Cripto-1 localizes to dynamic and shed filopodia associated with cellular migration in glioblastoma cells

Cripto-1 is a protein participating in tissue orientation during embryogenesis but has also been implicated in a wide variety of cancers, such as colon, lung and breast cancer. Cripto-1 plays a role in the regulation of different pathways, including TGF-β/Smad and Wnt/β-catenin, which are highly associated with cell migration both during embryonal development and cancer progression. Little is known about the detailed subcellular localization of cripto-1 and how it participates in the directional movement of cells. In this study, the subcellular localization of cripto-1 in glioblastoma cells was investigated in vitro with high-resolution microscopy techniques. Cripto-1 was found to be localized to dynamic and shed filopodia and transported between cells through tunneling nanotubes. Our results connect the refined subcellular localization of cripto-1 to its functions in cellular orientation and migration.     

The influence of contact guidance on the orientation of colonies of subcultured vascular smooth muscle cells

Summary Subcultures of smooth muscle cells derived from rat thoracic aorta were grown on plane plastic substrata and on plastic substrata having ridges molded in them by a heated, ruled template. The cells were found to have a very high degree of contact guidance when distributed sparsely on the ridged substrata. When the cell density increased multilayered, elongated colonies formed. On plane substrata these were irregular, curved, and disposed in all directions. On the ridged substrata, however, the colonies were straight, evenly spaced, and positioned at right angles to the ridges. Supported by Grant MT1011 from the Medical Research Council of Canada.     

Studies onMadhuca butyraceae seed proteins

DefattedMadhuca butyraceae seeds contain 24% of crude protein and 10.4% of saponins. The solubility ofMadhuca seed proteins was determined in water and NaCl as a function of pH and minimum solubility occurred at pH 4.0. The proteins consist of three components with S_20,w values of 2.2, 9.8 and 15.4. On gel filtration the proteins gave three peaks and on diethylaminoethyl cellulose chromatography they resolved into two components. Thein vitro digestibility ofMadhuca seed protein was found to be 69% when assayed with a pepsin-pancreatin system.     

Fine structure of early human embryos frozen with 1,2 propanediol

Previous studies by a French group (Fertil Steril 44:645–651, 1985) have shown that two-to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268–272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.     

Relative performance of monohaploid potato clones and their diploid parents at plant level and after protoplast isolation and subsequent fusion

Summary Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.     

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.     

In vitro reactivation of sarin-inhibited brain acetylcholinesterase from different species by various oximes

In vitro as well as in vivo evaluation of the reactivating efficacy of various oximes against nerve agent-inhibited acetylcholinesterase has been usually done with the help of animal experiments. Nevertheless, previously published data indicate that the reactivation potency of oximes may be different in human and animal species, which may hamper the extrapolation of animal data to human data. Therefore, to better evaluate the efficacy of various oximes (pralidoxime, obidoxime, HI-6, K033) to reactivate brain acetylcholinesterase inhibited by sarin by in vitro methods, human, rat and pig brain acetylcholinesterase were used to calculate kinetic parameters for the reactivation. Our results show differences among the species, depending on the type of oxime, and indicate that data from animal experiments needs to be carefully evaluated before extrapolation to humans.     

Effects of alginate and resistant starch on feeding patterns,behaviour and performance in ad libitum-fed growing pigs

This study assessed the long-term effects of feeding diets containing either a gelling fibre (alginate (ALG)), or a fermentable fibre (resistant starch (RS)), or both, on feeding patterns, behaviour and growth performance of growing pigs fed ad libitum for 12 weeks. The experiment was set up as a 2×2 factorial arrangement: inclusion of ALG (yes or no) and inclusion of RS (yes or no) in the control diet, resulting in four dietary treatments, that is, ALG−RS− (control), ALG+RS−, ALG−RS+, and ALG+RS+. Both ALG and RS were exchanged for pregelatinized potato starch. A total of 240 pigs in 40 pens were used. From all visits to an electronic feeding station, feed intake and detailed feeding patterns were calculated. Apparent total tract digestibility of energy, dry matter (DM), and CP was determined in week 6. Pigs’ postures and behaviours were scored from live observations in weeks 7 and 12. Dietary treatments did not affect final BW and average daily gain (ADG). ALG reduced energy and DM digestibility (P<0.01). Moreover, ALG increased average daily DM intake, and reduced backfat thickness and carcass gain : digestible energy (DE) intake (P<0.05). RS increased feed intake per meal, meal duration (P<0.05) and inter-meal intervals (P=0.05), and reduced the number of meals per day (P<0.01), but did not affect daily DM intake. Moreover, RS reduced energy, DM and CP digestibility (P<0.01). Average daily DE intake was reduced (P<0.05), and gain : DE intake tended to be increased (P=0.07), whereas carcass gain : DE intake was not affected by RS. In week 12, ALG+RS− increased standing and walking, aggressive, feeder-directed, and drinking behaviours compared with ALG+RS+ (ALG×RS interaction, P<0.05), with ALG−RS− and ALG−RS+ in between. No other ALG×RS interactions were found. In conclusion, pigs fed ALG compensated for the reduced dietary DE content by increasing their feed intake, achieving similar DE intake and ADG as control pigs. Backfat thickness and carcass efficiency were reduced in pigs fed ALG, which also showed increased physical activity. Pigs fed RS changed feeding patterns, but did not increase their feed intake. Despite a lower DE intake, pigs fed RS achieved similar ADG as control pigs by increasing efficiency in DE use. This indicates that the energy utilization of RS in pigs with ad libitum access to feed is close to that of enzymatically digestible starch.