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31.
The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have obtained rabbit antibodies that recognize the CYR1 protein. Antibodies were raised against synthetic oligopeptides and against a recombinant beta-galactosidase/CYR1 fusion protein. These antibodies have allowed the identification of the CYR1 gene product as a 205 kDa protein. Treatment with trypsin (2 micrograms/ml) reduced the size of the CYR1 protein from 205 to 155 kDa and produced an activated enzyme which no longer responded to guanine nucleotides. This result is consistent with a model in which adenylyl cyclase activity is regulated by an inhibitory domain near the amino-terminus of the CYR1 protein. This model is further supported by the finding that adenylyl cyclase activity is also markedly elevated and unresponsive to guanine nucleotides in mutant yeast strains that express only the carboxy-terminal half of the CYR1 protein. Treatment with high trypsin concentrations (greater than 10 micrograms/ml) caused release of adenylyl cyclase activity from the membrane. Comparison of immunoreactive CYR1 fragments released by trypsin and membrane bound genetically altered proteins suggests that the CYR1 protein is attached to the membrane via a separate trypsin sensitive anchoring protein rather than via a membrane anchoring domain. 相似文献
32.
33.
A. Birkenfeld Y. Ezra N. Ron D. Navot S. Granovsky J. G. Schenker I. S. Levij I. Vlodavsky 《In vitro cellular & developmental biology. Plant》1988,24(12):1188-1192
Summary The culturing of human endometrium in conventional plastic dishes and media is only partially successful, mainly because a
growth of a heterogeneous population of cells is achieved. Naturally produced extracellular matrix closely resembles the subepithelial
basement membrane and seems to affect both growth and differentiation of cells. These qualities of the extracellular matrix
(ECM) were applied for obtaining endometrial epithelial cultures. Endometrial tissue specimens were plated after slicing on
ECM-coated dishes and kept for up to 8 d. The growth of a confluent homogeneous tissue composed of polygonal epithelial-like
cells was demonstrated. To further characterize these cells, cultures were examined by scanning electron microscopy and transmission
electron microscopy. Scanning electron microscopy revealed flattened polygonal cells covered with microvilli, among which
ciliated cells were observed. By transmission electron microscopy the cells were seen as a monolayer, with some cells overlapping,
closely adherent to the matrix. Microvilli, as well as intracellular vacuoles and glycogen granules were observed. Cell type
specific cytoskeletal markers were demonstrated by antibodies to intermediate filament proteins (keratin and epithelial membrane
antigen). Taken together, the morphologic and immunohistochemical studies indicate that a selective growth of the epithelial
component of endometrial tissue was obtained after plating unprocessed endometrial tissue fragments on ECM-coated culture
dishes.
This work was supported by PHS grant no. CA 30289 to J.V. 相似文献
34.
The possibility of interaction of hepatocytes with the heparin binding domain of Fibronectin was examined. Rat hepatocytes
adhered to coverslips coated with the 33-kDa heparin binding fragment of the C-terminal region of plasma fibronectin. When
different concentrations of the heparin binding fragment were used to coat coverslips and used as substratum, cell attachment
showed saturation kinetics. Half the maximum attachment was observed at 30–40 min after seeding of cells. The cells became
flat after 2–3 h indicating that they spread on the heparin binding domain as they do on intact fibronectin. Among the different
glycosaminoglycans tested, maximum inhibition of attachment was observed for heparin. However it was not possible to completely
inhibit attachment even at high concentrations. These results indicate that hepatocytes interact with fibronectin not only
through the Arg-Gly-Asp-containing cell binding fragment, but also through the heparin binding domain of fibronectin and,
further, that there exist heparin-dependent and heparin-independent mechanisms of interaction of cells with the 33-kDa heparin
binding fragment of fibronectin 相似文献
35.
Roman G. Efremov Dmitry I. Gulyaev Gerard Vergoten Nikolai N. Modyanov 《Journal of Protein Chemistry》1992,11(6):665-675
A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na+, K+-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na+, K+-ATPase is discussed in the final part of the work. 相似文献
36.
