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P S James

Publications and source records attributed to P S James.

At least 19 recordsLinked to original sources
PubMed2001

Lipid dynamics in the plasma membrane of ram and bull spermatozoa after washing and exposure to macromolecules BSA and PVP.

Seminal plasma proteins and macromolecules in the external medium have a major influence on the functionality of sperm plasma membranes. In this investigation we have examined their effects on lipid diffusion in the surface membrane of ram and bull spermatozoa as measured by fluorescence recovery after photobleaching (FRAP). Results show that progressive removal of seminal plasma from ram spermatozoa by repeated centrifugation and resuspension in media +/- 4% bovine serum albumin (BSA) or 0.4% polyvinlypyrrolidone (PVP) causes a reduction in lipid diffusion in all regions of the membrane. By contrast, bull sperm membranes respond with an increase in diffusion in all regions. Repeated washing of bull spermatozoa whose membranes were previously immobile (i.e., showed no recovery after FRAP) restored lipid diffusion suggesting an inhibitory effect of seminal plasma proteins. Further analysis by atomic force microscopy revealed a close association between BSA and the plasma membrane. It is concluded that diffusion of lipids in the plasma membrane of ejaculated ram and bull spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium. Mol. Reprod. Dev. 59:306-313, 2001.

Animals↗
PubMed2001

Diffusion barriers in ram and boar sperm plasma membranes: directionality of lipid diffusion across the posterior ring.

The plasma membrane of mammalian spermatozoa, like that of other differentiated cells, is compartmentalized into discrete regions or domains that are biochemically and functionally distinct from one another. Physical structures within the membrane, such as the posterior ring at the juncture of the sperm head and tail, have long been thought to act as diffusion barriers to help segregate important molecules required for fertilization within specific domains and to regulate migration of molecules between domains. In this investigation, we used a quantitative photobleaching technique (video-FRAP) to assess the efficacy of the posterior ring as a barrier to exchange of lipids between the postacrosomal and midpiece plasma membranes. A lipid reporter probe (1,1'-diduodecyl-3,3,3', 3'-tetramethylindocarbocyanine; DiIC(12)) was incorporated into the plasma membrane of live ram and boar spermatozoa, and the directionality of its diffusion across the posterior ring was measured by line-profile analysis. Results showed that DiIC(12) was able to traverse the posterior ring from the direction of the postacrosomal plasma membrane and to diffuse onto the midpiece plasma membrane. These results suggest that the posterior ring is not an immutable barrier to lipid exchange in mature spermatozoa and that there are other mechanisms for maintaining in-plane lipid asymmetry, such as differential phase behavior and interaction with the submembranous cytoskeleton.

Acrosome↗
PubMed1999

Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching.

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5-(octa-decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity.

Animals↗
PubMed1999

Lipid dynamics in the plasma membrane of fresh and cryopreserved human spermatozoa.

Preserving the integrity of the plasma membrane of spermatozoa is crucial for retention of their fertilizing capacity, especially after stressful procedures such as freezing and storage. In this investigation we have measured lipid diffusion in different regions of the plasma membrane of fresh and cryopreserved human spermatozoa using a sensitive, high resolution fluorescence photobleaching technique (FRAP) with 5-(N-octadecanyl)aminofluorescein as reporter probe. Results show that diffusion was significantly faster on the plasma membrane overlying the acrosome and decreased progressively in the postacrosome, midpiece and principal piece. The midpiece plasma contains a higher proportion of immobile lipids than other regions. In cryopreserved spermatozoa, lipid diffusion in the plasma membrane was significantly reduced on the acrosome, postacrosome and midpiece relative to fresh spermatozoa. Diffusion, however, could be restored to normal levels by washing spermatozoa in a medium containing 0.4% polyvinylpyrrolidine but not in medium alone or in medium containing 0.4% albumin. These results suggest that (i) lipid dynamics in the plasma membrane of human spermatozoa varies significantly between surface regions; (ii) in-plane diffusion is adversely affected by cryopreservation; and (iii) washing frozen spermatozoa in 0.4% polyvinylpyrrolidine restores membrane lipid fluidity to normal levels. The latter finding has important implications for improving the fertility of human spermatozoa following cryopreservation.

