-inositol in seeds maturing at optimum temperatures was lower than in seeds maturing at both high and high-low temperature regimes. It was shown, that temperature conditions — constant high temperature, or physiologically optimal thermal oscillations (24 °/19 °C) or high-low temperature regime — differently affect the contents of six soluble carbohydrates in maturing seeds of yellow lupin.     

Dry weight accumulation and starch-synthesis enzymes in grain of cultured ears of three winter wheat varieties

Detached ears of three winter wheat ( Triticum aestivum L.) varieties were cultured in solution for 12 days with sucrose levels varying from 36.5 to 292 m M. The dry weight and starch content of grains increased asymptotically with the sucrose level in the solution. At 4 days of culture, glucose phosphate isomerase (EC 5.3.1.9) activity grain⁻¹ was lower with 36.5 m M than with higher sucrose levels in the medium; at 8 days, adenosinc diphosphoglucose pyrophosphorylase (EC 2.7.7.27) and (soluble plus bound) starch synthase (EC 2.4.1.21) activities grain⁻¹ were higher with 146 and 292 m M sucrose than with 36.5 and 73 m M sucrose. The multiple regression of starch content over these enzyme activities showed that starch synthase was relatively more important as an independent variable. The dry weight and starch content of grains were higher in the variety Maris Huntsman than in Splendeur and Hobbit. The water content of grains was lower in Splendeur than in the other two varieties. At 4 days the glucose phosphate isomerase, adenosine diphosphoglucose pyrophosphorylase and starch synthase activities grain⁻¹ were smaller in Splendeur than in Hobbit and Maris Huntsman and al 8 days they were higher in Maris Huntsman than in Hobbit and Splendeur. The varietal differences in starch content of grains were related to the activities of glucose phosphate isomerase and especially of starch synthase.     

Enhanced energy metabolism during cold hypoxic organ preservation: studies on rat liver after pyruvate supplementation

Previous studies have indicated that pyruvate is able to reduce ischaemia/reperfusion (I/R) injury in a variety of tissues, but a full understanding of the effects is lacking. In this current preliminary study, magnetic resonance spectroscopy (MRS) was used to investigate the biochemical effects of differing concentrations of pyruvate (3 and 15mM) on liver metabolism during the cold hypoxic preservation period itself, in order to gain insight into possible mechanisms. Hepatic lactate, alanine, and succinate levels were increased in livers preserved with 15mM pyruvate added to the University of Wisconsin (UW) solution and were generally elevated (but to a lesser degree) in livers flushed with 3mM pyruvate, compared to those cold stored in UW alone. Further, from enzymatic assays of adenine nucleotides, 15mM levels of pyruvate were found to maintain higher ATP levels during short periods (up to 4h) of cold hypoxic storage than in UW stored livers, whilst energy charge ratios (after 4 and 24h) were also higher (P<0.01 in each case). This may arise from enhanced glycolysis secondary to an improved redox status in the pyruvate-treated livers, as evident by the increase in the levels of lactate.     

Changing desiccation tolerance of pea embryo protoplasts during germination

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.     

Cryopreservation of epididymal sperm collected postmortem in the Tasmanian devil (Sarcophilus harrisii)

