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P J Booth

Publications and source records attributed to P J Booth.

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

Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: work in progress.

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

Animals↗
PubMed1999

High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins.

We describe a bovine embryo culture system that supports repeatable high development in the presence of serum or BSA as well as under defined conditions in the absence of those components. In the first experiment, embryo development in SOF with amino acids (SOFaa), sodium citrate (SOFaac) and myo-inositol (SOFaaci) and with BSA or polyvinyl alcohol (PVA) was compared with that in a M199 granulosa cell co-culture (M199 co-culture). Subsequently, development and cell numbers of blastocysts cultured under defined conditions in SOFaaci with PVA (SOFaaci-PVA), or under undefined conditions in SOFaaci with 5% cow serum (SOFaaci-CS) or M199 co-culture were compared. The repeatability of culture results in SOFaaci-CS was checked by weekly replicates (n = 30) spread over 11 months. The viability of embryos developed in SOFaaci-PVA was estimated by transfer of morphologically good blastocysts (n = 10) to synchronized recipients. In the second experiment, the effect of omitting CS or BSA from IVM and IVM-IVF on subsequent embryo development in SOFaaci-PVA or in SOFaaci-CS was investigated. Blastocyst development in SOFaa-PVA, SOFaac-PVA, SOFaa-BSA and M199 was 16 +/- 3b, 23 +/- 2ab, 30 +/- 8a and 36 +/- 7a%, respectively (Pab < 0.05). Additional inclusion of myoinositol resulted in 42 +/- 1a% blastocysts in SOFaaci-PVA vs 19 +/- 3b% in SOFaac-PVA, 47 +/- 7a% in SOFaac-BSA, and 36 +/- 7a% in M199 co-culture, respectively (Pab < 0.01). In 30 replicates, the average cleavage and blastocyst rates of oocytes in SOFaaci-CS were 87 +/- 4 and 49 +/- 5%, respectively. Five normal calves were produced after transfer of 10 blastocysts developed in defined culture medium (i.e., SOFaaci-PVA). Defined IVM or IVM-IVF (i.e., in absence of CS and BSA) reduced cleavage rates (83 +/- 3 and 55 +/- 3% vs 90 +/- 1% in presence of CS; P < 0.01). Subsequent embryo development in SOFaaci-CS was not affected in either of these defined conditions. However, cleavage and blastocyst rates under completely defined IVP conditions were 54 +/- 7 and 19 +/- 4%, respectively. It was concluded that under defined culture conditions, addition of citrate and myo-inositol improved blastocyst development to rates comparable to those obtained with serum, BSA or co-culture and that the quality of blastocysts was not affected by the absence of serum or BSA. However, serum was essential during IVM/IVF for normal fertilization and subsequent high blastocyst development.

Amino Acids↗
PubMed1999

Membrane protein folding.

Investigating the in vitro refolding of proteins that naturally reside in biological membranes is a notoriously difficult task. Biophysical studies on model systems are beginning to provide a sound physical basis for membrane protein folding that should help to alleviate this problem. Highlights of these studies include insights into the interaction of transmembrane alpha helices, as well as into the important role that membrane lipids play in folding.

Bacteriorhodopsins↗
PubMed1998

Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos.

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.

Animals↗
PubMed1998

Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos.

Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000 degrees C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over -180 degrees C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty-one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction.

Animals↗
PubMed1997

Intermediates in the assembly of bacteriorhodopsin investigated by time-resolved absorption spectroscopy.