Adult↗
PubMed1999

Up-regulation of microsphere transport across the follicle-associated epithelium of Peyer's patch by exposure to Streptococcus pneumoniae R36a.

Transport of antigens through the follicle-associated epithelium (FAE) of Peyer's patch (PP) is the critical first step in the induction of mucosal immune responses. We have previously described that short-term exposure to Streptococcus pneumoniae R36a induced dramatic morphological alterations of the FAE in rabbit PP. These results prompted us to investigate whether the pneumococci-induced modifications were accompanied by enhanced ability of the FAE to transport antigens. We addressed this problem by evaluating the ability of the FAE to bind, internalize, and transport fluorescent polystyrene microparticles, highly specific to rabbit M cells, after exposure to S. pneumoniae. Quantitative study revealed a marked increase in the number of microspheres in PP tissues exposed to S. pneumoniae compared to tissues exposed to either phosphate-buffered saline or Escherichia coli DH5alpha as controls. No sign of bacterially induced damage to the epithelial barrier was observed. Further confocal microscopy analysis of the FAE surface showed that a significant increase in the number of cells that showed both morphological and functional features of M cells took place within pneumococci-treated PP tissues. These data provide the first direct evidence that the FAE-specific antigen sampling function may be manipulated to improve antigen and drug delivery to the intestinal immune system.

Animals↗
PubMed1998

Regionalized lipid diffusion in the plasma membrane of mammalian spermatozoa.

The plasma membrane of mammalian spermatozoa shows pronounced lateral asymmetry with many glycoproteins restricted to specific domains. Some of these antigens are freely diffusing throughout the membrane whereas others appear static in position. It is not clear whether these concepts also apply to membrane lipids. In this investigation we have used fluorescence recovery after photobleaching (FRAP) techniques to spatially resolve lipid dynamics in various surface domains of 5 species of mammalian spermatozoa (bull, boar, ram, mouse, and guinea pig). Sperm plasma membranes were loaded with 5-(N-octadecanoyl)aminofluorescein (ODAF) reporter probe, and its diffusion was measured in various domains by FRAP analysis. Results showed that in live bull, boar, ram, and mouse spermatozoa, diffusion coefficients (D) were significantly higher over the acrosome and postacrosome than on the midpiece and principal piece of the tail. In dead or permeabilized cells, on the other hand, large immobile phases developed, particularly on the sperm tail, that severely reduced D values. ODAF diffusion was also sensitive to temperature and cross-linking of protein components within the membrane with paraformaldehyde. Guinea pig spermatozoa were different in almost all respects from those of the other species tested. It is concluded that lipid diffusion in the plasma membrane of live spermatozoa varies significantly between surface domains, because of either compositional heterogeneity, or differences in bilayer disposition, or the presence of intramembranous barriers that impede free exchange between domains. This study emphasizes the important role of membrane lipids in regulating polarized migration of sperm surface antigens during developmental processes such as maturation and capacitation.

Acrosome↗
PubMed1997

Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death.

