Rapid Recovery of Escherichia coli from Estuarine Water

The efficiencies of two 24-hr elevated-temperature tests to recover Escherichia coli from estaurine water were compared simultaneously with the 72-hr standard methods procedure of the American Public Health Association (APHA). From 1,710 tubes, E. coli was recovered 222 times in lauryl tryptose medium incubated at 44 ± 0.2 C for 24 hr, 261 times in an experimental medium incubated at 44.5 ± 0.2 C for 24 hr, and 257 times by the 72-hr APHA method. The number of false positives enumerated was similar in all three tests. The data indicated that E. coli in raw seawater could be determined in 24 hr without a significant loss of accuracy.     

Comparison of cold storage and perfusion of dog livers on function of tissue slices

We compared how two methods of hypothermic preservation affect physiological functions of tissue slices of dog liver. Livers were preserved by either (i) cold storage (CS) in Collins' solution or (ii) continuous perfusion (P) with a perfusate, containing hydroxyethyl starch, sodium gluconate, adenosine, and potassium phosphate, recently developed in our laboratory. Livers were cold stored for 6 to 8, 24, or 48 hr, and perfused for 24 or 72 hr. Tissue slices of preserved livers were incubated at 30 degrees C and analyzed for volume control, electrolyte-pump activity (K and Na), and adenine nucleotide concentration. Also, mitochondria were isolated after preservation to quantify respiratory activity. Slice functions of livers preserved for short periods (6 to 8 hr by CS and 24 hr by P) were similar to those for control livers. After normothermic incubation, the mean (+/- SD) water content of tissue (expressed per unit dry mass of tissue) was 2.3 +/- 0.3 kg/kg for control, 2.6 +/- 0.4 kg/kg for 6- to 8-hr CS, and 2.5 +/- 0.5 kg/kg for 24-hr P. Longer periods of preservation resulted in cell swelling, and water content was 3.3 +/- 0.4 kg/kg for 24- to 48-hr CS and 2.8 +/- 0.3 kg/kg for 72-hr P. The mean (+/- SD) K/Na ratio was nearly normal for livers preserved for short periods: 3.7 +/- 0.5 for control, 4.1 +/- 0.2 for 6- to 8-hr CS, and 3.3 +/- 0.4 for 24-hr P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coliform Behavior in Frozen Foods: I. Rapid Test for the Recovery of Escherichia coli from Frozen Foods

An assortment of 496 samples of frozen foods consisting of fish or marine products, variety types, and cream pie desserts were subjected to four parallel examinations for the recovery of Escherichia coli. The test procedures consisted of two low-temperature (35 C) and two high-temperature (44 C) presumptive tests, followed by an E C confirmatory test at 45.5 C. Of all the test methods examined, a single Lauryl Sulfate Tryptose (LST) presumptive test at 44 C gave best E. coli recovery (425). This recovery compared favorably with the lengthier Association of Official Analytical Chemists test with which only 420 E. coli cells were recovered. The LST (44 C) test saves much time, since it renders a follow-up 48-hr confirmatory test unnecessary. Moreover, since 96% of all the E. coli are recovered within 24 hr by LST (44 C), it is essentially a 24-hr test. The results of this study also confirmed earlier findings, in that it is possible to describe a specific coliform bacteriological test method by simple reproducible productivity ratios. E. coli recovery dilution data and coliform group behavior were also examined.     

Basal and HCG-stimulated testosterone production by interstitial cells of the Mongolian gerbil (Meriones unguiculatus)--I. Influence of preincubation length, medium composition and temperature

