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B J Fuller

Publications and source records attributed to B J Fuller.

At least 37 records · Page 2Linked to original sources
PubMed1999

Incorporation of metabolite prior knowledge for data analysis: biochemical implications of dynamic 31P NMR ex vivo pig liver studies.

A semi-automated, metabolite prior-knowledge-based, lineshape fitting analysis has been developed to assess the dynamic biochemical changes found in ex vivo 31P NMR pig liver preservation studies. Due to the inherent experimental limitations of the ex vivo study and the complexity of the composite phosphorus resonances, metabolite information obtained in vitro was incorporated into the ex vivo analysis. This approach has allowed complete metabolite analysis (phosphomonoesters, inorganic phosphate, phosphodiesters and nucleotide triphosphates) in over 2000 spectra in a fraction of the time compared with more conventional analysis methods. The developed analysis will enable complete and rapid assessment of the biochemical changes in ongoing cold preservation studies of the pig liver which will result in thousands of ex vivo 31P NMR spectra. It is also envisaged that comparative studies on human donor livers will be carried out, in which this type of analysis would be the method of choice. Moreover, this kind of analysis approach could be advantageous in many complex in vivo NMR spectroscopy applications.

Animals↗
PubMed1999

Cryopreservation of bovine ovarian tissue: structural normality of follicles after thawing and culture in vitro.

The recovery of viable follicles from cryopreserved ovarian tissue would be of benefit in many areas of assisted reproduction. Structural integrity needs to be maintained following cryopreservation of ovarian tissue in order to retrieve healthy follicles which can then be cultured in vitro to produce viable oocytes. We have assessed the effect of in vitro culture of bovine tissue for 0, 1, 4, 24, or 48 h after exposure to, or cryopreservation in, dimethylsulphoxide. Immediately after freezing, normality of primary and preantral follicles within the tissue was significantly lower than for tissue exposed to the cryoprotectant without freezing or for control tissue. After 4 h in culture, cryopreserved tissue appeared to have recovered from damage caused by freezing, although the percentage of tissue with normal morphology declined after 24 and 48 h of culture. There was no significant difference between percentage normality in control tissue and tissue exposed to the cryoprotectant without freezing for any of the culture times studied. These data indicate that it is possible to freeze/thaw bovine ovarian tissue while retaining a reasonable yield of morphologically intact follicles and that a short period of post-thaw culture may enhance follicle recovery.

Animals↗
PubMed1999

Energy metabolism following prolonged hepatic cold preservation: benefits of interrupted hypoxia on the adenine nucleotide pool in rat liver.

The ability of brief hypothermic reperfusion (HtR) to restore hepatic energy metabolism following periods of cold hypoxic preservation was studied in isolated rat livers after storage times of 5, 10, and 24 h. In addition, investigations were performed on the effects of HtR used to restore liver oxidative metabolism in the middle of a prolonged (24 h) hypoxic preservation period. A histidine-lactobionate-raffinose solution was used for the initial cold portal flush in all groups. Results showed that cold hypoxia for either 5 or 10 h yielded livers capable of similar recoveries of ATP, energy charge, and total adenine nucleotides, but that HtR after 24 h cold preservation resulted in reduced regeneration of ATP, a lower energy charge, and a fall in tissue adenine nucleotides. When livers were stored for 24 h but subjected to brief HtR after either 5 or 10 h before return to hypoxic storage, improved recoveries of the energy metabolites were seen over those recorded after 24 h hypoxia alone. The fact that these improvements were not due to an improved supply of adenine nucleotide precursors was demonstrated by studying groups which were given HtR with perfusate containing precursors of adenine nucleotides (adenosine, adenine, and inosine) after 24 h cold hypoxia. These data are consistent with the hypothesis that poor metabolic recovery after long-term hepatic cold preservation results more from decreased mitochondrial oxidative phosphorylation than from a lack of precursors for adenine nucleotide resynthesis. In addition, restoring oxidative metabolism at hypothermia for brief periods can to some extent protect final metabolic status after prolonged storage.

