Biomedical subjects

A J Travis

Publications and source records attributed to A J Travis.

10 recordsLinked to original sources

PubMed2001

Expression and localization of caveolin-1, and the presence of membrane rafts, in mouse and Guinea pig spermatozoa.

In somatic cells, caveolin-1 plays several roles in membrane dynamics, including organization of detergent-insoluble lipid rafts, trafficking of cholesterol, and anchoring of signaling molecules. Events in sperm capacitation and fertilization require similar cellular functions, suggesting a possible role for caveolin-1 in spermatozoa. Immunoblot analysis demonstrated that caveolin-1 was indeed present in developing mouse male germ cells and both mouse and guinea pig spermatozoa. In mature spermatozoa, caveolin-1 was enriched in a Triton X-100-insoluble membrane fraction, as well as in membrane subdomains separable by means of their light buoyant densities through sucrose density gradient centrifugation. These data indicated the presence of membrane rafts enriched in caveolin-1 in spermatozoa. Indirect immunofluorescence analysis revealed caveolin-1 in the regions of the acrosome and flagellum in sperm of both species. Confocal immunofluorescence analysis of developing mouse male germ cells demonstrated partial co-localization with a marker for the acrosome. Furthermore, syntaxin-2, a protein involved in acrosomal exocytosis, was present in both raft and nonraft fractions in mature sperm. Together, these data indicated that sperm membranes possess distinct raft subdomains, and that caveolin-1 localized to regions appropriate for involvement with acrosomal biogenesis and exocytosis, as well as signaling pathways regulating such processes as capacitation and flagellar motility.

Acrosome↗

PubMed2001

Real time distributed processing of multiple associated pulse pattern sequences.

A Real Time Distributed Associative Memory Artificial Neural Network (RTANN) is described. This network associates groups of pulse pattern sequences. The subsequent reoccurrence of some sequences will cause the remainder to be regenerated. Training is carried out in real time simply by feeding pattern sequences directly into the network. The connections between units incorporate a wide range of transmission delays. During training the network enhances connection weights on units where coincidences occur between input and delayed pulses. Pattern regeneration utilises the reoccurrence of coincidences between delayed pulses. The simulation of an RTANN is presented. Continuous dual pattern sequences from notional sensors monitoring the shape and colour of an object were associated directly with a third dual pattern sequence having the form 'These objects look colour'. After training the network was able to correctly generate sentences describing combinations of object and colour not encountered during training.

Artificial Intelligence↗

PubMed2000

Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa.

Spermatozoa are highly polarized cells with specific metabolic pathways compartmentalized in different regions. Previously, we hypothesized that glycolysis is organized in the fibrous sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a recent report suggested that glucose is not essential for murine sperm capacitation, we demonstrated that glucose (but not lactate or pyruvate) was necessary and sufficient to support the protein tyrosine phosphorylation events associated with capacitation. The effect of glucose on this signaling pathway was downstream of cAMP, and appeared to arise indirectly as a consequence of metabolism as opposed to a direct signaling effect. Moreover, the phosphorylation events were not affected by uncouplers of oxidative respiration, inhibitors of electron transfer, or by a lack of substrates for oxidative respiration in the medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either at the level of substrate availability or by downstream enzymes. These data support the hypothesis that ATP specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum, as opposed to ATP generated by mitochondria in the mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system for the study of integration of compartmentalized metabolic and signaling pathways.

Adenosine Triphosphate↗

PubMed1999

A novel NH(2)-terminal, nonhydrophobic motif targets a male germ cell-specific hexokinase to the endoplasmic reticulum and plasma membrane.

