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Query: UNIPROT:P06889 (Mol)
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The ovarian granulosa cell has previously been shown to be a site of insulin-like growth factor (IGF) I production, reception, and action. It is the objective of this study to characterize in greater detail the soluble IGF binding activity released by this cell type. To this end, use was made of granulosa cells from immature diethylstilbestrol-treated rats. Serum-free media conditioned for 72 h by cultured untreated cells acquired polyethylene glycol (PEG)-precipitable [125I]IGF-I binding activity. The latter proved cell density-dependent, displaying a minimal inoculum requirement of less than or equal to 3 x 10(5) cells/culture. The daily elaboration of IGF-I binding activity appeared constant throughout the 72 h experimental period, the overall time-dependent accumulation of binding activity (over the same time period) proving virtually additive. Scatchard analysis of detailed competition studies with IGF-I suggests that the latter ligand binds to granulosa cell-derived IGF binding protein(s) (IGFBPs) with an apparent affinity of 3 x 10(-10) M. Qualitatively similar results were obtained when using [125I]IGF-II suggesting that the IGFBPs in question are not IGF-I-selective. In fact, specificity studies using either [125I]IGF-I or [125I]IGF-II revealed a rank order of competitive potencies compatible with that observed in other tissues so studied (IGF-II greater than IGF-I much greater than insulin). The proteinacious nature of the acid-stable IGF binding activity under study was indicated by its sensitivity to relatively low concentrations of cycloheximide, its apparent deactivation following repeated cycles of freezing and thawing, and its virtual elimination when subjected to boiling or trypsin treatment. Cycloheximide-induced blockade of protein biosynthesis also revealed that the IGF binding activity is subject to measurable turnover thereby suggesting that its accumulation represents the balance struck between synthetic and degradative processes. Western ligand blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated media revealed a non-glycosylated major band doublet of 28-29 kDa. A single minor IGFBP species represented by a 23 kDa band was also appreciated in some but not all experiments. Taken together, these findings document the ability of ovarian granulosa cells to secrete a heterogenous mix of low molecular weight, high-affinity IGF-selective binding species. As such, these observations are in keeping with the concept of a complete intraovarian IGF system replete with ligands, receptors, and soluble binding activity.
Mol Cell Endocrinol 1990 Dec 21
PMID:Ovarian granulosa cell-derived insulin-like growth factor (IGF) binding proteins: release of low molecular weight, high-affinity IGF-selective species. 171 Jan 90

Cell suspension-derived rice (Oryza sativa L.) protoplasts were transformed by direct gene uptake. PEG-mediated transformation was more efficient than electroporation. Plasmid DNA containing a hygromycin phosphotransferase (HPT) gene (which confers hygromycin resistance) driven by the CaMV 35S promoter and a beta-D-glucuronidase (GUS) gene under control of the 1', 2' double promoter of the mannopine synthase (mas) locus of Agrobacterium tumefaciens was introduced into rice protoplasts. Southern analysis of DNA from transformed cell lines showed that the HPT and GUS genes were present intact. Both genes were expressed in transgenic cell suspensions. GUS activity was detected by histochemical staining of the cells and by enzyme assays. During a 12-day culture period the proportion of stained cells rose to a maximum and then decreased again. Considerably higher numbers of blue-stained cells were obtained when the transgenic cell lines were grown in the presence of 5-azacytidine. Transcripts of the GUS gene could not be detected, in contrast with the HPT gene. Plantlets were regenerated from one transgenic cell line. GUS activity was found in both leaf and root tissues of these plants, particularly, but not exclusively, in vascular bundles. A mouse dihydrofolate reductase coding sequence (DHFR), conferring methotrexate resistance, fused to the CaMV 35S promotor and the wild-type nopaline synthase (NOS) gene of A. tumefaciens were also introduced into rice protoplasts. Stable integration of both genes was confirmed by Southern analysis. Expression of the DHFR gene was demonstrated by high levels of resistance to methotrexate of the transgenic cell suspensions and by the presence of DHFR transcripts. Expression of the NOS gene at enzyme or RNA level was not detected. Southern analysis suggests that this gene was probably either methylated or scrambled in these lines.
Plant Mol Biol 1991 May
PMID:Transgenic rice cell lines and plants: expression of transferred chimeric genes. 171 79