Adenine Nucleotides↗
PubMed1999

Temperature dependence of Kedem-Katchalsky membrane transport coefficients for mature mouse oocytes in the presence of ethylene glycol.

Ethylene glycol (EG) is the emerging cryoprotectant of choice for preservation of mammalian embryos but has not been widely used for oocyte preservation. Techniques for oocyte cryopreservation need to be improved before they can be incorporated into routine clinical practice. Hence the permeability characteristics of oocytes in the presence of EG have been determined in order to facilitate the design of cryopreservation protocols using this cryoprotectant. Individual mouse oocytes were held using negative pressure applied to the zona pellucida by means of a micropipet. Each oocyte was perfused with 1 ml 1.5 mol L(-1) EG at 30, 19, or 10 degrees C, a total of 10 oocytes being perfused at each temperature. The osmotic response of each oocyte before, during and after perfusion was recorded on videotape. Measurements of mean cell diameter across three axes were used to calculate oocyte volume, assuming them to be spherical, and, using mathematical modeling, values for hydraulic conductivity (L(p)) were found to be 0.91 +/- 0.05, 0.51 +/- 0.02, and 0.18 +/- 0.01 microm min(-1) atm(-1); cryoprotectant permeability (P(EG)) was 0.24 +/- 0.01, 0.09 +/- 0.005, and 0.03 +/- 0.004 microm s(-1); and reflection coefficient (sigma) was 0.98 +/- 0.005, 0.96 +/- 0.01, and 0.97 +/- 0.01 at 30, 19, and 10 degrees C, respectively. The activation energy (E(a)) of L(p) was 14. 0 kCal mol(-1) and of P(EG) was 16.4 kCal mol(-1).

Animals↗
PubMed1999

A method for differentiating nonunique estimates of membrane transport properties: mature mouse oocytes exposed to glycerol.

Measurement of the osmotic response of a cell in the presence of cryoprotectant facilitates the determination of permeability coefficients which, in turn, can be used to design cryopreservation protocols which minimize osmotic stress. One problem encountered in determining permeability coefficients, using the Kedem-Katchalsky (K-K) model of membrane permeability, is that several combinations of the three passive coupled transport coefficients, namely, hydraulic permeability (L(p), microm min(-1) atm(-1)), solute permeability (P(gly), microm s(-1)), and the reflection coefficient (sigma), can give a similar fit to the measured data. A method for determining the "correct" set of coefficients is suggested. The osmotic response of 10 metaphase II mouse oocytes was measured on perfusion with 1.5 mol L(-1) glycerol at 24 degrees C. For 8 of 10 oocytes perfused, two combinations of L(p), P(gly), and sigma gave a predicted response which closely matched the measured osmotic response, depending upon the initial estimates supplied to the software for these parameters. For the remaining two oocytes, similar values for the permeability coefficients were generated regardless of the initial estimates. To determine the correct set of parameters, the K-K equations were used to predict experimental conditions for which volumetric histories would be distinctly different for the two sets of "best-fit parameters," and then additional experimental data were compared to these predictions. Thus a further three oocytes were perfused with 0.2 or 0.5 mol L(-1) glycerol in the absence of nonpermeating solute. In the presence of both 0.2 and 0.5 mol L(-1) glycerol, L(p) = 2.11 +/- 0.69, P(gly) = 0.0016 +/- 0.0015, and sigma = 0.44 +/- 0.11 yielded a very poor fit to the measured response while L(p) = 0.98 +/- 0.70, P(gly) = 0. 0031 +/- 0.0021, and sigma = 0.91 +/- 0.15 yielded a close fit to the measured response. Thus the latter combination of coefficients was taken to be correct.

Animals↗
PubMed1999

Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide.