Although three germ cell-specific transcripts of type 1 hexokinase exist in murine male germ cells, only one form, HK1-sc, is found at the protein level. This single isoform localizes to three distinct structures in mouse spermatozoa: the membranes of the head, the mitochondria in the midpiece, and the fibrous sheath in the flagellum (Travis, A. J., Foster, J. A., Rosenbaum, N. A., Visconti, P. E., Gerton, G. L., Kopf, G. S., and Moss, S. B. (1998) Mol. Biol. Cell 9, 263-276). The mechanism by which one protein is targeted to multiple sites within this highly polarized cell poses important questions of protein targeting. Because the study of protein targeting in germ cells is hampered by the lack of established cell lines in culture, constructs containing different domains of the germ cell-specific hexokinase transcripts were linked to a green fluorescent protein and transfected into hexokinase-deficient M+R42 cells. Constructs containing a nonhydrophobic, germ cell-specific domain, present at the amino terminus of the HK1-SC protein, were targeted to the endoplasmic reticulum and the plasma membrane. Mutational analysis of this domain demonstrated that a complex motif, PKIRPPLTE (with essential residues italicized), represented a novel endoplasmic reticulum-targeting motif. Constructs based on another germ cell-specific hexokinase transcript, HK1-sa, demonstrated the specific proteolytic removal of an amino-terminal domain, resulting in a protein product identical to HK1-SC. Such processing might constitute a regulatory mechanism governing the spatial and/or temporal expression of the protein.

Amino Acid Motifs↗

PubMed1999

Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation.

Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO(3) are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO(4) inhibited protein tyrosine phosphorylation, as well as capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin or OH-propyl-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and capacitation.

Biological Transport↗

PubMed1998

Targeting of a germ cell-specific type 1 hexokinase lacking a porin-binding domain to the mitochondria as well as to the head and fibrous sheath of murine spermatozoa.

Multiple isoforms of type 1 hexokinase (HK1) are transcribed during spermatogenesis in the mouse, including at least three that are presumably germ cell specific: HK1-sa, HK1-sb, and HK1-sc. Each of these predicted proteins contains a common, germ cell-specific sequence that replaces the porin-binding domain found in somatic HK1. Although HK1 protein is present in mature sperm and is tyrosine phosphorylated, it is not known whether the various potential isoforms are differentially translated and localized within the developing germ cells and mature sperm. Using antipeptide antisera against unique regions of HK1-sa and HK1-sb, it was demonstrated that these isoforms were not found in pachytene spermatocytes, round spermatids, condensing spermatids, or sperm, suggesting that HK1-sa and HK1-sb are not translated during spermatogenesis. Immunoreactivity was detected in protein from round spermatids, condensing spermatids, and mature sperm using an antipeptide antiserum against the common, germ cell-specific region, suggesting that HK1-sc was the only germ cell-specific isoform present in these cells. Two-dimensional SDS-PAGE suggested that all of the sperm HK1-sc was tyrosine phosphorylated, and that the somatic HK1 isoform was not present. Immunoelectron microscopy revealed that HK1-sc was associated with the mitochondria and with the fibrous sheath of the flagellum and was found in discrete clusters in the region of the membranes of the sperm head. The unusual distribution of HK1-sc in sperm suggests novel functions, such as extramitochondrial energy production, and also demonstrates that a hexokinase without a classical porin-binding domain can localize to mitochondria.

Amino Acid Sequence↗

PubMed1996

Properties and localization of a tyrosine phosphorylated form of hexokinase in mouse sperm.

