Co-culture of two-cell rat embryos on cell monolayers

Summary Monolayers of 2 different populations of uterine cells and of fetal fibroblasts were evaluated for the support of rat embryo development in vitro. Compared to controls, cultures performed in Earle's buffered saline solution (EBSS) alone, the cleavage rate of 2-cell embryos to the 4-cell stage was significantly increased when the embryos were cocultured for 24 h with mixed uterine stromal and myometrial cells (70.7 vs. 56.0%;P<0.01). Coculture of 2-cell embryos with either uterine epithelial-stromal or stromal-myometrial cells in medium TC 199 (M199) for 24 h significantly increased the cleavage rate to the 4-cell stage compared to controls in the same medium (respectively, 78.3 and 77.6 vs. 49.9%;P<0.01). The development was not improved when fibroblasts were used as feeder cells. After 48 h, the proportion of 4-cell embryos showing cellular fragmentation was significantly decreased in the presence of either epithelial-stromal or stroma-myometrial cells in M199 compared to controls (respectively, 18.4 and 20.0 vs. 43.8%;P<0.01). Coculture in EBSS or with fibroblasts failed to prevent embryo degeneration. In one coculture with stromal-epithelial cells in M199, 6/11 embryos proceeded beyond the 4-cell stage, two of them reaching the 8-cell stage. No embryo developed beyond that stage in our study. Although considerable efforts remain necessary to achieve further growth, these results suggest that coculture offers promise as a means of supporting the in vitro development of rat embryos.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Relationship between Vitreous Levels of Matrix Metalloproteinases and Vascular Endothelial Growth Factor in Proliferative Diabetic Retinopathy

To investigate which matrix metalloproteinases (MMPs) are more likely to be involved in the angiogenic process in proliferative diabetic retinopathy (PDR), we measured the levels of MMPs in the vitreous fluid from patients with PDR and controls and correlated these levels with the levels of vascular endothelial growth factor (VEGF). Vitreous samples from 32 PDR and 24 nondiabetic patients were studied by mosaic multiplex MMPs enzyme-linked immunosorbent assay (ELISA), single ELISA, Western blot and zymography analysis. Epiretinal membranes from 11 patients with PDR were studied by immunohistochemistry. MMP-8 and MMP-13 were not detected. ELISA, Western blot and gelatin ymography assays revealed significant increases in the expression levels of MMP-1, MMP-7, MMP-9 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls, whereas MMP-2 and MMP-3 were not upregulated in vitreous samples from PDR patients. Significant correlations existed between ELISA and zymography assays for the quantitation of MMP-2 (r=0.407; p=0.039) and MMP-9 (r=0.711; p<0.001). Significant correlations were observed between levels of VEGF and levels of MMP-1 (r=0.845; P<0.001) and MMP-9 (r=0.775; p<0.001), and between levels of MMP-1 and MMP-9 (r=0.857; p<0.001). In epiretinal membranes, cytoplasmic immunoreactivity for MMP-9 was present in vascular endothelial cells and stromal monocytes/macrophages and neutrophils. Our findings suggest that among the MMPs measured, MMP-1 and MMP-9 may contribute to the angiogenic switch in PDR.     

Eight new microsatellite markers in Atlantic cod (Gadus morhua L.) derived from an enriched genomic library

One hundred and fifty random clones from an enriched genomic library of Atlantic cod were sequenced. Primer pairs were designed for 15 microsatellites containing perfect di‐, tri‐, tetra‐ and hexanucleotide repeats and characterized in 96 unrelated fish. Eight markers were successfully amplified, with the number of alleles ranging from two to nine per locus and observed heterozygosity ranging from 0.341 to 0.977. Loci Gmo‐G13 and Gmo‐G14 had a significant excess of homozygotes. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

The Archaeal P-Type ATPases

A phylogenetic analysis was carried out of a total of 58 P-type ATPases encoded within the genomes of 20 archaea species. Members from six subfamilies were identified including: putative metal-, proton-, calcium-, sodium/potassium-, potassium-, and magnesium/nickel-transporting ATPases. Six novel putative proton-ATPases from archaea species growing under different temperature and pH conditions were shown to have shorter N- and C-termini than those of orthologous yeast or plant proton-ATPases. Moreover recent biochemical data are reviewed that report functional expression of putative archaea metal- or proton-ATPases in bacteria or yeast.     

The law and the local fairness of care practice. Reflections on the ethnographic field investigation concerning the failure of the Bopz Act in psychogeriatric nursing home care

Ten years after its introduction the Special Admissions to Psychiatric Hospitals Act (Bopz) still awaits full implementation in the field of psychogeriatric nursing home care. Ethnographic research into moral problems related to care giving in dementia patients in Dutch nursing homes yielded several reasons for this discrepancy between law and care practice. Firstly, practical effectuation of this law rests predominantly on the shoulders of nurses and nurse assistants who are mainly inspired by moral motives such as carefulness and providing safety and who are ill informed about the law and its prerequisites. Secondly, there is the problem of the loss of a common shared world of meaning, which in relational terms is typical for the process of dementia. As a result of this, crucial concepts of the Bopz loose their applicability as the disease progresses. Finally, there is an immanent tension between the anthropology of care and care giving and the anthropological presuppositions of the law, health law included, which on a fundamental level contributes to the resistance of the care practice to the legal procedures of the Bopz. These factors must be accounted for in new legislation or revision of the current law. In the mean time we urge the necessity to develop a moral code concerning how to deal with resistive behaviour and opposition to care of demented nursing home patients.     

Phylogenetic classification of transporters and other membrane proteins from Saccharomyces cerevisiae

On the basis of functional and phylogenetic criteria, we have identified a total of 229 subfamilies and 111 singletons predicted to carry out transport or other membrane functions in Saccharomyces cerevisiae. We have extended the Transporter Classification (TC) and created a Membrane Classification (MC) for non-transporter membrane proteins. Using the preliminary phylogenetic digits X, Y, Z (for new families, subfamilies, and clusters, respectively), we allocated a five-digit number to 850 proteins predicted to contain more than two transmembrane domains. Compared with a previous TC of the yeast genome, we classified an additional set of 538 membrane proteins (transporters and non-transporters) and identified 111 novel phylogenetic subfamilies. Electronic Publication