Equilibration of oocytes with cryoprotectants is a prerequisite of low temperature storage. However, cryoprotectant exposure may induce damage via osmotic stress. Knowledge of cell membrane permeability characteristics and their temperature dependence would facilitate the design of cryopreservation protocols in which osmotic stress is minimized and the incidence of intracellular freezing is reduced. To obtain such data, the volume change of donated human oocytes following exposure to cryoprotectant was measured at a variety of temperatures. After removal of cumulus cells, each oocyte was placed in a 5 microl droplet of phosphate-buffered medium. The oocyte was held in position by suction generated using a fine pipette and perfused with 1 ml 1.5 mol/l dimethylsulphoxide (DMSO) at 30, 24 or 10 degrees C. The volume of the oocyte before, during and after perfusion was recorded by videomicroscopy. Oocyte volume was calculated from radius measurements and the Kedem-Katchalsky (K-K) passive coupled transport coefficients, namely L(p) (hydraulic permeability), P(DMSO) (permeability to DMSO) and sigma (reflection coefficient) were derived. The resulting coefficients were L(p) = 1. 65 +/- 0.15, 0.70 +/- 0.06 and 0.28 +/- 0.04 microm/min.atm; P(DMSO) = 0.79 +/- 0.10, 0.25 +/- 0.04 and 0.06 +/- 0.01 microm/s and sigma = 0.97 +/- 0.01, 0.94 +/- 0.03 and 0.96 +/- 0.01 at 30, 24 and 10 degrees C respectively. The activation energy for L(p) was 14.70 and for P(DMSO) was 20.82 kcal/mol. The permeability parameters of human oocytes are higher than those of murine oocytes, suggesting that they require a shorter period of exposure to DMSO with concomitantly reduced toxic effects.

Cell Membrane Permeability↗
PubMed1999

Renal vasoconstriction induced by oxidized LDL is inhibited by scavengers of reactive oxygen species and L-arginine.

BACKGROUND: Low density lipoprotein (LDL) may be involved in the pathogenesis of glomerulosclerosis and progressive renal dysfunction associated with atherosclerotic renal artery stenosis (RAS). This study was undertaken to investigate the effects of native (n-LDL) and oxidized LDL (ox-LDL) on renal vascular response and function in an isolated perfused rat kidney (IPRK) model. MATERIAL AND METHOD: IPRK model was used for the study at a constant pressure of 100 mm of Hg in the renal artery with continuous monitoring of pressure and renal perfusate flow. Urine and perfusate samples were collected to determine [14C] Inulin clearance and fractional reabsorption of sodium. To elucidate the role of nitric oxide (NO) urinary c-GMP, nitrate and nitrite excretion were measured and the responses to the NO synthase inhibitor N-monomethyl-L-arginine (LNMMA) and the NO donor Nitroso-glutathione (GSNO) were assessed. The effect of L-arginine supplementation and the role of reactive oxygen species were also studied by adding superoxide dismutase (SOD) and catalase. RESULTS: Ox-LDL but not n-LDL caused vasoconstriction in IPRK, as evidenced by a significant dose dependent reduction in renal perfusate flow. [14C] Inulin clearance and fractional reabsorption of sodium were reduced during ox-LDL infusion whereas no significant change occured with n-LDL. There was a significant decrease in urinary excretion of c-GMP during ox-LDL infusion. 10 microM LNMMA significantly increased and GSNO (10 microM) significantly diminished the vasoconstrictory effect of ox-LDL. The presence of L-arginine (100 & 500 microM) significantly decreased ox-LDL induced vasoconstriction. SOD (150 U/ml) and catalase (1200 U/ml) both had a significant inhibitory effect and the combination of SOD and catalase almost completely abolished the vasoconstriction due to ox-LDL. CONCLUSION: These results suggest that ox-LDL induced vasoconstriction in IPRK is mediated by decreased activity of NO probably due to inactivation of NO by reactive oxygen species. The free radical scavengers SOD, catalase and L-arginine provided protection against ox-LDL induced vasoconstriction in this model.

