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1.
Cultured circular smooth muscle from the rabbit colon 总被引:1,自引:0,他引:1
H. W. Kao S. E. Finn A. M. Gown J. Lechago N. Lachant W. J. Snape Jr. 《In vitro cellular & developmental biology. Plant》1988,24(8):787-794
Summary Although cultured vascular smooth muscle cells have been extensively characterized and investigated, there are very few studies
of cultured intestinal smooth muscle cells. The aim of this study was to culture colonic smooth muscle (CSM) cells from the
rabbit colon. Freshly isolated CSM cells from the circular muscle layer of the distal colon were prepared by collagenase digestion.
In primary culture, CSM cells attached to the culture vessels by 48 to 72 h, proliferated by 3 to 7 d, and reached confluency
by 14 to 17 d with a “hill-and-valley” pattern. Spontaneous contractions were not observed at any time at 21° or 37° C. Confluent
primary cultures were greater than 95% CSM cells, as identified by intensely positive immunofluorescent staining to smooth
muscle actin-specific CGA7 and muscle-specific HHF-35 monoclonal antibodies. Transmission electron microscopy of freshly isolated
and proliferating CSM cells revealed ultrastructural features consistent with smooth muscle cells. We successfully cultured
CSM cells of the rabbit from freshly isolated cells and validated these CSM cells by electron microscopy and immunocytochemical
staining. These highly pure primary cultures may be used to investigate numerous aspects of CSM cell metabolism and physiology.
These studies were supported by the National Institutes of Health grant to the Inflammatory Bowel Disease Center (Bethesda,
MD) P30-AM-32200 and R01-DK-31147. Dr. Kao is the recipient of a Research Career Development Award from the National Foundation
for Ileitis and Colitis, Inc. A preliminary report of this work was presented at the American Motility Society Meeting, Houston,
TX, in October 1986, and appeared in abstract form inGastroenterology 91: 1057; 1986. 相似文献
2.
Jackie R. Vandenheede Sigrid Staquet Wilfried Merlevede 《Molecular and cellular biochemistry》1989,87(1):31-39
Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase FA-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M
r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF
Phenylmethanesulphonyl Fluoride
- TLCK
L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride
- TPCK
L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone 相似文献
3.
E. C. Foulkes 《Biological trace element research》1989,21(1):195-200
Review of the available evidence on the mechanism of cellular Cd uptake in the rat jejunum supports the concept that this
process consists of nonspecific binding to anionic sites on the membrane, followed by a temperature-dependent and rate-limiting
internalization step. Because temperature-sensitive transmembrane movement of Cd can be demonstrated also in isolated brush-border
vesicles and in erythrocyte ghosts, it is not likely to result from pinocytosis but may be related directly to membrane fluidity.
There is no need to assume the existence of saturable Cd carriers, or competition of Cd with essential polyvalent cations
for their specific transport systems. Uptake of Cd by tubular epithelium in the kidney of the intact rabbit appears to resemble
that described for the jejunum, with the internalization step limiting the rate of uptake. 相似文献
4.
Kunio Yonemasu Takako Sasaki Yoshiko Dohi Charles M. Lapière Betty Nusgens 《生物化学与生物物理学报:疾病的分子基础》1990,1096(1):47-51
C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q. 相似文献
5.
Hans Georg B?umert Akitsugu Kenmoku Gert Middelhoff Franz Ortanderl Alexander Thrun Heinz Faulstich Wolfgang Schiebler Hugo Fasold 《Journal of Protein Chemistry》1988,7(5):571-580
With the aid of tartryl-bis--aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited. The dimers are able to activate subfragment I ATPase of myosin and bind to DNase I with inactivation of the enzyme in the same manner as native G actin. Within the dimer, one ADP is immobilized and will exchange against ATP extremely slowly. The dimers do not bind to the mushroom toxin phalloidin. 相似文献
6.
Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes. 相似文献
7.
A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures 总被引:6,自引:0,他引:6
Alda Vidrich Rajeswari Ravindranath Kianbanoo Farsi Stephan Targan 《In vitro cellular & developmental biology. Plant》1988,24(3):188-194
Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions
as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth
requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development
of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of
homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the
cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now
be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture.
This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant
AM 27806 from the National Institutes of Health, Bethesda, MD. 相似文献
8.
The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ. 相似文献
9.
Many studies have established a correlation of differences in the activities of various muscle types with differences in the expression of myosin isoforms. In this paper we report the sequence determination of myosin light chain-2 from rabbit slow skeletal (LC2s) and ventricular (LC2v) nmscles. We sequenced tryptic peptides from LC2v which account for all except a few terminal amino acid residues. The major part (87 residues) of the rabbit LC2s sequence, obtained from tryptic and cyanogen bromide (CNBr) peptides, was found to be identical to rabbit LC2v. Our results provide the first sequence information on LC2s from any species, and lend strong support to the hypothesis that LC2s and LC2v are identical. Comparisons of rabbit LC2v and LC2s with rabbit LC2f (from fast skeletal muscle), and also with chicken LC2f and LC2v, show clearly that LC2s and LC2v from mammalian and avian species are more closely related to each other than they are to LC2f isoforms from the same species. 相似文献
10.
A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10µMolar region could be accurately and reproducibly measured using this technique.
The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen. 相似文献
The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen. 相似文献