Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Characteristics of galactomannanase for degrading konjac gel

Galactomannanase (Glmnase) is an enzyme product derived from Aspergillus niger. The activity of Glmnase degrading (hydrolyzing) the konjac gel were investigated. Significant loss in the enzyme activity was found when the temperature above 60 °C. Similar observations were obtained when the reaction pH above 5. Further increase in the pH value resulted in entirely loss of enzyme activity at the alkaline pH region (pH 8.0 and above). The optimal hydrolyzing temperature and pH were at 60 °C and 5.0, respectively. For the stability test, the purified Glmnase increased its thermostability up to 70 °C at pH 5.0, but it retained only about 60% activity after 60 min incubation at this temperature and its activity became zero after 20 min incubation at 80 °C. The Glmnase was stable at the pH range from 3.0 to 7.0 at room temperature and retained at least 80% activity for 60 min. For the storage temperature test, the lyophilized Glmnase still conserved about 90% activity during 7 days at 30 °C, and was higher than about 80% at 4 °C. The K_m and V_max, were 0.018 mg/ml konjac powder and 0.20 mg/ml reducing sugar per min, respectively.     

Ischemic preconditioning in a rodent hepatocyte model of liver hypothermic preservation injury

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.     

Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions

A uricase-producing bacterium was isolated from soil with a medium containing uric acid as the only carbon source. Based on its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the genus Microbacterium. After heat treatment at 70 °C for 30 min, the uricase retained about 100% of the initial activity. The enzyme activity remained largely unchanged when it was stored in borate buffer at pH 8.5 at 37 °C for 40 days. The effects of different factors on the enzyme production were studied. Maize milk was the best C and N resources, and the uric acid showed to be an inducer for uricase production. When the strain was cultured at 30 °C at pH 7.5 for 30–36 h, the uricase activity peaked at 1.0 U/ml.     

Primary throughput screening of protectants for hypothermic preservation of bioartificial liver in gel entrapped hepatocytes

Hypothermic preservation of hepatocytes has been widely studied for potential storage and transportation of bioartificial liver (BAL), but the liver-specific functions of hepatocytes are severely impaired by hypothermic treatment. A miniaturized gel entrapment-based BAL without circulation system was used to screen protectants from Chinese herbal medicines in this paper. Although anisodamine (ANI), matrine (MAT) and schisandrin B (Sch B) individually enhanced, to some extent, cell viability and liver-specific functions of hypothermically preserved hepatocytes, glycyrrhizic acid (GA), performed superior to these three extracts. The multieffect of GA on enhancement of mitochondrial membrane potential and inhibition of oxidative stress as well as lipid accumulation might determine its protection on hepatocytes from hypothermia-induced cell death. Furthermore, cell viability and intracellular glutathione (GSH) content decreased more dramatically at 6 h of the rewarming compared to those immediately after hypothermic preservation, indicating the aggravated cell injury by rewarming treatment. Therefore, gel entrapped hepatocytes in this study could be proposed for the throughput screening of desired conditions for hypothermic preservation of BAL.     

Body temperature dependency in baclofen-induced gastric acid secretion in rats relation to capsaicin-sensitive afferent neurons

Body temperature dependency in gastric functional responses to baclofen, a GABA(B) agonist, such as acid secretion, mucosal blood flow (GMBF) and motor activity, was examined in urethane-anesthetized rats under normal (37+/-1 degrees C) and hypothermic (31+/-1 degrees C) conditions. A rat stomach was mounted in an ex-vivo chamber, perfused with saline, and the acid secretion was measured using a pH-stat method, simultaneously with GMBF by a laser Doppler flowmeter. Gastric motility was measured using a miniature balloon as intraluminal pressure recordings. Intravenous administration of baclofen significantly increased acid secretion at the doses > 0.3 mg/kg under hypothermic conditions, yet it caused a significant stimulation only at doses > 10 mg/kg under normothermic conditions. The increases in gastric motility and GMBF were similarly induced by baclofen, irrespective of whether the animals were subjected to normothermic or hypothermic conditions. These functional responses to baclofen under hypothermic conditions were totally attenuated by either bilateral vagotomy or atropine (3 mg/kg, s.c.). Baclofen at a lower dose (1 mg/kg i.v.) significantly increased the acid secretion even under normothermic conditions when the animals were subjected to chemical deafferenation of capsaicin-sensitive neurons or pretreatment with intracisternal injection of CGRP8-37 (30 ng/rat). These results suggest that 1) gastric effects of baclofen are dependent on body temperature in stimulating acid secretion but not GMBF or motor activity, 2) the acid stimulatory action of baclofen is enhanced under hypothermic conditions, and 3) the suppression of baclofen-induced acid response under normothermic conditions may be related to capsaicin-sensitive afferent neuronal activity, probably mediated by central release     

Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

Summary In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.     