Animals↗
PubMed1998

Deuterium oxide-based University of Wisconsin solution improves viability of hypothermically stored vascular tissue.

BACKGROUND: Preservation of vascular function largely determines the outcome of transplantation. We have investigated replacing the water (H2O) in University of Wisconsin (UW) solution with deuterium oxide (D2O) in an attempt to improve vascular function after hypothermic storage. METHODS: Rat aortic segments were stored in UW solutions based on 100% H2O, 25% D2O, 50% D2O, and 100% D2O at 4 degrees C for 24, 48, or 72 hr. Vascular function was measured via contraction and endothelium-dependent relaxation after stimulation with phenylephrine and acetylcholine. RESULTS: UW solution with 25% D2O gave a significant (P<0.05) improvement of contraction and relaxation in comparison with H2O-based UW solution and other concentrations of D2O. CONCLUSIONS: Low concentrations (25%) of D2O-UW solution are significantly superior to the H2O-based (i.e., commonly used) equivalent at up to 72 hr. These results suggest that low concentrations of D2O-UW solution can improve the quality of hypothermic storage.

Adenosine↗
PubMed1998

Impairment of hepatic mitochondrial respiratory function following storage and orthotopic transplantation of rat livers.

Prolonged storage of organs for transplant results in tissue damage which may be compounded on reperfusion of the graft tissue. The effect of storage times was examined on hepatic mitochondrial oxygen consumption and activities of complexes I, II-III, IV, and V in mitochondria isolated from rat liver isografts stored for 25 min and 24 h pre- and posttransplantation. While Complex I activity was significantly (P < 0.05) inhibited under all the conditions studied, Complex II-III activity was only significantly (P < 0.05) reduced following transplantation of 24-h stored tissue. Complex IV activity remained unchanged under all the conditions studied. Although Complex V activity was significantly damaged within the first 25 min of ischemia, activity values were partially recovered to control levels following 3 h of reperfusion after transplantation. Prolonged (24 h) storage induced decreases in Complex V activity which were irrecoverable. Mitochondria subjected to 25 min ischemia alone also showed a significant (P < 0.01) decrease in NAD(+)-linked respiratory control indices due to a stimulated state 4 rate. The 24-h storage and transplantation brought about a significantly (P < 0.001) greater inhibition of respiratory control and state 3 respiration. FAD-linked respiration parameters were significantly (P < 0.05) affected in livers subjected to prolonged (24 h) storage or transplantation. These data suggest that a loss of membrane integrity coupled with an inhibition of Complexes I and V and an involvement of Complex II-III in 24-h stored hepatic transplants accounts for mitochondrial respiratory dysfunction in hepatic transplantation injury. No indication of Complex IV damage was found in this study. This study shows that damage to specific mitochondrial complexes occurs as a consequence of hypothermic ischemic injury.

Adenosine Triphosphate↗
PubMed1998

Metabolic effects of citrate in liver during cold hypoxia studied by 1H NMR spectroscopy.