Effective sperm cryopreservation protocols are limited to a small number of marsupial species. In this study, postmortem gamete rescue (PMGR) epididymal sperm samples from Tasmanian devils (N = 34) euthanized due to the fatal Devil Facial Tumor Disease were used to develop long-term sperm storage techniques for the species. Cryoprotectant toxicity associated with equilibration of sperm samples in a TEST yolk diluent (TEST; 189 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 85 mM Trizma base [Tris], 11 mM glucose, 20% vol/vol egg yolk; pH 7.1, and 315.0 ± 5.0 mOsm/kg) with a final concentration of 0.06 M trehalose, or 4%, 10%, and 18% vol/vol of either glycerol or dimethyl sulfoxide (DMSO), was examined over 12 h at 15 °C. Trehalose supplementation resulted in an immediate decline (P < 0.05) of total motility. After 12 h, total motility was reduced (P < 0.05) in treatments containing 18% glycerol, and 10% and 18% dimethyl sulfoxide. The effects of final glycerol concentration (4% and 10%), glycerol equilibration duration (10 min 1 h, or 3 h) prefreeze, freezing rate and the addition of 0.10 M lactose or a combination of 0.10 M lactose and 0.11 M raffinose were assessed during three experiments on the cryopreservation of postmortem gamete rescue samples in TEST. In all experiments, motility and viability were reduced (P < 0.01 postthaw). Samples cryopreserved in TEST supplemented with lactose or lactose with raffinose using a fast freezing rate (−8 °C/min from 4 to −40 °C, then −65 °C/min until −165 °C) produced the highest (P < 0.05) postthaw motility (18.6 ± 5.5% and 16.9 ± 8.5%, respectively), which represented 35% to 48% retention of prefreeze motility. These results apparently were the best postthaw results of dasyurid sperm reported to date and will help lay the foundations for developing assisted reproductive technologies for marsupial species.     

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells (P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.     

Glutathione oxidation and activation of pentose phosphate cycle during hydroperoxide metabolism. A comparison of livers from fed and fasted rats

Perfusion of livers from fed and fasted rats with 0.07--0.1 mM t-butyl hydroperoxide for 15 min decreased the levels of reduced glutathione (GSH) by 1.5 mumol/g liver in both nutritional states. Glutathione disulfide (GSSG) was increased by 70 and 140 nmol/g liver and glutathione mixed disulfides enhanced by 45 and 150 nmol/g liver in livers from fed and fasted animals, respectively. The ratio of GSH/GSSG was decreased from 243 to 58 in fed animals, and from 122 to 8 in fasted animals. The increase of GSSG and the mixed disulfides was nearly parallel until an apparently critical low GSH content of 1.5 mumol/g was reached. Only in livers from fasted rats 14CO2-production from [1-14C]glucose was stimulated upon t-butyl hydroperoxide infusion at the employed rates. Flux of glucose through pentose phosphate cycle rose from 8 to 12% of glucose utilization via glycolysis, whereas in livers from fed animals this portion remained unchanged at 8% Dithio-erythritol reversed pentose phosphate cycle activity as well as GSSG and protein-bound glutathione contents to the original levels. In livers from fasted rats the activity of glucose-6-phosphate dehydrogenase was increased by 34% by t-butyl hydroperoxide infusion.     

Integral kinetics of alpha-galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose

alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.     

The role of the visceral yolk sac in hyperglycemia-induced embryopathies in mouse embryos in vitro.

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia-induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4-6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S-methionine and 35S-cysteine-labeled hemoglobin (35S-Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16-18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S-labeled products was observed at shorter exposure periods (12-24 and 24-36 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Microvascular changes of the liver preserved in UW solution. Pathological and immunohistochemical examination.

Rat livers preserved in University of Wisconsin (UW) solution for 24 h were compared with those preserved in Euro-Collins (EC) solution before and after liver transplantation using an immunohistochemical method. Tissue ATP and total tissue adenine nucleotide (TAN) were measured using HPLC. The levels of TAN in the UW group or the EC group were significantly low compared with the control group (no preservation) after 24-h storage. In the EC group, the levels of tissue adenine nucleotides (TAN) decreased 1 h after reperfusion and never reached control levels. In the UW group, the levels of TAN increased a little 1 h after reperfusion and increased more 3 h after reperfusion. After 24-h preservation, the expression of factor VIII-related antigen (FRA) in endothelial cells of central veins was weak in the EC group; in the UW group, FRA was clearly detected in these cells. After reperfusion, although severe endothelial cell damage to the central veins and numerous FRA-positive substances were observed in EC group, endothelial cells of central veins retained their normal structure and FRA-positive substances were rarely noted in the UW group. In both groups, no endothelial changes were detected in portal veins. From these results, it is concluded that UW solution prevents endothelial cell damage and microcirculatory injury in zone III during the preservation period resulting in prevention of initial graft nonfunction. Also, measurement of the TAN level after reperfusion is useful to predict the function of the graft.     