Compartmentalization of surface membrane antigens into discrete regions or domains is a characteristic feature of differentiated cells. In mammalian spermatozoa at least 5 surface domains are known, implying the presence of barriers or boundaries within the plasma membrane. Using the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusibility of fluorescent lipid analogues 1,1'-dihexadecyl-3,3,3'3'-tetramethylindocarbocyanine (DiIC[16]) and 5-(N-octa-decanoyl) aminofluorescein (ODAF), we have investigated lipid topology and dynamics in the plasma membrane of ejaculated bull spermatozoa. Contrary to reports in the literature, we have found that DiIC(16) stains only dead or damaged spermatozoa whereas ODAF intercalates into the plasma membrane of both live and dead cells, each type showing a distinctive staining pattern. FRAP analysis with ODAF revealed that diffusion coefficients on live spermatozoa are significantly faster on the acrosome and postacrosome (29.3x10(-9) cm2/second) than on the midpiece and principal piece (11.8x10(-9) cm2/second). Recovery (R) is >90% in all domains. ODAF diffusion also shows regionalized temperature-sensitivity with a 4-fold increase over the sperm head and a 1.8-fold increase on the tail between 20 degrees C and 37 degrees C. Remarkably, dead or permeabilized spermatozoa rapidly develop a large immobile phase (R<25%) over the whole plasma membrane. This rigidification is temperature insensitive and irreversible suggesting major changes in the physical state of membrane lipids. It is concluded that lipid diffusion in the plasma membrane of live bull spermatozoa is rapid and varies significantly between surface domains. Following permeabilization or cell death, however, a large immobile phase develops indicating substantial changes in membrane lipid disposition.

Animals↗
PubMed1994

A mammalian homologue of Drosophila heterochromatin protein 1 (HP1) is a component of constitutive heterochromatin.

The Drosophila HP1 gene contains a highly conserved sequence, the chromobox, which can be used to isolate HP1-like genes from both mouse (M31 and M32) and man (HSM1) (Singh et al., 1991). Here we report that a monoclonal antibody (MoAb) raised against the M31 protein recognises a 26-kDa protein in murine and human nuclear extracts and localises to large masses of condensed chromatin within murine interphase nuclei, some of which are associated with the nucleoli. At metaphase, the MoAb binds to the centromeres of both human and murine chromosomes. The evolutionary conservation of this chromosomal localisation indicates that the M31 protein is likely to be important in the packaging of mammalian chromosomal DNA into constitutive heterochromatin.

3T3 Cells↗
PubMed1993

Molecular cloning and tissue distribution of pig transforming growth factor alpha.

Transforming growth factor alpha (TGF alpha) was originally identified as a product of tumour tissues and transformed cells in culture. Although it is now clear that expression of this factor is not restricted to neoplastic cells, there remains relatively little information about the sites of expression of TGF alpha in normal tissues. Therefore, an amplified DNA fragment encoding the pig TGF alpha precursor was cloned by reverse transcription-PCR (RT-PCR) using RNA isolated from normal skin tissue as the template. Nucleotide sequence analysis predicts a 160-residue transmembrane polypeptide that differs from the rat, mouse and human TGF alpha precursors at 14, 15 and six sites respectively. The distribution of TGF alpha mRNA in a wide variety of pig tissues was analysed by RT-PCR, using oligonucleotide primers based on the pig TGF alpha cDNA sequence. TGF alpha transcripts were detected in RNA isolated from 17 of the 22 tissues analysed, including four previously unreported sites. Using an antibody raised against a synthetic TGF alpha peptide, we have immunolocalized TGF alpha protein to cells within the red pulp of the spleen and to the distal convoluted tubules of the kidney.

Amino Acid Sequence↗
PubMed1993

Distribution of saccharides in pig lymph-node high-endothelial venules and associated lymphocytes visualized using fluorescent lectins and confocal microscopy.

The distribution of saccharides in pig lymph nodes, particularly on high-endothelial venule (HEV) endothelium and on lymphocytes in these vessels, was studied by examining the binding of fluorescent conjugates of 18 different lectins. Eight of the lectins, particularly with glycan specificity restricted to mannose and polyacetyllactosamine determinants, were found to bind with a high affinity to these structures. Competitive inhibition experiments revealed that polylactosamine-containing glycans were present on endothelia and lymphocytes using lectins from Lycopersicon esculentum and Solanum tuberosum, the latter lectin reacting with lymphocytes only when apparently adherent to the luminal endothelium. The The absence on pig endothelium of the Ulex europaeus binding, shown by human endothelia due to the presence of certain fucose epitopes, was confirmed. Pig lymph-node endothelium, however, bound the fucose-specific lectin of Tetragonolobus purpureas, indicating the presence of fucose on pig endothelia in a different conformation to that seen on human endothelia. The results suggested that pig lymph-node HEV endothelium expressed a core fucosylated tri- or tetra-antennary complex glycan with polylactosamine extensions and expressing an Ley determinant.