We propose the use of 1H nuclear magnetic resonance (NMR) spectroscopy to investigate metabolite fluxes in the mammalian liver during cold hypoxia. Rat livers were flushed with one of four different preservation solutions and stored on ice in the same solution. The preservation solutions were: Marshall's hypertonic citrate (HC); carnosine modified HC (HC-C); modified University of Wisconsin (mod UW); and Bretschneider's histidine--typtophan--ketoglutarate (HTK). Liver biopsies were taken before and at 1, 2.5. 4, 24, and 48 h after storage, and freeze-clamped. The liver was extracted with perchloric acid and analyzed by 1H NMR spectroscopy. Components of the individual preservation solutions, such as citrate, histidine, mannitol, and raffinose, were detected in the extracts. Lactate was increased over the first 4 h in all stored livers, but only continued to increase in those stored in HC-C and HTK, reaching significantly high levels of 15 and 14 mumol/g, respectively, by 48 h storage (P < 0.05 and P < 0.01, respectively). Levels of succinate and fumarate in all livers were generally unchanged in the first 0-4 h of storage. However, after 4 h of storage, succinate levels rose in the HC and HC-C livers, while remaining unchanged in mod UW and HTK livers. The presence of citrate in the preservation solutions appeared to enhance the late hepatic synthesis of succinate. Fumarate levels were significantly decreased by 48 h of cold storage, indicating continued fumarate consumption at low temperatures. Despite cold hypoxic conditions, some carbon-substrate cycling appears to continue in mammalian liver via pathways other than glycolysis, and citrate from the preservation solution appears to influence this.

Adenosine↗
PubMed1998

Vitrification of mature mouse oocytes in a 6 M Me2SO solution supplemented with antifreeze glycoproteins: the effect of temperature.

Oocytes have been successfully cryopreserved using rapid and slow freezing procedures. However, variability in the success of replicates has limited its practical application. In the present study, mature mouse oocytes were vitrified in 6 M dimethyl sulfoxide supplemented with 1 mg/ml antifreeze glycoproteins (AFGP) (solution known as VSD + AFGP) from the blood of Antarctic notothenioid fish. Such AFGPs have been used to protect mammalian cells during hypothermia and cryopreservation. However, the degree of protection afforded is a contentious issue. Stepwise addition of cryoprotectant was performed either at room temperature (19-21 degreesC) or on ice (2-4 degreesC), at the final stage of which oocytes were pipetted into 0.25 ml plastic insemination straws and held in liquid nitrogen vapor at -140 degreesC for 3 min before being plunged into liquid nitrogen. Thawing involved holding the straw in the air for 10 s and then in water at 20 degreesC for 10 s before dilution of the VSD solution with 1 M sucrose. Viability was assessed by in vitro fertilization; results have been quoted as median (range). Statistical analyses were performed using Kruskall-Wallis and Mann-Whitney U tests (P < 0.05). Of the oocytes cryopreserved following exposure to VSD + AFGP at room temperature (n = 518, 15 experimental runs), 78% (0-94%) retained normal morphology and, of these, 53% (0-100%) cleaved to two cells. Of these two-cell embryos, 56% (0-100%) went on to develop to blastocyst. The overall percentage development to blastocyst, i.e., number of blastocysts/total number of oocytes treated x 100, was 20% (0-76%). Exposure of oocytes to the VSD + AFGP on ice prior to cryopreservation yielded significantly improved rates of fertilization (94%, 82-100%) and overall development to blastocyst (66%, 24-89%) when compared with oocytes cryopreserved following exposure to the VSD + AFGP at room temperature. Rates of normality (86%, 35-95%) and development to blastocyst (89%, 64-100%) were also improved. Cryopreservation in 6 M dimethyl sulfoxide supplemented with 1 mg/ml AFGP resulted in poor rates of survival which were highly variable when exposure to cryoprotective agent (CPA) was performed at room temperature. Lowering the temperature of exposure to CPA prior to cryopreservation resulted in improved viability.

Animals↗
PubMed1998

Differential effects of cryopreservation on nuclear or cytoplasmic maturation in vitro in immature mouse oocytes from stimulated ovaries.