In the absence of HCG, production of testosterone by whole testes superfused in vitro was quite constant during the 5-hr superfusion period. Addition of 23-184 mIU/ml HCG caused a significant increase of testosterone production which was apparent from 30 min after start of superfusion. Basal and HCG-stimulated testosterone production by whole testes was significantly higher (400, 1950 ng/testis/5 hr, without and with 100 mIU HCG) than by isolated cells (200, 1350 ng/testis/5 hr). Incubation of isolated interstitial cells in medium 199 supplemented with fetal calf serum (FCS), (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid, HEPES) and 3-isobutyl-methylxanthine (MIX), and in medium 199 without FCS, HEPES or MIX, gave similar testosterone responses. While centrifugation at 8000 g for 2 min drastically diminished testosterone formation by isolated interstitial cells, production was similar by cells incubated in either 0.5, 1.0 or 1.5 ml medium. A significant decrease of testosterone synthesis by isolated interstitial cells was found when cells were stored at 4 degrees C for 2 days and then were incubated at 35 degrees C for 6 hr without or with 1-1000 microIU HCG. While isolated interstitial cells incubated at 5 degrees C did not produce testosterone at all, testosterone production increased to 49.5 +/- 3.9 ng/10(5) cells (30 degrees C) and 24.1 +/- 1.1 ng/10(5) cells (40 degrees C), respectively. HCG-stimulated testosterone production was maximal when interstitial cells were incubated at 34 degrees C.     

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).     

Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation

Effects of various preservation solutions were compared in an experimental hypothermic preservation model using cultured rat hepatocytes. Hepatocytes prepared by the collagenase perfusion method were cultured for 48 hr, then the medium in each culture dish was exchanged for various preservation solutions, and the dishes were hypothermically (0-2 degrees C) stored in a refrigerator for 12-72 hr. After the preservation period, the hepatocytes were cultured again at 37 degrees C for 2 hr. Hepatocytes' viability after 18-hr preservation and reculture was greater when they were preserved in "intracellular" rather than "extracellular" solutions. Even with Euro-Collins solution (intracellular solution), hepatocyte viability decreased to approximately 20% after 24-hr preservation, and an increase in the cellular lipid peroxide content was observed. However, when this solution contained a submillimolar concentration of calcium, lipid peroxidation was significantly suppressed and hepatocyte viability was dramatically improved. Vitamin E was almost equally effective and a marked synergistic effect was observed with calcium. Calcium was found to be capable of maintaining the cellular glutathione level during cold storage, which seems to suppress lipid peroxidation and consequently improve hepatocyte survival.     

Effect of drug treatment on liver-slice function following 72-hour hypothermic perfusion

The viability of hypothermically perfused dog liver was evaluated with a tissue-slice technique. After being preserved for 72 hr, slices of liver were incubated at 30 degrees C for as long as 2 hr; then water content, K+/Na+ ratio, and ATP concentration were measured. Dog livers were assigned to the following experimental groups: Group 1 (no preservation; control); Group 2 (livers preserved for 72 hr); Group 3 (donor animals pretreated with 3.5 mg/kg of chlorpromazine (CPZ) and 20 mg/kg of methylprednisolone (MP), and livers preserved for 72 hr); Group 4 (livers pretreated with 2-deoxycoformycin (2-DOC), 50 mg/liter, and preserved for 72 hr); and Group 5 (combination of Group 3 and Group 4 treatments). Livers in Groups 2, 3, and 4 lost K+ during preservation, and the mean K+/Na+ ratio significantly decreased from a control value of 4.2 +/- 0.4 to 1.5-1.9 (P less than 0.05). Group 5 livers did not lose K+; mean K+/Na+ ratio was 3.9 +/- 0.5. Fresh livers (no preservation) rapidly reaccumulated K+ when the tissue slices were incubated for 2 hr at 30 degrees C; mean K+/Na+ ratio was 3.7 +/- 0.5. Tissue slices from Group 2 livers (72 hr preservation), and livers pretreated with CPZ-MP (Group 3) or pretreated with 2-DOC (Group 4) did not significantly reaccumulate K+ at 30 degrees C; mean K+/Na+ ratio was 1.7-2.1. Only slices prepared from liver pretreated with both CPZ-MP and 2-DOC reaccumulated K+; mean K+/Na+ ratio was 4.6 +/- 1.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and Destruction of Salmonella typhimurium in Egg White Foam Products Cooked by Microwaves