Temperature and carbon source affect the production and secretion of a thermostable β-xylosidase by Aspergillus fumigatus

The effect of the temperature of growth and carbon source on the production and secretion of β-xylosidase (EC 3.2.1.37) by the thermotolerant fungi Aspergillus fumigatus was studied in submerged cultures. In cultures developed at optimal temperature (30 °C), the enzyme was predominantly cell-bound, while in cultures developed at higher temperature (42 °C), the β-xylosidase activity was predominantly found in the cell-free filtrates. The use of corn cob powder instead of xylan as substrate increased considerably the secretion of enzyme. The highest level of extracellular β-xylosidase (45 U/ml or 360 U/mg protein) was obtained in 3% corn cob cultures grown at 42 °C for 72 h. The partially purified enzyme was active and stable at high temperatures. The presence of high titres of β-xylosidase activity in association with xylanase in the culture filtrates enhanced the efficiency of the pulp hydrolysis process.     

Effect of low temperature preservation and cell density on metabolic function in a bioartificial liver

Difficulties associated with bioartificial liver (BAL) preservation limit not only the commercialization of BAL, but also its clinical trials. In this study, the possibility of cold preservation of BAL cartridges containing porcine hepatocytes was examined at 4 °C. In anin vitro perfusion culture system, BAL cartridges maintained cytochrome P450 metabolic function for at least 50 days. However, all BAL cartridges completely lost their ammonia eliminating ability when stored at 4 °C. We also studied the effect of cell density on the maintenance of BAL liver function in a highly differentiated and healthy state. As expected, BALs containing a larger number of hepatocytes demonstrated higher metabolic functions. When metabolic functions were compared per gram of hepatocytes, no large differences were observed between devices containing different densities of hepatocytes. Decreased cell density did not successfully prolong BAL function. The viability and function of isolated hepatocytes highly depend on the culture conditions, such as cell density, substrata, culture media, and additives to the culture media. Perfusion culture of BAL cartridges at 4°C gave a promosing result with respect to the maintenance of P450 activity. However, as indicated by the rapid loss of ammonia metabolic activity, many factors still remain to be optimized for preservation of BAL keeping high metabolic functions for a longer time.     

The influence of hypothermia on the restoration of neural activity of brain slices from hibernating ground squirrels

1. 1. Neural activity was recorded in hippocampal slices from deep hibernating Yakut ground squirrels and in hippocampal and septal slices from non-hibernating animals.

2. 2. Slices were placed immediately after preparation in hypothermic conditions (3–4°C). Their activity was tested under standard conditions at 31°C in the incubation chamber. Some of the prepared slices were tested after maintenance in hypothermia for 2 or 24 h.

3. 3. In the hippocampal slices of hibernating ground squirrels, neural activity was present, irrespective of the period in hypothermia.

4. 4. Slices from guinea-pigs and hamsters did not possess neural activity after either 2 or 24 h of hypothermic treatment.

     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Perinatal epigenetic temperature adaptation in avian species: comparison of turkey and Muscovy duck

1. Heat production (HP) and body core temperature (CT) where measured in 1- to 10-day old Muscovy ducklings and turkey chick, incubated during the last week before hatching at a lower (34.5 °C, LT-group) or at higher (38.5 °C, HT-group), than the normal temperature of 37.5 °C (control C-group).

2. In Muscovy ducklings, on the 1st day post-hatching HP was affected by exposure to low T_a of 10 °C T_a 28.2±3.9 W kg⁻¹ in the LT-group vs. 18.1±2.4 W kg⁻¹ in normal controls. On the same day, CT was higher (39.5±1.1 °C) in the HT- than in the CT-group (37.5±2.9 °C).

3. In turkeys, the relationships between T_a and HP could be described by parabola-like functions. Apart from the first day of life, the HP of the LT-group and the HT-group was higher than of the CT-group.