Avermectins are a family of potent broad-spectrum anthelmintic compounds, which bind with high affinity to membranes isolated from the free-living nematode Caenorhabditis elegans. Binding of avermectins is thought to modulate chloride channel activity, but the exact mechanism for anthelmintic activity remains to be determined. In this report, the properties of an avermectin-sensitive membrane current were evaluated in Xenopus laevis oocytes that were injected with poly(A)+ RNA from C. elegans. In such oocytes, avermectins increased inward membrane current at a holding potential of -80 mV. An avermectin analog without anthelmintic activity had no effect. Half-maximal activation of current was observed with 90 nM avermectin. The reversal potential for avermectin-sensitive current was -19.3 +/- 1.9 mV, and it shifted with external chloride, as expected for a chloride current. Avermectin increased membrane current in C. elegans-injected oocytes that were also injected with the Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The response to avermectin was greatest in the 1.0-2.5-kilobase class of size-fractionated C. elegans poly(A)+ RNA. Oocytes that responded to avermectin were insensitive to gamma-aminobutyric acid and the avermectin-induced current was blocked by picrotoxin.
Mol Pharmacol 1991 Sep
PMID:Avermectin-sensitive chloride currents induced by Caenorhabditis elegans RNA in Xenopus oocytes. 171 30

Polychlorinated hydrocarbons known to be nongenotoxic carcinogens were screened as activators of protein kinase C (PKC)-beta 1 either at high concentrations of Ca2+ or in the absence of Ca2+ (i.e., with 1 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N',-tetraacetic acid). Of those compounds tested, kepone and dicofol significantly stimulated PKC activity in the absence, but not the presence, of Ca2+. PKC activation was most pronounced in the presence of phosphatidylserine. Kepone and dicofol stimulated PKC activity 26% and 13%, respectively, as compared with the PKC activity (100%) stimulated by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Northern blot analysis of expression of TPA-inducible genes by kepone showed slight expression of phorbin and ornithine decarboxylase in murine embryo fibroblasts. Future studies are required to determine the relevance of PKC activation by kepone and dicofol to the known carcinogenicity of these compounds.
Mol Carcinog 1991
PMID:Two polychlorinated hydrocarbons cause phospholipid-dependent protein kinase C activation in vitro in the absence of calcium. 172 72

The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for beta-glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB x 26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adh1 gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 bp (HindIII-PvuII) fragment of the Adh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3' end of the pB x 26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.
Mol Gen Genet 1991 Nov
PMID:Homologous recombination between plasmid DNA molecules in maize protoplasts. 174 30

The very high concentration of macromolecules within cells can potentially have an overwhelming effect on the thermodynamic activity of cellular components because of excluded volume effects. To estimate the magnitudes of such effects, we have made an experimental study of the cytoplasm of Escherichia coli. Parameters from cells and cell extracts are used to calculate approximate activity coefficients for cytoplasmic conditions. These calculations require a representation of the sizes, concentrations and effective specific volumes of the macromolecules in the extracts. Macromolecule size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous solution or by using the molecular mass distribution of macromolecules from gel filtration. Macromolecule concentrations in cytoplasm are obtained from analyses of extracts by applying a correction for the dilution that occurs during extraction. That factor is determined from experiments based upon the known impermeability of the cytoplasmic volume to sucrose in intact E. coli. Macromolecule concentrations in the cytoplasm of E. coli in either exponential or stationary growth phase are estimated to be approximately 0.3 to 0.4 g/ml. Macromolecule specific volumes are inferred from the composition of close-packed precipitates induced by polyethylene glycol. Several well-characterized proteins which bind to DNA (lac repressor, RNA polymerase) are extremely sensitive to changes in salt concentration in studies in vitro, but are insensitive in studies in vivo. Application of the activity coefficients from the present work indicates that at least part of this discrepancy arises from the difference in excluded volumes in these studies. Applications of the activity coefficients to solubility or to association reactions are also discussed, as are changes associated with cell growth phase and osmotic or other effects. The use of solutions of purified macromolecules that emulate the crowding conditions inferred for cytoplasm is discussed.
J Mol Biol 1991 Dec 05
PMID:Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. 174 95