The in vitro folding and assembly kinetics of bacteriorhodopsin have been studied by absorption spectroscopy. Folding is initiated by rapid stopped-flow mixing of denatured apoprotein (bacterio-opsin) in SDS micelles and mixed dimyristoylglycerophosphocholine/Chaps micelles containing retinal. The apparent mixing rate of the two types of micelles has been determined by time-resolving the changes in light scattering by the micelles. Micelle mixing appears to occur in two stages: a fast phase with an apparent rate constant of about 420 s-1, and a second phase with an apparent rate constant of about 10 s-1. A rate constant of similar magnitude to the latter has previously been assigned to a protein-folding event on the basis of protein fluorescence studies [Booth, P. J., Farooq, A. & Flitsch, S. L. (1996) Biochemistry 35, 5902-5909]. However the results presented here show that this rate constant may be associated with a rearrangement of the mixed detergent/lipid micelles. When the changes in the retinal absorption band are time-resolved during assembly of bacteriorhodopsin, a retinal-protein intermediate, with an absorption maximum of about 430 nm, has been identified. This absorption maximum lies between that of unbound retinal (at about 380 nm) and the native chromophore (at about 560 nm). A comparison of fluorescence and absorption data, together with previous evidence [Booth, P. J., Flitsch, S. L., Stern, L. J., Greenhalgh, D. A., Kim, P. S., & Khorana, H. G. (1995) Nat. Struct. Biol. 2, 139-143], suggests that the covalent Schiff-base link to retinal is not formed in the 430-nm-absorbing intermediate.

Bacteriorhodopsins↗
PubMed1997

Slow alpha helix formation during folding of a membrane protein.

Very little is known about the folding of proteins within biological membranes. A "two-stage" model has been proposed on thermodynamic grounds for the folding of alpha helical, integral membrane proteins, the first stage of which involves formation of transmembrane alpha helices that are proposed to behave as autonomous folding domains. Here, we investigate alpha helix formation in bacteriorhodopsin and present a time-resolved circular dichroism study of the slow in vitro folding of this protein. We show that, although some of the protein's alpha helices form early, a significant part of the protein's secondary structure appears to form late in the folding process. Over 30 amino acids, equivalent to at least one of bacteriorhodopsin's seven transmembrane segments, slowly fold from disordered structures to alpha helices with an apparent rate constant of about 0.012 s-1 at pH 6 or 0.0077 s-1 at pH 8. This is a rate-limiting step in protein folding, which is dependent on the pH and the composition of the lipid bilayer.

Bacteriorhodopsins↗
PubMed1997

Evidence that bilayer bending rigidity affects membrane protein folding.

The regeneration kinetics of the integral membrane protein bacteriorhodopsin have been investigated in a lipid-based refolding system. Previous studies on bacteriorhodopsin regeneration have involved detergent-based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles. Here, we show that the short chain lipid dihexanoylphosphatidylcholine (DHPC) can be substituted for the detergent CHAPS and that bacteriorhodopsin can be regenerated to high yield in mixed DMPC/DHPC micelles. Bacteriorhodopsin refolding kinetics are measured in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is employed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectroscopy to follow changes in protein fluorescence during folding. Essentially identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein reaction is identified, in which retinal binds to a partially folded apoprotein intermediate, and the free energy of this retinal binding reaction is found to be the same in both types of mixed micelles. Formation of the partially folded apoprotein intermediate is a rate-limiting step in protein folding and appears to be biexponential. Both apparent rate constants are found to be dependent on the relative proportion of DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermediate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the two apparent rate constants associated with formation of this intermediate from 0.29 and 0.031 to 0.11 and 0.013 s-1, respectively. Formation of the intermediate also slows with increasing pH, from 0.11 and 0.013 s-1 at pH 6 to 0.033 and 0.0053 s-1 at pH 8. Since this pH change has no known effect on the phase behavior of lecithins, this is more likely to represent a direct effect on the protein itself. Thus, it appears to be possible to control the rate-limiting process in bacterioopsin folding through both bilayer bending rigidity and pH.

Bacteriorhodopsins↗
PubMed1997

Folding alpha-helical membrane proteins: kinetic studies on bacteriorhodopsin.

The correct folding and assembly of proteins within biological membranes is essential for membrane biogenesis and function. In contrast to the large body of work on water-soluble protein folding, however, very little is known about how membrane proteins fold to their final structures. Recent biophysical studies on membrane-protein folding in vitro are beginning to shed light on this problem. In particular, the forces that the membrane lipids impose on the folding protein appear to control certain events. The seven-helix transmembrane protein bacteriorhodopsin has been the focus of much attention and kinetic studies on the folding of this protein form the basis of this review.