Currently, in both home and institutional food preparation, attempts are being made to produce high quality foods with a minimum of time and effort. Research is being carried out to develop equipment capable of cooking foods in a fraction of the time required by conventional methods; as a result, the problem arises as to the bacteriological safety of these products. We investigated the microbiological aspects of lemon and chocolate foam pies before and after cooking by microwaves for less than 2 min. Pies prepared with sterile equipment under sanitary conditions were inoculated with washed cells from a 24-hr broth culture of Salmonella typhimurium and were incubated for 24, 48, and 72 hr at 33 C. The same procedures were followed in model systems to determine the effects of various sugar and pH levels on the survival of S. typhimurium. No S. typhimurium was detected in inoculated cooked or uncooked lemon pies by the plating method; with the Lactose Broth pre-enrichment method, survivors were detected in lemon pies immediately after preparation. After electronic cooking, no survivors were detected in lemon pies by plate counts, whereas cells were recovered from chocolate pies by the Lactose Broth method. Both chocolate and lemon pies had lower counts throughout the 72-hr incubation period than the model systems compared to them. With the model systems, at pH 7.3, media containing sugar inhibited the growth of S. typhimurium but did not cause a significant reduction in counts during the incubation times studied. At pH 3.7, media without sugar yielded no cells with the Lactose Broth pre-enrichment method after 48 hr of incubation, whereas media with sugar were not sterile until after 72 hr of incubation. Apparently, the presence of sugar in the medium had a protective influence which made the lethal effect of the low pH less severe.     

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.     

In vitro protein synthesis during germination and vernalization in winter wheat embryos

Protein synthesis during germination at 24?C and vernalizationat 4?C in winter wheat embryos were investigated with a cell-freesystem. During germination, the capacity for protein synthesisincreased in the early stage between 12 and 36 hr of imbibitionthen declined to a final low level between 48 and 72 hr. Thistransition was due to quantitative changes of the activitiesof ribosomal and supernatant fractions in the early stage andmainly to those of the supernatant fraction in the later stage.During vernalization, the capacity for protein synthesis continuedto decline over 15 to 60 days at 4?C. This transition was dueto the change in activity of the supernatant fraction; the activityof the ribosomal fraction was nearly constant. Electrophoretic analysis of in vitro products indicated thatthe high molecular weight proteins present in 12-hr embryoshad disappeared in 48-hr germinated wheat embryos and that theproducts in 24- and 36-hr embryos were types intermediate betweenthose of 12- and 48-hr embryos. The products in each vernalizedembryo resembled those in 24- and 36-hr germinated embryos.Therefore, it was concluded that the mRNA species for translationchanged during germination and vernalization in winter wheatembryos. (Received January 20, 1977; )     

Myristic acid, unlike palmitic acid, is rapidly metabolized in cultured rat hepatocytes

This study was designed to examine and compare the metabolism of myristic and palmitic acids in cultured rat hepatocytes. [1-(14)C]-Labeled fatty acids were solubilized with albumin at 0.1 mmol/L in culture medium. Incubation with 24-hr cultured hepatocytes was carried out for 12 hr. Myristic acid was more rapidly (P < 0.05) taken up by the cells than was palmitic acid (86.9 +/- 0.9% and 68.3 +/- 5.7%, respectively, of the initial radioactivity was cleared from the medium after 4 hr incubation). Incorporation into cellular lipids, however, was similar after the same time (33.4 +/- 2.8% and 34.9 +/- 9.3%, respectively, of initial radioactivity). In the early phase of the incubation (30 min), myristic acid was more rapidly incorporated into cellular triglycerides than was palmitic acid (7.4 +/- 0.9% and 3.6 +/- 1.9%, respectively, of initial radioactivity). However, after 12 hr incubation, the radioactivity of cellular triglycerides, cellular phospholipids, and secreted triglycerides was significantly higher with palmitic acid as precursor. Myristic acid oxidation was significantly higher than that of palmitic acid (14.9 +/- 2.2% and 2.3 +/- 0.6%, respectively, of the initial radioactivity was incorporated into the beta-oxidation products after 4 hr). Myristic acid was also more strongly elongated to radiolabeled palmitic acid (12.2 +/- 0.8% of initial radioactivity after 12 hr) than palmitic acid was to stearic acid (5.1 +/- 1.3% of initial radioactivity after 12 hr). The combination of elongation and beta-oxidation results in the rapid disappearance of C14:0 in hepatocytes whereas C16:0 is esterified to form glycerolipids. This study provides evidence that myristic acid is more rapidly metabolized in cultured hepatocytes than is palmitic acid.     