4. The low prenatal temperature of incubation resulted in a decrease of the preferred temperature in the LH-group and in an increase in the HT-group.

5. It is concluded that changes in incubation temperature at the end of embryonic development may induce an epigenetic temperature adaptation, which results in a long-lasting cold- and warm-adaptation in ducks but not in turkeys.

Production, optimization growth conditions and properties of the xylanase from Aspergillus carneus M34

Growth conditions, including incubation times, temperature, agitation rate and initial pH of medium, that affect xylanase production by Aspergillus carneus M34 were studied sequentially use the classical “change-one-factor-at-a-time” method. Our results showed that there was a similar trend between cellular xylanase activity and extracellular xylanase activity. The optimal conditions for xylanase production, different from their cell growth, were on the third day, 30 °C, 100 rpm and pH 4, respectively, in this test. Response surface methodology (RSM) was further introduced to optimize the cultivation conditions and to evaluate the significance of these factors. The optimal cultivation conditions predicted from canonical analysis of this model were achieved by incubation at 35.08 °C with an agitation rate of 111.9 rpm and an initial pH of 5.16. In addition, temperature was the most critical factor for xylanase production by A. carneus M34. Xylanase activity of 22.2 U/mL was verified using the predicted optimal conditions and confirmed the fitness and applicability of the model. The optimal temperature and pH of the crude xylanase activity was observed at 60 °C and acidic pH, respectively. Sustained xylanase activity in the crude extract was also detected over a broad range of pH from 3 to 10. Considering its higher specificity toward agricultural wastes, especially corn cob and coba husk, this strain can be used to develop low-cost media for the mass-production of xylanase.     

Hypothermic preservation of hepatocytes : I. Role of cell swelling

Hepatocytes from isolated rat livers were hypothermically incubated (5 degrees C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation (r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermic incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.     

Liquid storage of Asian elephant (Elephas maximus) sperm at 4 degrees C

The Asian elephant (Elephas maximus) population in the wild has been in decline for several decades and breeding in captivity has not been self-sustaining. The use of artificial insemination (AI) can help overcome many of the difficulties associated with breeding elephants in captivity; however, the ability to store semen for extended periods of time is critical to the successful application of AI to elephants. The objective of the present study was to assess the effects of four different semen extenders and the presence of egg yolk on the viability and motility of Asian elephant semen stored at 4 °C. High quality ejaculates (n=4) were collected from two Asian elephant bulls by rectal massage. Aliquots of each ejaculate were extended in four different diluents (Beltsville thawing solution (BTS); Tris–citric acid (TCA)/fructose-based; Beltsville F5 (BF5); dextrose-supplemented phosphate-buffered saline (PBS)) with or without egg yolk then cooled and stored at 4 °C. The percentages of viable (viability) and motile (motility) sperm were evaluated at 8, 24 and 48 h following collection. The addition of egg yolk significantly reduced the percentage loss in viability from initial collection to 48 h compared to extenders without egg yolk (17.0±8.2 versus 32.6±8.9 decline in percent viable sperm in the population, respectively; P<0.05). Extender and egg yolk affected (P<0.005) total motility and percent progressively motile sperm at all evaluation times during incubation. TCA + egg yolk maintained higher (P<0.05) levels of progressive motility compared to other extenders supplemented with egg yolk. These results indicate that Asian elephant semen extended in TCA diluent supplemented with egg yolk can maintain at least 50% viability and motility when stored at 4 °C for 48 h.     

Production and partial characterization of bacteriocin-like pepitdes by Bacillus licheniformis ZJU12

Bacillus licheniformis ZJU12, which was isolated from soil, could produce bacteriocin-like peptides that exhibited a broad spectrum of antagonistic activity against various species of Gram-positive bacteria and fungal pathogens, but not against Gram-negative bacteria tested except Xanthomonas oryzae pv. oryzae, a rice pathogen. The bacteriocin-like peptides were sensitive to proteinase K and trypsin. The activity was stable during temperature exposure up to 100 °C for 30 min, but lost completely at 121 °C for 15 min. The cell-free supernatant of B. licheniformis ZJU12 was shown to retain the activity within the pH range of 2–9, and the optimum pH for the activity was about 6.5. No adverse effect of the antagonistic compound to mice was observed in acute toxicity tests with the dose of 0.8 mg/20 g.     