The IIA constituent of the lactose permease from Staphylococcus aureus has been crystallized in two different forms. Crystals of form I have been grown from polyethylene glycol 4000 with beta-octyl glucoside. They diffract to 3.0 A resolution and belong to space group C2 with unit cell dimensions a = 141.7 A, b = 130.7 A, c = 96.5 A and beta = 96.2 degrees. Form II crystals have been obtained from a solution containing polyethylene glycol 400, ammonium sulfate and manganese chloride. They diffract to at least 2.8 A resolution and belong to space group P2(1)2(1)2(1) with unit cell dimensions a = 89.9 A, b = 101.5 A and c = 90.9 A.
J Mol Biol 1991 Dec 20
PMID:Crystallization and preliminary X-ray analysis of the lactose-specific phosphocarrier protein IIAlac of the phosphoenolpyruvate: sugar phosphotransferase system from Staphylococcus aureus. 176 52

The negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa has been purified from an over-expressing clone and crystalized. Crystals of diffraction quality were obtained from polyethylene glycol 4000 and ammonium sulphate. AmiC crystallizes in P4(2)2(1)2 (a = 104.4 A, c = 66.6 A) with one subunit in the asymmetric unit. Crystals diffract beyond 2.8 A.
J Mol Biol 1991 Dec 20
PMID:Crystallization of and preliminary X-ray data for the negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa. 176 55

A stable transformation procedure has been developed for Phytophthora infestans, an oomycete fungus that causes the late blight diseases of potato and tomato. This is the first description of reliable methods for transformation in an oomycete pathogen. Drug-resistant transformants were obtained by using vectors that contained bacterial genes for resistance to hygromycin B or G418 fused to promoters and terminators from the Hsp70 and Ham34 genes of the oomycete, Bremia lactucae. Using polyethylene glycol and CaCl2, vector DNA was introduced into protoplasts as a complex with cationic liposomes or with carrier DNA only. Transformants were obtained at similar frequencies with each combination of promoter and selectable marker and were confirmed by DNA and RNA hybridization and phosphotransferase assays. Transformation occurred through the integration of single or tandemly repeated copies of the plasmids into genomic DNA, conferring mitotically stable drug-resistant phenotypes. The sizes of the marker gene mRNAs in each transformant and the results of transcript mapping studies were consistent with the function of the B. lactucae regulatory sequences in P. infestans. A hygromycin-resistant transformant was tested and found to maintain pathogenicity, indicating that the gene transfer procedure will be useful for the molecular analysis of genes relevant to disease.
Mol Plant Microbe Interact
PMID:Transformation of the oomycete pathogen, Phytophthora infestans. 180 4

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C12E8) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca2+ only. The membrane fraction has also found to contain Mg(2+)-dependent Ca(2+)-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca2+ required for maximum activity is 3 mM for both Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca(2+)-uptake study shows that the uptake in the presence of Ca2+ and ATP is higher than in the presence of Mg2+, Ca2+ and ATP. The findings lead us to believe that the Mg(2+)-independent Ca(2+)-ATPase has some role in Ca2+ transport like Mg(2+)-dependent enzyme.
Mol Cell Biochem 1991 May 15
PMID:Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa. 183 Jan 26


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