Daily alterations in plasma testosterone in boars at different ages

Eighteen purebred Yorkshire boars, reared outdoors on concrete, were randomly assigned in equal numbers to three age groups (150 +/- 7, 200 +/- 7 and 250 +/- 7 days of age) for the purpose of examining endogenous testosterone concentrations over a 24-hr period. Blood was obtained at 30-min intervals for 24 hr, and plasma testosterone concentrations were quantitated by radioimmunoassay. For each set of 24-hr samples, mean concentration (C), frequency (F) and magnitude (M) of secretory spikes of testosterone were determined. There was no difference (P>.10) in C, F and M across the three age groups. When averaged over ages, testosterone mean (+/- SEM) for C, F and M was 1.4 +/- .1 ng/ml, 2.9 +/- .5 and 3.0 +/- .3 ng/ml, respectively. The results suggest that testosterone secretory patterns are established in boars by five months of age and that these patterns may be influenced by degree of daylinght.     

Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin-like growth factor binding proteins.

Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin-like growth factor binding proteins (IGF-BPs). IGF-II tracer binding activity in conditioned media is two to three times greater than that of IGF-I. Under reducing SDS-PAGE conditions, 125I-IGF affinity-crosslinked binding protein (BP) is visualized as a broad band between 36 +/- 2.9 and 49 +/- 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non-reducing conditions (33-45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity-crosslinked binding protein exhibits a complex pattern of non-uniform densities that suggests structural or functional IGF-BP micro-heterogeneity. IGF-BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid-sized or smaller IGF-BPs exhibit side chains linked to the core protein with N-glycosidic linkage. None of the crosslinked IGF-BPs exhibit O-linked side chains. Long-term (12, 24, 48 hr) conditioning studies revealed that IGF-BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short-term (12 hr) experiments indicate that, in fresh medium, the levels of IGF-BP increase during the first 6-8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF-BP, aliquots of 8-hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37 degrees C for 16 hr. Importantly, it was found that incubation at 37 degrees C resulted in a near total loss of binding activity. This is the first report of IGF-BP degrading activity in a cell culture system. These findings indicate that 1) primate RPE cells rapidly secrete a complex mixture of N-glycosylated and non-glycosylated IGF-BPs, and 2) the steady state levels of secreted IGF-BP are tightly regulated at least in part through a concomitant IGF-BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF-BP synthesis, secretion, and degradation at the cellular level.     

Growth and survival of Escherichia coli O157: H7 in meat,poultry and vegetables mixed with different concentrations of mayonnaise

In the last 20 years Escherichia coli O157: H7 has emerged as a new pathogen, causing worldwide disease, death and economic loss. Different studies have revealed important survival characteristics of this pathogen, although there are divergent criteria about its ability to survive in various mayonnaise formulations. We studied the effect of different mayonnaise concentrations (0%, 18%, 37% and 56%) (weight/weight) over the survival of the bacterium in common foods from a neotropical environment (Costa Rica). High [10(7)-10(8) Colony Forming Units (CFU)/ml] and low E. coli populations (10(4)-10(6) CFU/ml) were inoculated, (three replicates) in meat, chopped cabbage and poultry, and mixed with commercial mayonnaise to obtain the concentrations specified. They were incubated at 12 degrees C for 24, 48 and 72 hr. The E. coli O157: H7 enumeration was done according to a standard methodology. Populations of E. coli O157: H7 showed an increasing trend during the first incubation period (48 hr), in all the preparations, regardless of the fat concentration used. Our data indicate that E. coli O157: H7 is capable of surviving and growing in meat, cabbage and poultry mixed with mayonnaise, independently of its concentration.     

