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51.
52.
Summary The anastomosing ER system of epidermal cells of onion bulb scales is composed of three modifications: lamellar and tubular elements, located in the cell periphery, and long tubular stands located deeper in the cytoplasm. Cytoplasmic acidification of epidermal cells by loading with weak organic acids like acetic or propionic acid causes the decay of the lamellar elements and the disappearance of long tubular strands. Organelle movement is also inhibited. The effects depend on the pH of the incubation medium and on the administered acid concentration, and are characterized by a distinct lag phase of about 7 min. The induced ER changes are transient with adaptation starting after about 50min. Buffer components alone have little influence on the cellular ER organization within a pH-range of 4.0–8.0. However, the pH of the medium strongly affects the time course of the effects as well as recovery after omitting the administered acid. Both modulation and recovery occur more rapidly at neutral or slightly alkaline pH. Actin filaments, which play a major role in ER organization and organelle movement, are not affected by cytosolic acidification.Dedicated to the memory of Professor Oswald Kiermayer 相似文献
53.
Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related. 相似文献
54.
Hillary A. Hahm Margot M. Ip Kathleen Darcy Jennifer D. Black Wendy K. Shea Suzanne Forczek Masami Yoshimura Takami Oka 《In vitro cellular & developmental biology. Plant》1990,26(8):803-814
Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within
a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and
secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within
an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting
of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor,
bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic
level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins.
Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was
observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of
casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot
analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein
mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels.
Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike
colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the
RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs
from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid
when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve
as an excellent model in which the regulation of mammary development and gene expression can be investigated.
This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health,
Bethesda, MD. 相似文献
55.
Mouse submandibular salivary epithelial cell growth and differentiation in long-term culture: Influence of the extracellular matrix 总被引:7,自引:0,他引:7
Elisa M. Durban 《In vitro cellular & developmental biology. Plant》1990,26(1):33-43
Summary The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal
cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors
of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular
salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation
procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor
synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional
collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic
analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of
granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature
of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor
as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen
matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in
the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from
collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein
with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor
was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation
of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and
its receptor and signal transduction pathway.
This work was supported by Public Health Service grant DE07766 from the National Institute of Dental Research, National Institutes
of Health, Bethesda, MD. 相似文献
56.
Roberto F. Nicosia Antonio Ottinetti 《In vitro cellular & developmental biology. Plant》1990,26(2):119-128
Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional
networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural
studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The
angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma
clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly
smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly
reduced. Autoradiographic studies of3H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid
endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from
Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network
when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation,
and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that
the basement membrane is a potent regulator of microvascular growth and morphogenesis.
This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes
of Health, Bethesda, MD. 相似文献
57.
James H. Fitzpatrick Jr. Douglas Kintner †Mark Anderson †William M. Westler ‡Sherrie E. Emoto David D. Gilboe 《Journal of neurochemistry》1996,66(6):2612-2620
Abstract: The inorganic phosphate (Pi) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of Pi. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated Pi spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of Pi buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the Pi peaks arising from the three compartments with different pH values if each peak made up 10–35% of total Pi area. In vivo, we identified the plasma compartment by intraarterial infusion of Pi. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that Pi compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pHi. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger Pi peaks represented intracellular spaces. 相似文献
58.
Paracrine functions of the coronary vascular endothelium 总被引:5,自引:0,他引:5
Ingrid Fleming Johann Bauersachs Rudi Busse 《Molecular and cellular biochemistry》1996,157(1-2):137-145
59.
Paul D. Roepe LiYong Wei Mary M. Hoffman Friederike Fritz 《Journal of bioenergetics and biomembranes》1996,28(6):541-555
Overexpression of the MDR protein, or p-glycoprotein (p-GP), in cells leads to decreased initial rates of accumulation and altered intracellular retention of chemotherapeutic drugs and a variety of other compounds. Thus, increased expression of the protein is related to increased drug resistance. Since several homologues of the MDR protein (CRP, ltpGPA, PDR5, sapABCDF) are also involved in conferring drug resistance phenomena in microorganisms, elucidating the function of the MDR protein at a molecular level will have important general applications. Although MDR protein function has been studied for nearly 20 years, interpretation of most data is complicated by the drug-selection conditions used to create model MDR cell lines. Precisely what level of resistance to particular drugs is conferred by a given amount of MDR protein, as well as a variety of other critical issues, are not yet resolved. Data from a number of laboratories has been gathered in support of at least four different models for the MDR protein. One model is that the protein uses the energy released from ATP hydrolysis to directly translocate drugs out of cells in some fashion. Another is that MDR protein overexpression perturbs electrical membrane potential () and/or intracellular pH (pHi) and therebyindirectly alters translocation and intracellular retention of hydrophobic drugs that are cationic, weakly basic, and/or that react with intracellular targets in a pHi, or -dependent manner. A third model proposes that the protein alternates between drug pump and Cl– channel (or channel regulator) conformations, implying that both direct and indirect mechanisms of altered drug translocation may be catalyzed by MDR protein. A fourth is that the protein acts as an ATP channel. Our recent work has tested predictions of these models via kinetic analysis of drug transport and single-cell photometry analysis of pHi, , and volume regulation in novel MDR and CFTR transfectants that have not been exposed to chemotherapeutic drugs prior to analysis. This paper reviews these data and previous work from other laboratories, as well as relevant transport physiology concepts, and summarizes how they either support or contradict the different models for MDR protein function. 相似文献
60.
Slow wave potentials (SWPs) are transient depolarizations which propagate substantial distances from their point of origin. They were induced in the epidermal cells of pea epicotyls by injurious methods such as root excision and heat treatment, as well as by externally applied defined steps in xylem pressure (Px) in the absence of wounding. The common principle of induction was a rapid increase in Px. Such a stimulus appeared under natural conditions after (i) bending of the epicotyl, (ii) wounding of the epidermis, (iii) rewatering of dehydrated roots, and (iv) embolism. The induced depolarization was not associated with a change in cell input resistance. This result and the ineffectiveness of ion channel blockers point to H(+)-pumps rather than ion channels as the ionic basis of the SWP. Stimuli such as excision, heat treatment and pressure steps, which generate SWPs, caused a transient increase in the fluorescence intensity of epicotyls loaded with the pH-indicator DM-NERF, a 2',7'-dimethyl derivative of rhodol, but not of those loaded with the pH indicator 2',7'bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Matching kinetics of depolarization and pH response identify a transient inactivation of proton pumps in the plasma membrane as the causal mechanism of the SWP. Feeding pump inhibitors to the cut surface of excised epicotyls failed to chemically simulate a SWP; cyanide, azide and 2,4-dinitrophenol caused sustained, local depolarizations which did not propagate. Of all tested substances, only sodium cholate caused a transient and propagating depolarization whose arrival in the growing region of the epicotyl coincided with a transient growth rate reduction. 相似文献