The aim of this study was to develop a maturation protocol for immature oocytes and assess the protocol with cryopreserved oocytes. Nuclear maturation (mature spindle and aligned chromosomes) occurred irrespective of the treatment regime: 71-89% of oocytes matured in vitro had a normal spindle and chromosomes compared with 87% matured in vivo. Fertilization rates were not significantly different from those of in-vivo matured oocytes. Of the maturation treatment regimes investigated, the initial treatment producing best development to blastocyst (cytoplasmic maturation) involved a 2 h incubation in standard maturation medium (SMM) containing 7.5 IU follicle stimulating hormone (FSH) followed by 14 h in SMM plus 7.5 IU FSH:luteinizing hormone with follicular cells [62% (range 49-69)]. The addition of 1 ng/ml epidermal growth factor (EGF) in this protocol resulted in development [75% (range 71-81)] that was not significantly different from in-vivo matured oocytes [82% (range 73-90)]. Exposure of the oocytes to 1.5 M dimethylsulphoxide (DMSO) did not affect fertilization or development rates. Following a slow-cool/thaw freezing regime, 81% (range 74-89) of the oocytes were morphologically normal, i.e. had a spherical shape with an intact zona and oolemma; they had, however, lost their previously attached cumulus and corona cells. Maturation of frozen-thawed oocytes in the presence of EGF gave good fertilization rates but poor development rates [80% (range 77-86) and 37% (range 33-40) respectively]. In conclusion, the best maturation, both nuclear and cytoplasmic, occurred in the presence of a combination of gonadotrophins, EGF and follicular cells. Oocytes cryopreserved using a slow-cool/thaw regime can be matured to produce blastocysts after in-vitro fertilization.

Animals↗
PubMed1997

Assessment of quantitative artificial neural network analysis in a metabolically dynamic ex vivo 31P NMR pig liver study.

Quantitative artificial neural network analysis for 1550 ex vivo 31P nuclear magnetic resonance spectra from hypothermically reperfused pig livers was assessed. These spectra show wide ranges of metabolite concentrations and have been analyzed using metabolite prior knowledge based lineshape fitting analysis which had proved robust in its biochemical interpretation. This finding provided a good opportunity to assess the performance of artificial neural network analysis in a biochemically complex situation. The results showed high correlations (0.865 < or = R < or = 0.992) between the lineshape fitting and artificial neural network analysis for the metabolite values, and the artificial neural network analysis was able to fully represent the trends in the metabolic fluctuations during the experiments.

Animals↗
PubMed1997

Temperature dependence of mature mouse oocyte membrane permeabilities in the presence of cryoprotectant.

Knowledge of cell membrane permeability characteristics facilitates the design of cryopreservation protocols which minimize damage from osmotic stress and reduce the incidence of intracellular freezing. Such permeability characteristics can be determined for oocytes from volume measurements taken during exposure to cryoprotectant. Individual mouse oocytes were held using negative pressure applied to the zona pellucida by means of a micropipet. Each oocyte was perfused with 1 ml 1.5 mol liter-1 dimethyl sulfoxide (Me2SO) or propane-1,2-diol at 30, 23, or 10 degrees C. The osmotic response of each oocyte before, during, and after perfusion was recorded by videomicroscopy until equilibrium was reached. Mean cell diameter across three axes was used to calculate oocyte volume, assuming sphericity, and, using mathematical modeling, values for hydraulic conductivity (Lp) were found to be 0.64, 0.41, and 0.20 micron min-1 atm-1 in the presence of Me2SO and 0.53, 0.36 and 0.15 in the presence of propane-1,2-diol at 30, 23, and 10 degrees C, respectively. Cryoprotectant permeability (omega) was 0.37, 0.16, and 0.035 for Me2SO and 0.43, 0.24, and 0.04 for propane-1,2-diol, while the reflection coefficient was 0.98, 0.94, and 0.99 (Me2SO) and 0.76, 0.99, and 0.95 (propane-1,2-diol) all at 30, 23, and 10, respectively. The corresponding activation energies (Ea) were 11.65 and 12.23 kCal mol-1 for Lp and 23.52 and 22.48 kCal mol-1 for omega, in the presence of Me2SO and propane-1,2-diol, respectively. Values generated for Lp and associated Ea were similar to those found for mouse oocytes in the absence of cryoprotectant, while omega and its Ea were similar to those found for oocytes of other species.

Animals↗