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81.
Summary An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation. 相似文献
82.
Germinal vesicle migration (GVM) and dissolution (GVD) were studied in goldfish oocytes treated with 17-α,20–β-dihydroxyprogesterone (DHP) and/or demecolcine (DE; a colchicine derivative also known as colcemid) in vitro. DE (100 μg/ml) in the presence of DHP, enhanced steroid-induced GVM, after both 24 and 48 hr of incubation and significantly reduced the DHP ED50 value for GVM. Similarly, administration of DE alone elicited a significant, dose-related increase in GVM after 24 or 48 hr of incubation. The presence of DE, either alone or in combination with DHP, was without effect on GVD. The effect of DE was also tested on ooplasmic viscoelasticity in goldfish follicles subjected to a centrifugal force (160g for 1 min). Preincubation (24 hr) of goldfish follicles in DE significantly influenced the direction and the extent of the centrifugally induced GV movement along the axis of centrifugal force in a dose-related fashion. The present results provide support for the hypothesis that cytoskeletal components, such as microtubules that are sensitive to DE, are involved in the mechanism of GVM in goldfish oocytes. 相似文献
83.
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
84.
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
85.
Claudio Nicolini Andrew S. Belmont Antonietta Martelli 《Cell biochemistry and biophysics》1986,8(2):103-117
Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology
and autoradiography grain patterns between middle G1 and middle S phases: Our results show two distinct [3H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in
contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of
very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the
nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of
the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of
nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size
(reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel
experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms
the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles
our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near
the nuclear periphery. 相似文献
86.
Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli 总被引:11,自引:0,他引:11
J R Guest H M Lewis L D Graham L C Packman R N Perham 《Journal of molecular biology》1985,185(4):743-754
The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain. 相似文献
87.
Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking 总被引:33,自引:0,他引:33
The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs. 相似文献
88.
Association of Serotonin, Dopamine, or Noradrenaline with an Actin-Like Component in Pheochromocytoma (PC12) Cells 总被引:1,自引:1,他引:0
A rat pheochromocytoma (PC12) cell line was used to examine the possibility that 5-hydroxytryptamine (serotonin), 3,4-dihydroxyphenylethylamine (dopamine), or noradrenaline may be associated with cytoplasmic actin, as was suggested by previous in vitro binding studies on an actin-like protein from rat brain synaptosomes. When PC12 cells were incubated with [3H]serotonin. [3H]dopamine, or [3H]noradrenaline for 30 min at 37 degrees C, approximately 2-4% of the radioactivity present in the cells was found to be associated with a high-molecular-weight (actin-like) component in supernatant fractions. Evidence relating this monoamine binding component to actin filaments includes: (a) its strong absorption by myosin filaments at low ionic strength: (b) a decrease in its affinity for myosin in the presence of 1 mM ATP, which lowers the affinity of authentic actin for myosin: (c) displacement of bound [3H]serotonin from it by DNase I, which binds strongly to actin and which inhibits [3H]serotonin binding to actin in vitro; (d) an increase in its binding of each monoamine (by 25-40%) after PC12 cells were preincubated with 10 microM cytochalasin B (a drug that induces depolymerization of F-actin). These findings suggest that serotonin, dopamine, or noradrenaline may associate with actin filaments in vivo. 相似文献
89.
Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca2+. To determine if Ca2+ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca2+, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca2+, was observed only in the perfusion solution containing Ca2+, indicating that myosin is responsible for the inhibitory effect of Ca2+ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca2+ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP
adenosine 5-triphosphate
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid) 相似文献
90.
Milan Höfer Klaas Nicolay George Robillard 《Journal of bioenergetics and biomembranes》1985,17(3):175-182
The electrochemical gradient of protons,
, was estimated in the obligatory aerobic yeastRhodotorula glutinis in the pH0 range from 3 to 8.5. The membrane potential, , was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP+) for negative above pH0 4.5, and thiocyanate (SCN–) for positive below pH0 4.5. The chemical gradient of H+ was determined by measuring the chemical shift of intracellular Pi by31P-NMR at given pH0 values. The values of pHi increased almost linearly from 7.3 at pH0 3 to 7.8 at pH0 8.5. In the physiological pH0 range from 3.5 to 6,
was fairly constant at values between 17–18 KJ mol–1, gradually decreasing at pH0 above 6. In deenergized cells, the intracellular pHi decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH0 4.5 or 7.5. There was no membrane potential detectable in deenergized cells. 相似文献