INDUCING TIME FOR NEURAL TISSUES IN GASTRULA ECTODERM OF CYNOPS PYRRHOGASTER

The inducing times for spinal cord and deuterencephalon in Cynops gastrula were determined by the sandwich method. The extreme posterior of the archenteron roof at the slit-blastopore stage (tail organizer) was used as an inducer. First, the presumptive ectoderm of the earliest gastrula (0-hr stage) was put in contact with the organizer for 6 to 24 hr. Spinal cord and deuterencephalon were induced in almost all explants after 24 and 21 hr of contact, respectively, indicating that 24 hr is enough time for differentiations of both spinal cord and deuterencephalon. Next, presumptive ectoderm of 6- to 21-hr exogastrulae was put in contact with the organizer until the 24-hr stage. Results showed that the net inducing times for spinal cord and deuterencephalon were 18 and 15 hr, respectively, and that neural competence appeared in the presumptive ectoderm at the 6-hr stage (straight-blastopore stage).     

Uptake and effect of praziquantel and the major human oxidative metabolite, 4-hydroxypraziquantel, by Schistosoma japonicum

After exposure to praziquantel in vitro at a concentration of 1 microgram/ml for 0.5-2 hr, amounts of praziquantel in Schistosoma japonicum varied from 2.1 +/- 1.2 to 3.7 +/- 1.6 ng/male worm and 1.3 +/- 1.2 to 2.2 +/- 1.5 ng/female worm during the time studied. At 30 micrograms/ml, praziquantel amounts were 11-33-fold higher. However, within 2 hr after removal from a medium containing 30 micrograms/ml praziquantel, 95% of the drug was released from the parasites. When S. japonicum worm pairs were incubated in vitro with 1, 10, and 30 micrograms/ml of 4-hydroxypraziquantel, the major human oxidative metabolite of praziquantel, 0.2 +/- 0.2, 3.8 +/- 1.3, and 7.4 +/- 1.3 ng/worm pair, respectively, were found after a 2-hr incubation. 15-30-fold lower than corresponding worm pair amounts of praziquantel. In vivo, when 4- or 5-wk S. japonicum-infected mice were treated orally with praziquantel (300 mg/kg), peak concentrations of praziquantel in plasma determined by high pressure liquid chromatography were 14.7 +/- 1.5 micrograms/ml (4-wk infection) and 16.7 +/- 2.8 micrograms/ml (5-wk infection) 15 min after treatment. Corresponding in vivo worm praziquantel amounts were 1.8 +/- 0.4 ng/male worm and 2.4 +/- 1.1 ng/female worm, respectively, in the 4-wk infection and 4.6 +/- 1.6 ng/male worm and 5.6 +/- 1.2 ng/female worm in the 5-wk infection. Peak plasma concentrations of 4-hydroxypraziquantel were similar but corresponding in vivo worm amounts were 1-20-fold lower, depending on the time after drug administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Behavior of lambs in rest pens during long-distance transport

The aim of this study was to determine how one group of lambs utilized 2 consecutive rest periods in novel environments with access to food and water that occurred during 22 hr of motor transport. The 18.5 +/- 0.6 kg lambs (n = 15) were transported for 8 hr and then unloaded for a 6-hr rest period. After 6 hr, the lambs were reloaded for another 8 hr of transport followed by a 24-hr rest period. Reloading for a second 8 hr of transport followed the initial rest period. The percentage of lambs engaged in drinking, eating, lying, playing, or "other" was determined at 15-min intervals. During the 6-hr rest period, peak lying behavior occurred during the 2nd and 6th hr of the period. During the first 6 hr of the 24-hr rest period, the percentage of lambs lying increased while the percentage of lambs eating decreased. In addition, the percentage of lambs lying during the first 6 hr of the 24-hr rest period was greater than during the 6-hr rest period. Lying down had a greater priority than eating during the second (24-hr) rest period.     

Significance of anaerobic preincubation of Helicobacter pylori for measuring metronidazole susceptibility by the Etest.

