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Biomedical subjects

S J Kimber

Publications and source records attributed to S J Kimber.

47 records · Page 3Linked to original sources
PubMed1984

Oligosaccharides containing fucose linked alpha(1-3) and alpha(1-4) to N-acetylglucosamine cause decompaction of mouse morulae.

Two- to four-cell and eight-cell mouse embryos were incubated in various fucosylated and unfucosylated oligosaccharides, fucose binding protein, and fucosylated BSA. Compaction at the eight-cell stage was reversed by a mixture containing the oligosaccharides lacto-N-fucopentaose II (80-90%), in which fucose is linked alpha(1-4) to N-acetylglucosamine, and lacto-N-fucopentaose III (10-20%), in which fucose is linked alpha(1-3) to N-acetylglucosamine. Pure lacto-N-fucopentaose III (LNFP III) and 3-fucosyl lactose (containing fucose alpha(1-3)glucose) had a similar effect. All three molecules affected blastocyst formation. Various closely related fucosylated and unfucosylated oligosaccharides did not induce decompaction or inhibit blastocyst formation. The proportion of embryos incubated from the two- to four-cell stage in LNFP II/III which reached the eight-cell stage and formed blastocysts was reduced. Those which formed compact morulae subsequently decompacted. Precompact or early compacting eight-cell embryos incubated in LNFP II/III compacted normally but subsequently decompacted and failed to form blastocysts. Decompaction of eight-cell embryos in LNFP II/III occurred during a specific period of development (80-90 hr post-hCG) and was reversible up to 84-86 hr post-hCG, but not by 92 hr post-hCG. The period of sensitivity to LNFP II/III was associated with the decrease in the ability of calcium-free medium to cause decompaction. It appears that LNFP II/III interferes with a later calcium-independent phase of compaction and we propose that LNFP III and II inhibit an endogenous lectin-saccharide interaction between membranes involved in the stabilization of compaction.

Acetylglucosamine↗
PubMed1983

Mevalonate reverses the developmental arrest of preimplantation mouse embryos by Compactin, an inhibitor of HMG Co A reductase.

Hydroxymethyl glutaryl Co A reductase (HMG Co A reductase) is the key regulatory enzyme in the conversion of acetate to mevalonate. Mevalonate is the precursor for sterol and non-sterol isoprenes involved in membrane biogenesis, DNA replication and protein glycosylation. The influence of two inhibitors of HMG Co A reductase, Compactin (or ML236B) and an oxygenated sterol, Diosgenin, were tested on preimplantation development of mouse embryos. Compactin arrested development at about the 32-cell stage, leaving the blastomeres decompacted. Ultrastructural examination of the embryos revealed reduced membrane apposition but no major effects on cell organelles. There was however a predominance of nuclei with highly condensed chromatin. Glycosylation of proteins also appeared to be inhibited as shown by reduced incorporation of sugar precursors but not that of amino acids. The influence of Compactin was judged to be highly specific since only 10 micrograms/ml (0.08 mM) mevalonic acid abolished the effects of Compactin. Mevalonate in embryos may not be primarily utilized in the synthesis of sterols since a specific inhibitor of cholesterol synthesis, DL-4,4,10-beta-trimethyl-trans-decal-3-beta-ol had no detectable effect on development. The non-sterol isoprenes of mevalonate such as dolichol and isopentenyl adenine may play a more significant role during early development since the influence of Compactin resembled that previously described using tunicamycin, a specific inhibitor of dolichol mediated synthesis of N-glycosidically linked glycoproteins. Hence, lack of dolichol may partly be the cause of arrest of embryonic development by Compactin. Diosgenin caused embryonic arrest at about the 16-cell stage and the influence was not reversible by mevalonic acid. Cholesterol was able to rescue 50% of the embryos but the effect of Diosgenin could be non-specific and probably caused by its entry into the plasma membrane.

Animals↗
PubMed1982

Evidence on the role(s) of ATP in the mechanism of nitrogenase, from proton NMR relaxation studies on metal and nucleotide binding to the molybdenum-iron protein.