Sertoli cells in culture produce two isoforms of proteoglycans which are found in the culture medium and associated with the cell membrane. The amount of both types of proteoglycans increased when Sertoli cells were plated on type I collagen-coated dishes as compared to uncoated dishes. The effect is due to an increase in the synthesis of proteoglycans rather than a diminished rate of degradation of these molecules. The collagen substrate also affects the distribution of these macromolecules; an increase in the amount of membrane-associated proteoglycans occurs at the expense of the proteoglycans released to the culture medium. 相似文献
37.
Ellen M. Johnson Linda S. Schnabelrauch Barbara B. Sears 《Molecular & general genetics : MGG》1991,225(1):106-112
Summary A bovine tRNA gene cluster has been characterized and the sequences of four tDNAs determined. Two of the tDNAs could encode tRNASer
IGA, one tDNASer
UGA, and the fourth tRNAGln
CUG. The three serine tDNAs representing the UCN codon isoacceptor family are almost identical. However, the sequence of the tDNASer
TGA differs from a previously sequenced bovine tDNASer
TGA at 12 positions (ca. 14%). This finding suggests that in the bovine genome, two subfamilies of genes might encode tRNASer
UGA. It also raises the possibility that new genes for a specific UCN isoacceptor might arise from the genes of a different isoacceptor, and could explain previously observed differences between species in the anticodons of coevolving pairs of tRNAsSer
UCN. The gene cluster also contains complete and partial copies, and fragments, of the BCS (bovine consensus sequence) SINE (short interspersed nuclear element) family, six examples of which were sequenced. Some of these elements occur in close proximity to two of the serine tDNAs. 相似文献
38.
Philip Skehan James E. Thomas Susan J. Friedman 《In vitro cellular & developmental biology. Plant》1986,22(11):632-636
Summary Sarcoma 180 monolayers spontaneously shed single cells and small multicellular aggregates into the surrounding medium to produce
a dual population of floating and substratum-attached cells. Shedding was a motility-associated event that occurred when cells
attempted to migrate over one another. It resulted from a combination of cell shape change and active motility, which increased
sensitivity to fluid shear dislodgement by reducing a cell's surface area of adhesive contact and increasing strain tension
at its adhesive contact points. Shedding occurred at all phases of the cell cycle. Extracellular matrix but not conditioned
medium enhanced the floating subpopulation by slowing the kinetics of rattachment to plastic and cellular substrata. Although
sarcoma 180 cells are anchorage independent in the sense that they grow readily in single cell suspension, they nevertheless
exhibited anchorage modulation of their cell cycle. Short periods in suspension produced a mild G1 accumulation, whereas longer periods of anchorage deprivation led to a mild G2 accumulation which appeared to result from an interference with cytokinesis.
This work was supported by grants from the Medical Research Council of Canada, The National Cancer Institute of Canada, the
Alberta Heritage Savings and Trust Fund for Applied Cancer Research, and the Alberta Heritage Fund for Medical Research. 相似文献
39.
Michael M. Lipsky Talia R. Sheridan Richard O. Bennett Eric B. May 《In vitro cellular & developmental biology. Plant》1986,22(6):360-362
Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes.
Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency
(93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers,
while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all
substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment
in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of
tyrosine aminotransferase by hydrocortisone (200%).
This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency.
Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations
of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest
in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly
since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David
W. Barnes 相似文献
40.
Correlative light, high-voltage electron and conventional electron microscopic methods were used to investigate digestion in two allogromiid foraminiferans, Allogromia sp., strain NF, and A. laticollaris Arnold. Microscopic observations showed that bacterial prey are phagocytosed by reticulopodia and are transported to the allogromiid cell body within blister-like phagosomes. Larger prey (algae, diatoms) are transported along the reticulopodial surface and are either stored extrathalamously or phagocytosed at the oral opening (peduncle). Studies of allogromiids optimally fixed and labeled with an extracellular-space label (colloidal thorium) showed that phagocytosed prey are completely enclosed by a plasma membrane envelope; this finding was corroborated by a serial-section three-dimensional reconstruction of the oral zone of one allogromiid. Cytochemical staining for acid phosphatase showed that lysosomes are absent from reticulopods but abundant in the cell body, particularly in the oral zone cytoplasm. We conclude that digestion in allogromiid foraminiferans is accomplished by a vacuole-based digestive apparatus and not by extracellular digestion within a lacunary system, as has been suggested in earlier studies. 相似文献