Animals↗
PubMed1993

Selective binding and transcytosis of latex microspheres by rabbit intestinal M cells.

The interaction between polystyrene microspheres and the follicle-associated epithelium of rabbit Peyer's patches has been examined. Microspheres bind selectively to, and are transcytosed by, membranous or microfold (M) cells within the follicle-associated epithelium. M cells are able to transport, in 45 min, approximately 10(5) microspheres of 0.46 micron diameter across the epithelium overlying each lymphoid follicle dome of rabbit Peyer's patches. The high capacity of M cells for particulate transcytosis and the subsequent delivery of these particulates to the mucosal immune system highlights the potential importance of this portal in the delivery of antigens and drugs. In addition, the selective binding and uptake of microspheres may be utilised as a functional marker in the identification and isolation of M cells.

Animals↗
PubMed1993

Possible models describing enterocyte replacement in mouse Peyer's patch follicle-associated epithelial tissue.

Epithelial cells emerging from intestinal crypts surrounding Peyer's patch lymphoid follicles can be shown in chimaeric mice to adopt a wedge-like formation during migration to a central zone of cell extrusion (Schmidt et al. 1985). Similar tissue taken from normal mice has been used as a model in the present work to investigate how such a pattern of convergent migration might take place. Tissue treated cytochemically to reveal differences in surface alkaline phosphatase activity enabled a number of cell surface area measurements to be carried out by image analysis. The position where these measurements had been taken was then related to the geometrical centre of the tissue. Epithelial cell surface area halved during migration from edge to centre of these disc-like structures. An equation used to describe the positional dependence of this change had the form rho(r) = 6.86r(-0.24), where rho is cell density and r is cell distance from the centre measured in micrometers. Further calculation showed this decrease to be insufficient to explain all features associated with convergent migration. Differential changes occurring in the rate of cell migration and premature cell loss were also considered as possible factors affecting migration patterns. Mathematical modelling of these variables produced a migration pattern consistent with previous experimental findings. This model assumes that half the cell population is lost during migration and that cell migration rate near the centre is four times greater than at the edge. It should be possible to test this latter prediction in future experiments.

Alkaline Phosphatase↗
PubMed1992

Confocal microscopical analysis of epithelial cell heterogeneity in mouse Peyer's patches.

Cyanine dye fluorescence and alkaline phosphatase activities have been compared directly by confocal microscopy in a wide variety of cells present in the follicle-associated epithelium of the mouse Peyer's patch to test the hypothesis that antigen-transporting M cells have a low membrane potential. In order to make these comparisons it was first necessary to equilibrate living tissue with the membrane potential sensitive dye DIOC5(3), fix with glutaraldehyde and then incubate the fixed tissue with naphthol AS-BI phosphate, a substrate which is hydrolysed by alkaline phosphatase present in the luminal membrane of these epithelial cells. Naphthol AS-BI produced by this reaction is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. Selecting the 488 nm wavelength of the argon laser source then allows one to measure alkaline phosphatase activities as Fast Red absorbance and membrane potentials by DIOC5(3) fluorescence. Results obtained show a linear correlation between membrane potential and alkaline phosphatase activity. Relative lack of alkaline phosphatase activity, determined in fixed tissue, has been used previously to identify antigen-transporting M cells (Smith et al., 1987). The present work shows that it is now possible to recognize these cells in living tissue by measurement of DIOC5(3) fluorescence. The possible importance of this finding in providing a way to study cell surface-antigen interactions taking place in living tissue is discussed.

