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51.
A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin. 相似文献
52.
Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions 总被引:21,自引:14,他引:7
Abstract: Guanine nucleotides differentiate binding of tritium-labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [3 H]-labeled agonists by 20–60% and [3 H]-labeled antagonists by 0–20%. In the presence of 100 mM-NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high-affinity [3 H]dihydromorphine sites by 52% and low-affinity sites by 55%. Without sodium, GTP reduced high-affinity [3 H]-naloxone sites by 36%; in the presence of 100 mM-NaCl, GTP had no effect on either high- or low-affinity [3 H]naloxone sites. GTP increased the association rate of [3 H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [3 H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [3 H]-diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40- 120-fold, with met- and leu-enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [3 H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [3 H]-labeled agonist binding by stimulating membrane-bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors. 相似文献
53.
Opioid receptor-coupled second messenger systems 总被引:19,自引:0,他引:19
S R Childers 《Life sciences》1991,48(21):1991-2003
Although pharmacological data provide strong evidence for different types of opioid receptors (e.g., mu, delta, and kappa), they share many common properties in their ability to couple to second messenger systems. All opioid receptor types are coupled to G-proteins, since agonist binding is diminished by guanine nucleotides and agonist-stimulated GTPase activity has been identified in several preparations. Moreover, all three types inhibit adenylyl cyclase. This second messenger system has been identified for opioid receptors in both isolated brain membranes and in transformed cell culture. Studies with chronic treatment with opioid agonists suggest that the coupling of receptors with G-proteins and second messenger effectors may play important roles in development of opioid tolerance. 相似文献
54.
We have identified a novel N -acetylgalactosaminyltransferase activity in
lactating bovine mammary gland membranes. Acceptor specificity studies and
analysis of products obtained in vitro by 400 MHz1H-NMR spectroscopy
revealed that the enzyme catalyses the transfer of N - acetylgalactosamine
(GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal,
beta-linked N -acetylglucosamine (GlcNAc) residue and establishes a
beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N
'-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be
identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N-
acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles
invertebrate beta4-GalNAcT as well as mammalian beta4-
galactosyltransferase (beta4-GalT) in acceptor specificity. It can,
however, be clearly distinguished from the pituitary hormone-specific
beta4-GalNAcT by its incapability of acting with an elevated activity on a
glycoprotein substrate carrying a hormone-specific peptide motif.
Furthermore, the GalNAcT activity appeared not to be due to a promiscuous
action of a beta4-GalT as could be demonstrated by comparing the
beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine
colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc
and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting
antibody. Interestingly, under conditions where mammalian beta4-GalT forms
with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary
gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an
increased efficiency yielding the lactose analog GalNAcbeta1-->4Glc.
This enzyme thus forms the second example of a mammalian
glycosyltransferase the specificity of which can be modified by this milk
protein. It is proposed that the mammary gland beta4-GalNAcT functions in
the synthesis of lacdiNAc- based, complex-type glycans frequently occurring
on bovine milk glycoproteins. The action of this enzyme is to be considered
when aiming at the production of properly glycosylated protein
biopharmaceuticals in the milk of transgenic dairy animals.
相似文献
55.
The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding (Knd), and protein unfolding by urea (I50). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity. 相似文献
56.
57.
58.
Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges 总被引:13,自引:1,他引:13
The phylum Porifera (sponges) was the first to diverge from the common
ancestor of the Metazoa. In this study, six cDNAs coding for protein-
serine/threonine kinases (PS/TKs) are presented; they have been isolated
from libraries obtained from the demosponges Geodia cydonium and Suberites
domuncula and from the calcareous sponge Sycon raphanus. Sequence
alignments of the catalytic domains revealed that two major families of
PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the
cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC),
form two separate clusters. In each cluster, the sequence from S. raphanus
diverges first. To approach the question about the origin of
protein-tyrosine kinases (PTK), which are found only in Metazoa, we
analyzed two additional PS/TKs which have been cloned from S. domuncula:
the stress-responsive protein kinase (KRSvSD) and the
protein-kinase-C-related kinase (PRKvSD). The construction of the
phylogenetic tree, comprising the eight PS/TKs and the PTK cloned
previously from G. cydonium, revealed that the PTK derived from the branch
including the KRSvSD kinase. These data facilitate the first molecular
approach to elucidate the origin of metazoan PTK within the PS/TK
superfamily.
相似文献
59.
Quantitative and Qualitative Aspects of Dissolved Organic Carbon Leached from Senescent Plants in an Oligotrophic Wetland 总被引:1,自引:0,他引:1
Nagamitsu Maie Rudolf Jaffé Toshikazu Miyoshi Daniel L. Childers 《Biogeochemistry》2006,78(3):285-314
We conducted a series of experiments whereby dissolved organic matter (DOM) was leached from various wetland and estuarine
plants, namely sawgrass (Cladium jamaicense), spikerush (Eleocharis cellulosa), red mangrove (Rhizophora mangle), cattail (Typha domingensis), periphyton (dry and wet mat), and a seagrass (turtle grass; Thalassia testudinum). All are abundant in the Florida Coastal Everglades (FCE) except for cattail, but this species has a potential to proliferate
in this environment. Senescent plant samples were immersed into ultrapure water with and without addition of 0.1% NaN3 (w/ and w/o NaN3, respectively) for 36 days. We replaced the water every 3 days. The amount of dissolved organic carbon (DOC), sugars, and
phenols in the leachates were analyzed. The contribution of plant leachates to the ultrafiltered high molecular weight fraction
of DOM (>1 kDa; UDOM) in natural waters in the FCE was also investigated. UDOM in plant leachates was obtained by tangential
flow ultrafiltration and its carbon and phenolic compound compositions were analyzed using solid state 13C cross-polarization magic angle spinning nuclear magnetic resonance (13C CPMAS NMR) spectroscopy and thermochemolysis in the presence of tetramethylammonium hydroxide (TMAH thermochemolysis), respectively.
The maximum yield of DOC leached from plants over the 36-day incubations ranged from 13.0 to 55.2 g C kg−1 dry weight. This amount was lower in w/o NaN3 treatments (more DOC was consumed by microbes than produced) except for periphyton. During the first 2 weeks of the 5 week
incubation period, 60–85% of the total amount of DOC was leached, and exponential decay models fit the leaching rates except
for periphyton w/o NaN3. Leached DOC (w/ NaN3) contained different concentrations of sugars and phenols depending on the plant types (1.09–7.22 and 0.38–12.4 g C kg−1 dry weight, respectively), and those biomolecules comprised 8–34% and 4–28% of the total DOC, respectively. This result shows
that polyphenols that readily leach from senescent plants can be an important source of chromophoric DOM (CDOM) in wetland
environments. The O-alkyl C was found to be the major C form (55±9%) of UDOM in plant leachates as determined by 13C CPMAS NMR. The relative abundance of alkyl C and carbonyl C was consistently lower in plant-leached UDOM than that in natural
water UDOM in the FCE, which suggests that these constituents increase in relative abundance during diagenetic processing.
TMAH thermochemolysis analysis revealed that the phenolic composition was different among the UDOM leached from different
plants, and was expected to serve as a source indicator of UDOM in natural water. Polyphenols are, however, very reactive
and photosensitive in aquatic environments, and thus may loose their plant-specific molecular characteristics shortly. Our
study suggests that variations in vegetative cover across a wetland landscape will affect the quantity and quality of DOM
leached into the water, and such differences in DOM characteristics may affect other biogeochemical processes. 相似文献
60.