Hepatocyte NF-kappaB activation is hepatoprotective during ischemia-reperfusion injury and is augmented by ischemic hypothermia

The present study examined the role of hepatocyte NF-kappaB activation during ischemia-reperfusion injury. Second, we evaluated the effects of ischemic hypothermia on NF-kappaB activation and liver injury. C57BL/6 mice underwent 90 min of partial hepatic ischemia and up to 8 h of reperfusion. Body temperature was regulated during the ischemic period between 35 and 37 degrees C, 33 and 35 degrees C, 29 and 33 degrees C or unregulated, where temperature fell to <29 degrees C. Liver injury, as measured by serum alanine aminotransferase as well as liver histopathology, was inversely proportional to regulated body temperature, with the unregulated group (<29 degrees C) being highly protected and the normothermic group (35-37 degrees C) displaying the greatest injury. Inflammation, as measured by production of TNF-alpha and liver recruitment of neutrophils, was greatest in the normothermic groups and lowest in the ischemic hypothermia groups. Interestingly, hepatocyte NF-kappaB activation was highest in the hypothermic group and least in the normothermic group. Paradoxically, degradation of IkappaB proteins, IkappaB-alpha and IkappaB-beta, was greatest in the normothermic group, suggesting an alternate NF-kappaB regulatory mechanism during ischemia-reperfusion injury. Subsequently, we found that NF-kappaB p65 protein was increasingly degraded in normothermic versus hypothermic groups, and this degradation was specific for hepatocytes and was associated with decreased expression of the peptidyl-prolyl isomerase Pin1. The data suggest that NF-kappaB activation in hepatocytes is a protective response during ischemia-reperfusion and can be augmented by ischemic hypothermia. Furthermore, it appears that Pin1 promotes NF-kappaB p65 protein stability such that decreased expression of Pin1 during ischemia-reperfusion results in p65 degradation, reduced nuclear translocation of NF-kappaB, and enhanced hepatocellular injury.     

The activation of factor X by hepatocyte plasma membranes

Synthesis and secretion of blood coagulation factor X was studied during incubations of hepatocytes prepared by perfusion of rat livers with collagenase. The apparent molecular weight of factor X isolated from the incubation medium was about 14,000 less than factor X isolated from rat plasma. The extracellular form of factor X was a two-chain polypeptide and the observed difference in molecular weight was reflected in the heavy chain. Since these properties were more characteristic of factor Xa than factor X, experiments were designed to determine if factor X activation occurred during the incubations. Clotting factor assays indicated that factor X secreted by hepatocytes was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes the added factor X was converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium-dependent reaction. The results suggest that the activity is due to the presence of thromboplastin (tissue factor) and factor VII in the membrane preparations.     

Comparison of swimming speed and photophysiological responses to different external conditions among three Karenia brevis strains

Behavior, growth, and production are integral in the life history of Karenia brevis, an autotrophic, dinoflagellate HAB species, and are important variables in modeling blooms in the Gulf of Mexico. This study compares swimming speeds, growth rates, and photosynthetic responses of recent isolates of K. brevis (specifically the Apalachicola – APA, Manasota – MAN, and Jacksonville – JAX strains) over a range of light intensities and temperatures. Strain swimming speeds were similar and remained fairly constant from 17 to 30 °C, but decreased markedly at 13 °C. Photosynthetic responses of the strains to different acclimated temperatures had opposite trends with APA exhibiting higher electron transport rates (ETR) at higher temperatures and MAN exhibiting higher ETR at lower temperatures. In the light experiments, the cells’ internal physiological state (represented by photosynthetic yield, ETR, and neutral lipid reserves) and swimming capabilities were examined in the dark after 6 h incubations in the radial photosynthetron. For all strains, at initial incubation light intensities swimming speed decreased and ETR increased. As incubation light intensities increased, ETR decreased and swimming speed increased. At the highest incubation light intensities, ETR and swimming speed decreased. Neutral lipids followed a pattern similar to ETR, only lipids peaked after ETR at a light intensity that corresponded to the increase in swimming speed. The results suggest that cells may partition energy selectively depending on the needs of the cell. Information was combined to characterize a generalized species response to light and temperature ranges.