Interactions between the molybdenum-iron protein (Kp1) of nitrogenase (reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolysing,) EC 1.18.2.1) from Klebsiella pneumoniae and the divalent ions, Mn2+, Mg2+, Ca2+ and Ba2+, have been studied by monitoring the water proton NMR relaxation enhancement caused by the paramagnetism of Mn2+. We observed several binding sites for Mn2+, equivalent within experimental error (Kd = 209 +/- 23 microM), increasing in number from 1.0 to 2.9 per molecule in direct proportion to the specific activity of the protein. Metal binding sites on the MoFe protein are therefore essential to the enzymic function of nitrogenase. A maximum of four such sites is inferred for the fully active protein molecule. All manganese sites can alternatively bind the diamagnetic ions studied, the binding being one order-of-magnitude weaker (Kd = 2.2 +/- 0.3 mM for Mg2+; 1.6 +/- 0.2 mM for Ca2+; 3.4 +/- 0.3 mM for Ba2+), ATP and ADP form ternary complexes via Mn2+ with Kpl. The above data and other evidence on MgATP binding are discussed in terms of the site of hydrolysis of ATP during turnover and its possible bridging role between the two protein components of the enzyme.

Adenosine Triphosphate↗
PubMed1982

Spreading of blastomeres from eight-cell mouse embryos on lectin-coated beads.

Single blastomeres from 8-cell embryos adhere to lectin-coated agarose beads. The cells spread over the surface of peanut lectin (PNA), wheat germ lectin (WGA) and concanavalin A (Con A)-coated agarose beads, and this was prevented by sugars with high affinity for the appropriate lectin. Spreading was also prevented by cytochalasin D at a concentration (0.5-1.0 microgram/ml) known to inhibit compaction of whole 8-cell embryos. Colcemid (1-2 micrograms/ml) only depressed spreading, as measured by linear contact distance, to 70-80% of that for the controls, while removal of calcium from the medium with EDTA had little effect on spreading. Fucose promoted spreading on WGA beads. Cells from 8-cell embryos will not adhere to negatively charged G-Sephadex and CM-Sephadex beads. They do adhere to positively charged DEAE-Sephadex beads but do not spread on them. In an attempt to inhibit cell spreading at compaction in intact 8-cell embryos, these were disaggregated in the presence of various (34) saccharide-containing molecules. Neither any sugar-containing molecules alone, nor combinations of N-acetyl-glucosamine, galactose and mannose, fucose and galactose, and fucose and lactose were able to inhibit compaction at physiological concentrations. However, blastocyst formation was inhibited to various extents by high nonphysiological concentrations (50 mM) of D-glucose, D-mannose, L-fucose, D-arabinose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, alpha-lactose, 25 mM-alpha-melibionic acid and by 2 mg/ml fucoidin, ovine mucin and desialylated ovine mucin. Our results indicate that interaction of PNA, WGA and Con A with molecules on the cell surface that bind these lectins can trigger the cells to spread. However, a simple lectin-sugar interaction alone may not account for the spreading of cells on one another at compaction in whole 8-cell embryos.

Animals↗
PubMed1982

Interactions of blastomeres suggest changes in cell surface adhesiveness during the formation of inner cell mass and trophectoderm in the preimplantation mouse embryo.

Whole 8-cell morulae can be aggregated with isolated inner cell masses from blastocysts. On examining semithin light microscope sections of such aggregates we found that cells of the morula changed shape and spread over the surface of the ICM, thus translocating it to the inside of the aggregate. Using single cells from 8-cell embryos in combination with single cells from other stage embryos or isolated ICMs we show that 1/8 blastomeres spread over other cells providing a suitably adhesive surface. The incidence of spreading is high with inner cells from 16-cell embryos (56%) and 32-cell embryos (62%) and isolated inner cell masses (64%). In contrast, the incidence of spreading of 1/8 blastomeres is low over outer cells from 16-cell embryos (26%) and 32-cell embryos (13%). Blastomeres from 8-cell embryos do not spread over unfertilized 1-cell eggs, 1/2 or 1/4 cells or trophectoderm cells contaminating isolated ICMs. When 1/8 cells are aggregated in pairs they flatten on one another (equal spreading) as occurs at compaction in whole 8-cell embryos. However, if 1/8 is allowed to divide to 2/16 in culture one of the cells engulfs the other (51-62% pairs). Based on the ideas of Holtfreter (1943) and Steinberg (1964, 1978) these results are interpreted to indicate an increase in adhesiveness at the 8-cell stage as well as cytoskeletal mobilization. Following the 8-cell stage there is an increase in adhesiveness of inside cells while the outside cells decrease in adhesiveness. The difference in adhesiveness between inside and outside cells in late morulae is probably central to the divergent differentiation of (inner) ICM and (outer) trophectoderm cell populations.

Animals↗
PubMed1981

A 31P-NMR study of mono- and dimagnesium complexes of adenosine 5'-triphosphate and model systems.