Sucrose feeding effects inhibition of gamma-glutamyltranspeptidase in the liver of the rat: possible mediation by thyroid hormone

The effect of sucrose on Fischer 344 rat liver gamma-glutamyltranspeptidase (gammaGT) was studied: in adults fed sucrose for 3 weeks; and rats exposed to sucrose from the 18th day of gestation to the 40th day after birth. Rats fed regular rodent chow served as controls. Sucrose caused mild lipemia; and in the liver an increase in size and fat build-up without damage. In adult sucrose-fed rats, compared to controls, plasma glucose levels were increased: 1.12-, 1.40- and 1.13-fold after 24, 48h and 3 week consumption of sucrose, respectively. Insulin levels were unaltered for the first week of sucrose consumption but increased from control levels: 16% at 1 week, and 2.0-fold at 3 weeks. The T3 levels were comparable to control levels 24h after the sucrose was started and were increased: 1.22-, 1.13- and 1.12-fold at 48h, 1 and 3 weeks, respectively. The T4 levels were comparable at all time points between sucrose-fed and control rats. Liver gammaGT activity exhibited a steady decrease from control levels: after 24, 48h, 1 and 3 weeks of sucrose feeding the decrease was 5, 8, 21 and 37%, respectively in homogenates; and 10, 17, 24 and 41%, respectively in plasma membranes. Perinatal sucrose exposure effected in 40-day-old rats, compared controls: a 1.09-fold increase in plasma glucose; no change in plasma insulin; an increase of 1.15- and 1.39-fold in plasma levels of total and free T3, respectively; a decrease of 20 and 14% in plasma levels of total and free T4, respectively. gammaGT activity was decreased in liver plasma membranes isolated from sucrose-exposed rats relative to those of control: 80% in the male; 82% in the female. Relative specific activities of gammaGT were the same in both males: 15.4 and 16.1 in control and sucrose-exposed male rats, respectively; and females: 14.1 and 15.4 in control and sucrose-exposed female rats, respectively. gammaGT was 2-fold higher in the livers of female relative to male rats in sucrose-exposed and control groups. Kidney gammaGT activities were the same in control and sucrose-exposed rats. The involvement of T3 in the sucrose-induced decrease in liver gammaGT is discussed.     

Import of sucrose and its transformation to starch in the developing sorghum caryopsis

Levels of soluble and bound invertases and amylases were studied in relation to the changes in the free sugars and the accumulation of starch in the developing sorghum [Sorghum bicolor (L.) Moench, cv. spv. 351] caryopsis and its associated bractspedicel. Besides sucrose, glucose and fructose as the principal sugars, small amounts of sugars of the raffinose series were detected in the developing caryopsis. Through out the period of caryopsis development, the amount of reducing sugars was higher than that of sucrose. With the advancement in the development of the caryopsis, the contents and levels of sucrose rose with a concomitant fall in the activity of soluble acid (pH 4.8) invertase (EC 3.2.1.26) in the endosperm. In the pericarp-aleurone layer, the activity of soluble acid invertase predominated over soluble neutral (pH 7.5) invertase (EC 3.2.1.27). The activity of bound acid invertase declined with the ageing of the caryopsis. In bracts-pedicel, the activity of bound invertase and the levels of reducing sugars peaked around 18 days post anthesis. In these organs, the level of starch gradually decreased concomitantly with an increase in its level in the developing caryopsis. Amylases (EC 3.2.1.1 and 3.2.1.2) are distributed in the endosperm as well as in the pericarp-aleurone layer. On culturing detached ears in [U-¹⁴C]-sucrose solution for 6 h in the dark at 25°C, 80–90% of the ¹⁴C of extracted major sugars (i.e. sucrose + glucose + fructose) of the caryopsis appeared in sucrose alone. In comparison with the effects of glucose or fructose, transport into the caryopsis of ¹⁴C from [U-¹⁴C]-sucrose supplied to detached ears was promoted by the addition to the radiolabelled sucrose solution of 1% unlabelled sucrose. Addition to the [U-¹⁴C]-sucrose solution fed to the detached ears of 20 mM NaN₃ or HgCl₂ or galactose, lowered the amount of ¹⁴C in the free sugars and starch of the earyopsis.     

Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine.

An approach to broaden the product range of the ethanologenic, gram-negative bacterium Zymomonas mobilis by means of genetic engineering is presented. Gene alaD for L-alanine dehydrogenase (EC 1.4.1.1.) from Bacillus sphaericus was cloned and introduced into Z. mobilis. Under the control of the strong promoter of the pyruvate decarboxylase (pdc) gene, the enzyme was expressed up to a specific activity of nearly 1 mu mol . min -1 . mg of protein -1 in recombinant cells. As a results of this high L-alanine dehydrogenase activity, growing cells excreted up to 10 mmol of alanine per 280 mmol of glucose utilized into a mineral salts medium. By the addition of 85 mM NH4+ to the medium, growth of the recombinant cells stopped, and up to 41 mmol alanine was secreted. As alanine dehydrogenase competed with pyruvate decarboxylase (PDC) (EC 4.1.1.1.) for the same substrate (pyruvate), PDC activity was reduced by starvation for the essential PDC cofactor thiamine PPi. A thiamine auxotrophy mutant of Z. mobilis which carried the alaD gene was starved for 40 h in glucose-supplemented mineral salts medium and then shifted to mineral salts medium with 85 mM NH4+ and 280 mmol of glucose. The recombinants excreted up to 84 mmol of alanine (7.5 g/liter) over 25 h. Alanine excretion proceeded at an initial velocity of 238 nmol . min-1 . mg [dry weight]-1. Despite this high activity, the excretion rate seemed to be a limiting factor, as the intracellular concentration of alanine was as high as 260 mM at the beginning of the excretion phase and decreased to 80 to 90 mM over 24 h.     

Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine.

An approach to broaden the product range of the ethanologenic, gram-negative bacterium Zymomonas mobilis by means of genetic engineering is presented. Gene alaD for L-alanine dehydrogenase (EC 1.4.1.1.) from Bacillus sphaericus was cloned and introduced into Z. mobilis. Under the control of the strong promoter of the pyruvate decarboxylase (pdc) gene, the enzyme was expressed up to a specific activity of nearly 1 mu mol . min -1 . mg of protein -1 in recombinant cells. As a results of this high L-alanine dehydrogenase activity, growing cells excreted up to 10 mmol of alanine per 280 mmol of glucose utilized into a mineral salts medium. By the addition of 85 mM NH4+ to the medium, growth of the recombinant cells stopped, and up to 41 mmol alanine was secreted. As alanine dehydrogenase competed with pyruvate decarboxylase (PDC) (EC 4.1.1.1.) for the same substrate (pyruvate), PDC activity was reduced by starvation for the essential PDC cofactor thiamine PPi. A thiamine auxotrophy mutant of Z. mobilis which carried the alaD gene was starved for 40 h in glucose-supplemented mineral salts medium and then shifted to mineral salts medium with 85 mM NH4+ and 280 mmol of glucose. The recombinants excreted up to 84 mmol of alanine (7.5 g/liter) over 25 h. Alanine excretion proceeded at an initial velocity of 238 nmol . min-1 . mg [dry weight]-1. Despite this high activity, the excretion rate seemed to be a limiting factor, as the intracellular concentration of alanine was as high as 260 mM at the beginning of the excretion phase and decreased to 80 to 90 mM over 24 h.