The Etest is widely used for measuring the susceptibility of Helicobacter pylori to metronidazole. By using 55 H. pylori isolates from 55 patients and a standard H. pylori strain, NCTC11637, we compared metronidazole susceptibility results obtained from the Etest with or without anaerobic preincubation to those obtained from the agar dilution method. Mueller Hinton agar plates supplemented with 5% horse blood were used for both methods. For the Etest, plates were incubated for 72 hr at 35 C under microaerophilic conditions after 0-, 4- or 24-hr periods of anaerobic preincubation. For the agar dilution method, the plates were incubated at the same microaerophilic conditions as those for the Etest. Without anaerobic preincubation for the Etest, 39 of the 56 (70%) H. pylori isolates were categorized as resistant to metronidazole (minimal inhibitory concentration>8 mg/liter), whereas only one of the 56 (1.8%) isolates was resistant according to the agar dilution method. The resistant and susceptible agreement rate was 32%. Four-hour anaerobic preincubation did not alter the readings of the Etest significantly. However, when the Etest was performed with 24-hr anaerobic preincubation, the number of isolates categorized as resistant was reduced to six (11%), improving the agreement rate to 91%. For measuring the metronidazole susceptibility of H. pylori by the Etest, 24-hr anaerobic preincubation is necessary to agree with the results obtained by the agar dilution test.     

High developmental competence of cattle oocytes maintained at the germinal vesicle stage for 24 hours in culture by specific inhibition of MPF kinase activity

Roscovitine, a potent inhibitor of M-phase Promoting Factor (MPF) kinase activity, was used to maintain cattle oocytes at the germinal vesicle stage for a 24-hr culture period. A concentration of 25 microM of roscovitine was sufficient to reach the maximum level of meiotic resumption inhibition with 83 +/- 6% of the oocytes remaining at the germinal vesicle stage after the 24 hr of culture. The histone H1 kinase activity was maintained at a basal level after culture under roscovitine inhibition at any of the concentrations tested (12.5, 25, 50, and 100 microM). This inhibitory effect of roscovitine was fully reversible since 89 +/- 4% of the oocytes cultured for 24 hr in the presence of 25 microM of roscovitine reached the metaphase II stage after a further culture of 24 hr in permissive medium (TCM199 supplemented with 10 ng/ml EGF). The cleavage rate as well as the development to the blastocyst stage was not different for oocytes cultured for 24 hr under roscovitine (25 microM) inhibition and then matured for 24 hr in the presence of EGF as compared to oocytes not submitted to prematuration culture (82 +/- 8% cleavage and 41 +/- 4% blastocysts at 8 days post insemination for control oocytes compared to 90 +/- 7% and 36 +/- 7% respectively for roscovitine-treated oocytes). Roscovitine meiotic inhibition was also effective in the presence of EGF, and the final developmental potential as well as the kinetics of blastocyst formation were not affected after such prematuration treatment. The EGF induced cumulus expansion was also inhibited by roscovitine. These results indicate for the first time the feasibility of culturing cattle oocytes under meiotic inhibition without decreasing their resulting developmental potential.     

Pancreas preservation with TP-IV: a hyperosmolar colloid solution

This study compares the efficacy of a new hyperosmolar colloid solution (TP-IV) with Euro-Collins solution for long-term (72 hr) hypothermic storage of canine pancreas autografts. Four experimental recipient groups and their survival (30-day study period) results were as follows: Gr. I (n = 6) pancreatectomized controls, without autotransplant (X +/- SD = 5.83 +/- 3.06 days); Gr. II (n = 6) fresh nonpreserved autografts (X +/- SD = 23.83 +/- 10.12 days, 5 of 6 greater than 30 days); and Gr. III (n = 7) and Gr. IV (n = 5) receiving pancreas autografts stored at 4 degrees C for 72 hr in either Euro-Collins or TP-IV, respectively (Gr. III, 13.85 +/- 9.04 days; Gr. IV, 21.2 +/- 12.37 days). The results appear to indicate that TP-IV is superior to Euro-Collins solution for 72-hr hypothermic storage of pancreas grafts. In fact, survival in the TP-IV-presented group was comparable to that of fresh, non-preserved autografts.