Alkaline Phosphatase↗
PubMed1992

Confocal analysis of fluorescent bead uptake by mouse Peyer's patch follicle-associated M cells.

Latex beads coated with secretory immunoglobulin (IgA) instilled into mouse intestinal loops are taken up by M cells present in Peyer's patch follicle-associated epithelial tissue. Bead adsorption and uptake is greater at the edge compared with the apex of follicle domes. Coating beads with bovine serum albumin (BSA) causes a fourfold reduction in adsorption and a twentyfold reduction in uptake. Results demonstrate selectivity between adsorption and uptake and between the ability of different proteins to facilitate uptake.

Adsorption↗
PubMed1992

Expression of the genes for TGF alpha, EGF and the EGF receptor during early pig development.

Expression of mRNA for transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF) and the epidermal growth factor receptor (EGF-R) during early pig development was evaluated by reverse transcription-PCR. In the unfertilised pig oocyte, maternal transcripts for EGF, but not for TGF alpha or the EGF-R, were detected. Pig conceptuses were analysed at days 7, 8, 10, 12, 15, 17, 18 and 22 of pregnancy. EGF-R mRNA was detected at all stages of conceptus development analysed. Interestingly, TGF alpha mRNA was expressed by the developing blastocyst only at days 8, 10 and 12 of pregnancy, with the highest levels apparent at day 10. In contrast, EGF mRNA was first expressed by the post-elongation conceptus at around day 15 of pregnancy with levels continuing to increase up to day 22. In the day-18 and day-22 conceptuses, this EGF message was shown to be primarily a product of the embryo-amnion and not the placental membranes. Furthermore, EGF was immunolocalised in the day-22 embryo to the developing lung bud, gut loop and amnion. In summary, the expression pattern of TGF alpha mRNA during early pig development is coincident with the onset of blastocyst elongation and suggests a possible role for TGF alpha during this period of cellular remodelling. The temporal and spatial expression of EGF mRNA and protein suggests a possible involvement for EGF in the establishment of the early organ systems.

Animals↗
PubMed1991

Expression of epidermal growth factor and its mRNA in pig kidney, pancreas and other tissues.

The levels of epidermal growth factor (EGF) mRNA in a wide range of pig tissues were measured by a RNAase-protection assay. The highest levels were found in kidney and pancreas, with lower levels in submaxillary gland, prostate gland and seminal vesicles. Immunocytochemical staining using an antiserum raised against recombinant pig EGF localized expression of the factor to the distal convoluted tubules of the kidney and to the epithelial cells lining the pancreatic and salivary ducts. The observed expression of EGF mRNA and EGF in pig tissues is consistent with a possible role for EGF in the maintenance and repair of the epithelial lining of various ductal systems.

Animals↗
PubMed1991

Galactose effects on enterocyte differentiation in the mouse jejunum.

The present work investigates the ability of galactose to affect enterocyte differentiation during normal development in vivo. Energy intake has also been varied to take account of the fact that galactose is poorly metabolized in mice. Brush-border lactase, alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N, alkaline phosphatase and microvillus length were measured as markers of enterocyte differentiation in mice fed diets containing galactose (G diet), corn oil (E diet) or galactose + corn oil (G + E diet). Maintaining mice on a G instead of E diet reduced brush-border lactase activity and enterocyte migration rates; alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N and microvillus length expression increased and alkaline phosphatase activity remained unchanged. Feeding the G + E diet restored enterocyte migration rates, lactase, aminopeptidase N and dipeptidylpeptidase-IV activities to values found in mice fed the E diet. Galactose stimulation of alpha-glucosidase and microvillus length expression was, however, fully maintained in mice fed the G + E diet. Present results show that enterocyte differentiation is affected independently by varying dietary galactose and energy levels; that galactose effects always increase and energy effects usually decrease expression of enterocyte components and that energy stimulation of lactase activity is exceptional.

Animals↗