31P-NMR chemical shifts and spin-lattice relaxation times of ATP (adenosine 5'-triphosphate), ribose 5'-triphosphate and tripolyphosphate show closely similar behaviour in aqueous solution at pH 7.5 on titration with Mg2+. The results are interpreted in terms of formation of 1 : 1 and 2 : 1 (dimagnesium) complexes with Mg2+ bound exclusively to the triphosphate chain. Stability constants for these complexes are reported. It is suggested that the predominant form of the 1 : 1 complexes has Mg2+ bound in tridentate manner (via non-bridging oxygen) to the alpha, beta and gamma phosphorus atoms; whilst that of the 2 : 1 complexes has each Mg2+ bound in bidentate manner, one to the alpha and beta, and the other to the beta and gamma, phosphorus positions.

Adenosine Triphosphate↗
PubMed1981

The secretion of the eggshell of Schistocerca gregaria, analysis of the kinetics of secretion in vitro by light and electron microscope autoradiography.

The secretion of the 2 main layers (endochorion and exochorion) of the eggshell of the desert locust Schistocerca gregaria was investigated using light and electron microscope autoradiography. Follicles undergoing endochorion secretion were labelled using a 3 min 'pulse' of [3H]leucine in vitro followed by a 0-115 min non-radioactive 'chase'. Immediately after the pulse the silver grains were distributed over the cytoplasm and organelles including rough endoplasmic reticulum, while by 2 and 5 min Golgi bodies contained radioactivity. By 12 min from the beginning of the chase the cell apex containing small secretory vesicles was labelled. By 20 min most of the silver grains were over the endochorion. The half-transport time (t50) was 14--15 min (from mid pulse), the lag time was 9--10 min and the percentage transport rate was 14--15% per min. When a 3 min pulse of [3H]galactose was used to label exochorion precursors, the shorter t50 (11 min) and the clumped grain distribution in light microscope autoradiographs after 0-min chase suggested that galactose was incorporated in Golgi bodies. The secretion of exochorion precursors appears to occur at a similar rate to that of endochorion precursors (approximately 15% per min). The results indicate that the follicle cells are among the fastest secreting cells.

Animals↗
PubMed1981

Morphogenetic analysis of changing cell associations following release of 2-cell and 4-cell mouse embryos from cleavage arrest.

Two-cell and four-cell mouse embryos were cultured in Cytochalasin D (CD) for 40-48 h. They were fixed for light and electron microscopy at various times after washing off the CD. Cleavage-arrested embryos in CD had well separated blastomeres but by 1 h from washing the embryos had compacted, in most cases without undergoing cell division. By 2 h after release from arrest one blastomere of the 2-cell arrested embryos had become crescent shaped and at 4-5 h the crescent-shaped blastomere had started to spread over the surface of the other rounded blastomere. This process continued until by 16-24 h from explantation to fresh medium one blastomere had almost completely engulfed the other. A similar process occurred in 4-cell arrested and released embryos. At this stage the embryos had accumulated fluid and become blastocyst-like vesicles. In 20% of 2-cell and 4-cell embryos one or two blastomeres underwent one cell division after release from arrest. Serial sections of these embryos lead to the conclusion that one or both progeny of the first cell to divide tended to be engulfed by the later dividing or non-dividing cell(s). These results are discussed in relation to the differentiation of ICM and trophectoderm in blastocysts.

Animals↗
PubMed1980

The secretion of the eggshell of Schistocerca gregaria: ultrastructure of the follicle cells during the termination of vitellogenesis and eggshell secretion.

The secretion of the eggshell by the follicle cells in the desert locust, Schistocerca gregaria, was studied using the electron microscope. The 3 layers of the eggshell, the vitelline membrane, the endochorion, and the exochorion, are produced in sequence over a short period of about 30-36 h. The follicle cells contain little rough endoplasmic reticulum (RER) and small inconspicuous Golgi bodies during vitellogenesis. As eggshell secretion approaches there is an increase in the amount of RER and Golgi cisternae contain electron-dense product. At each stage of the 3-phase secretion cycle the follicle cells contain Golgi bodies and secretion vesicles with distinct morphology. The follicle cells increase in breadth and decrease in height between the beginning and end of eggshell secretion. The endochorion ridges arise at the junction between follicle cells and appear to be moulded by the microvilli formed at this position. In the ovary prior to ovulation, the eggshell consists of a thin (0.5 micrometer) electron-dense vitelline and an outer fibrillar exochorion layer, 20-30 micrometer thick. Further changes take place in the vitelline membrane and the endochorion after oviposition, and a layer of curly fibres, the extrachorion, is secreted in the oviduct.

Animals↗