Mouse sperm possess a phosphotyrosine-containing hexokinase type 1 (HK1) that is associated with the plasma membrane fraction of these cells (Kalab et al., 1994; J. Biol Chem 269:3810-3817). This apparent plasma membrane association appears unique, since somatic HK1 is normally cytoplasmic or bound to the outer mitochondrial membrane via contact sites with a voltage-dependent anion channel (porin) through a porin-binding domain. In male germ cells, three cDNA clones have been described that encode unique HK1 isoforms (HK1-sa, HK1-sb, HK1-sc) that do not contain porin binding domains (Mori et al., 1993: Biol Reprod 49:191-203). This suggests that these proteins might not be localized to the outer mitochondrial membrane and could have alternative functions in germ cells and/or sperm. We demonstrate in the mouse that male germ cells and sperm could potentially express four HK1 isoforms (HK1-sa, HK1-sb, HK1-sc, and the somatic HK1). At the protein level, at least one of the HK1 isoforms becomes phosphorylated on tyrosine residues during spermatogenesis. Treatment of sperm membrane fractions to dissociate the phosphotyrosine-containing HK1 (pY-mHK1) yields results demonstrating that pY-mHK1 has properties of an integral membrane protein. Indirect immunofluorescence using a monoclonal antibody to HK1 demonstrates specific staining both in the head and tail regions of sperm. Surface biotinylation of intact sperm followed by precipitation with either polyclonal HK1 antiserum or with avidin-Sepharose suggests that pY-mHK1 possesses an extracellular domain. These results suggest that mouse sperm contain at least one HK1 isoform that is present on the sperm head, has an extracellular domain, and behaves as an integral membrane protein.

Animals↗

PubMed1991

Effect of lactation on the decline of brush border lactase activity in neonatal pigs.

It has been shown that during the early phase of lactation porcine milk contains high concentrations of hormones and growth factors. The aim of the present investigation was to examine the hypothesis that the temporal coordination of intestinal maturation in piglets can be extrinsically regulated through changes in the composition of milk during the suckling period. Gut morphology and the ontogeny of brush border lactase activity were investigated in piglets reared on two suckling regimens designed to expose the animals to compositionally distinct milk. The first group of animals were cross-fostered onto postcolostrum sows and thereafter suckled normally for up to eight weeks. These normally suckled (N) animals consequently received both early and late lactation products. The second group of piglets were cross-fostered each week, for up to eight weeks, onto newly farrowed sows which were postcolostrum. As a result of this repeated cross-fostering (CF) these animals received only early lactation products. Animals were sacrificed at one, three, five, seven, and eight weeks postpartum. Biochemically active lactase decreased significantly (p less than 0.001) in both groups over eight weeks, but the rate of loss of activity was greater in the CF animals than in the N pigs by approximately 50% at week 3 and 25% at week 8. Quantitative histochemical analysis of lactase activity corroborated the biochemical data. At three weeks maximal enzyme activity was observed approximately 400 microns from the villus/crypt junction. Histochemically detected lactase decreased throughout the suckling period, but the intensity of reaction product was consistently weaker over the entire villus surface in the CF animals. Immunocytochemically detectable lactase was identified at the same sites as the histochemical reaction products. In addition, immunofluorescence microscopy showed the presence of histochemically undetectable enzyme on the basolateral and brush border membranes of both villus and crypt cells. Villus/crypt ratios were significantly lower (p<0.001) in the CF animals than in the N pigs between weeks 3 and 5. The results of this study suggest that lactation products can accelerate the loss of brush border lactase activity. The observed decline in biochemically and histochemically detected lactase was considered to be a consequence of reduced enterocyte lifespan, decreased synthesis of enzyme protein, or altered post-translational modification of enzyme protein, or a combination of there.

Animals↗

PubMed1990

A requirement for Zn2+ for the induction of thymidine kinase but not ornithine decarboxylase in 3T3 cells stimulated from quiescence.

In 3T3 cells stimulated from quiescence by serum, impaired thymidine incorporation caused by inadequate supply of Zn2+ was associated with both decreased thymidine kinase activity and a comparable decrease in its mRNA concentration. In contrast, the amount of mRNA for ribosomal protein S6 was not affected, nor was the earlier increase in the activity of ornithine decarboxylase.

Animals↗

PubMed1989

An automatic objective estimation of vascularization of normal and tumor-invaded brain tissue using image analysis.

A rapid method for the estimation of vascularization of normal and tumor-invaded brain tissue is described. The method involves immunostaining of cryostat sections using antibodies directed against a vascular protein (type IV collagen) and an objective measurement of these vascular elements using an automatic image processing system. Significant differences between tissues of normal brains and two tumors were shown. The potential of this method with regard to forming an index of malignancy is discussed.

Animals↗