Bacteriorhodopsins↗
PubMed1996

Retinal binding during folding and assembly of the membrane protein bacteriorhodopsin.

The factors driving folding and assembly of integral membrane proteins are largely unknown. In order to determine the role that the retinal chromophore plays in assembly of bacteriorhodopsin, we have determined the kinetics and thermodynamics of retinal binding during regeneration of bacteriorhodopsin, from denatured apoprotein, in vitro. Regeneration is initiated by rapid, stopped-flow, mixing of the denatured apoprotein bacterioopsin in sodium dodecyl sulfate micelles with mixed detergent/lipid micelles containing retinal. Regeneration kinetics are measured by time-resolving changes in protein fluorescence. The dependence of each kinetic component on retinal concentration is determined. Only one experimentally observed rate constant is dependent on retinal concentration, leading to identification of only one second-order reaction involving retinal and bacterioopsin. This reaction occurs after a rate-limiting step in bacterioopsin folding, and results in formation of a noncovalent retinal/protein complex. The free energy change of this retinal binding step is determined, showing that thermodynamic information can be obtained on transient intermediates involved in membrane protein regeneration.

Bacteriorhodopsins↗
PubMed1996

Assembly of light-harvesting chlorophyll a/b complex in vitro. Time-resolved fluorescence measurements.

The assembly kinetics of a pigment-binding membrane protein, the light- harvesting chlorophyll a/b complex of green plants, have been determined in vitro. Time-resolved fluorescence spectroscopy, with millisecond time resolution, has been used to monitor changes in both protein and chlorophyll (Chl) fluorescence, as well as in energy transfer from Chl b to Chl a, during complex assembly. Three reaction steps could be resolved after rapid, stopped-flow, mixing of the apoprotein (light-harvesting Chl a/b protein, LHCP) and pigments, solubilized in sodium dodecyl sulfate (SDS) and octyl glucoside (OG), respectively. A fast step in the range of 10 ms was detected regardless of whether the reaction mixture contained pigments or proteins, or both, and is interpreted as being connected with the formation of mixed SDS and OG detergent micelles. Two further steps were resolved: one with a time constant of about 1 min and another, slow step with a time constant of several minutes. Both of these were dependent on the presence of protein, Chls, and xanthophylls. Most, if not all, of the energy transfer from Chl b to Chl a was established during the slow step, indicating the the juxtaposition of these pigments, either by a structural rearrangement of the complex of by additional pigment binding, is the final stage in LHCII assembly in vitro.

Apoproteins↗
PubMed1996

Endocrine and metabolic responses to realimentation in feed-restricted prepubertal gilts: associations among gonadotropins, metabolic hormones, glucose, and uteroovarian development.

Temporal relationships among plasma LH, FSH, metabolic hormones, and glucose concentrations were studied in nutritionally manipulated prepubertal gilts. Twenty-four growth-matched littermate gilts (73.6 +/- 1.2 kg) were fed at maintenance for 7 d (d 1 to 7). From d 8 to 14, littermates were divided between two treatments and were either fed twice daily to appetite or continued on feed restriction. Feeding to appetite increased episodic LH secretion during the first 6 h of sampling on d 8 (P < .05). Plasma FSH concentrations were not affected by treatment between d 8 and 14. Nocturnal increases in plasma LH (P < .01) and FSH (P < .002) concentrations were detected in feed-restricted gilts but not realimented gilts. Mean total plasma IGF-I concentrations gradually increased in gilts fed to appetite (d 12, P < .02; d 13 to 14, P < .001). Mean postprandial insulin concentrations were also greater in gilts fed to appetite (P < .01). Treatment differences in mean postprandial glucose concentrations on d 9 and 10 indicate that normoglycemia was established by d 9 in gilts fed to appetite. At slaughter on d 15, numbers of ovarian follicles (P < .01), follicular volume (P < .001), and uterine weight (P < .05) were greater in gilts fed to appetite. In conclusion, uteroovarian responses to realimentation after short-term feed restriction in prepubertal gilts are mediated by enhanced LH secretion. This rapid enhancement of LH secretion in response to dietary repletion might have been mediated by changes in glucose and insulin status. Ovarian responses to gonadotropins may also be potentiated by observed increases in plasma glucose, insulin, and IGF-I. During feed restriction, a distinct diurnal rhythm in gonadotropin secretion was evident.

Aging↗
PubMed1995

Intermediates in the folding of the membrane protein bacteriorhodopsin.

Assembly of proteins within lipid bilayers is essential for the biogenesis and function of biological membranes. Little is known, however, about the underlying mechanism of assembly, and it is not clear whether it is possible to observe individual folding steps for integral membrane proteins either in vivo or in vitro. Fluorescence spectroscopy is used here to follow the time course of folding events for bacteriorhodopsin in mixed detergent/lipid micelles. Transient folding-intermediates are detected and binding of the retinal chromophore occurs at a late stage, when it binds to an apoprotein intermediate.

Apoproteins↗
PubMed1995

Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle.

Large-scale in vitro bovine embryo production systems commonly use genital tracts obtained from an abattoir as a source of both cumulus-oocyte complexes and co-culture feeder cells. Tissues derived from this source may be contaminated with non-cytopathogenic bovine viral diarrhoea virus (BVDV) since, in several countries surveyed, approximately 1% of animals tested are persistently infected with this pathogen. Therefore, the use of such material in in vitro fertilization systems presents a potential risk for the transmission of BVDV to bovine embryos and via embryo transfer. This potential was investigated by obtaining oviduct epithelial cells and granulosa cells, which are commonly used as feeder cells, from cattle persistently infected with BVDV and examining them for the presence of BVD viral antigen (p80 non-structural protein and gp53 envelope glycoprotein) by indirect immunofluorescent histochemistry, and also viral RNA (encoding the p80 region) by in situ hybridization. In addition, titres of virus present in oviduct, ovary and blood were assayed by immunodetection on calf testis cell cultures. Luminal epithelial cells from the oviduct and primary cultures of granulosa cells and oviduct epithelial cells from such cattle were shown to contain both viral antigen and RNA. The susceptibility of both cell types to BVDV infection was further established by inoculating primary cell cultures of cells derived from cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV (Pe515). RNA encoding BVDV and the antigen were detected 12 h after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Nutritional manipulation of growth and metabolic and reproductive status in prepubertal gilts.

Reproductive status, metabolic state, and carcass composition were compared in prepubertal littermate gilts of 85 kg BW and identical ages after allocation to one of two feeding regimens at 75 kg, either twice-daily feeding to appetite up to 85 kg and then maintenance feeding until slaughter (Group 85AR) or an initial period of maintenance feeding at 75 kg and then feeding to appetite to reach 85 kg (Group 85RA). Other littermates were studied at the start of treatment (Group 75C) and after the initial period of feeding to appetite (Group 85A) or maintenance feeding (Group 75R). In the absence of differences in backfat depth or longissimus muscle area, Group 85RA gilts showed increased follicular development (P < .001) compared with their feed-restricted Group 85AR littermates. Plasma LH (P < .001) and FSH (P < .02) responses to an LHRH challenge differed between these same groups. Feed restriction reduced preprandial glucose, postprandial cortisol, and insulin (P < .05), total plasma IGF-I, and basal and postprandial free triiodothyronine (T3; all P < .001) at all stages of the experiment. Postprandial insulin, pre- (P < .001) and postprandial free T3, and plasma IGF-I (P < .001) increased in response to refeeding (Group 75R vs 85RA). These data indicate that changes in metabolic status can mediate short-term nutritional effects on reproductive function, in the absence of changes in age, weight, and widely used indices of body composition, and also establish the short-term, feed-restricted, prepubertal gilt as an effective model for studying the mechanisms mediating nutrition-reproduction interactions.

Animal Feed↗