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M F Lopez

Publications and source records attributed to M F Lopez.

34 records · Page 2Linked to original sources
PubMed1996

Insulin-like growth factor II affects the appearance and glycogen content of glycogen cells in the murine placenta.

The phenotype of mice with a targeted disruption of the insulin-like growth factor II gene (IGF-II null mice) is growth retardation of both fetus and placenta during the last two thirds of gestation (1). We have compared the placenta of IGF-II null and wild-type mice from days 9-18 of gestation. No morphological differences were detected until after day 12 of gestation, when a new population of placental cells, the glycogen cells, normally first appears. Fewer glycogen cells were present in the null placenta compared to the wild-type placenta on days 13, 15, and 18 of gestation. By day 15, glycogen cells constituted approximately 50% of the basal zone cells in the wild-type placenta, but only 20% of the basal zone cells in the null placenta (P < 0.01). By contrast, spongiotrophoblasts constituted 40% of the basal zone cells in the wild-type placenta and 70% of the basal zone cells in the null placenta. There were no differences in cell size at any time. These results suggest that glycogen cells do not efficiently differentiate in the absence of IGF-II. The differentiation of glycogen cells did not appear to be simply delayed in the null placenta, as the number of glycogen cells in the null placenta did not increase between days 15-18, and there was no change in the ratio of glycogen to spongiotrophoblasts in the basal zone (P > 0.50). The glycogen content of both spongiotrophoblasts and glycogen cells was significantly reduced in the null placenta, suggesting that IGF-II may be an important regulator of glycogen synthesis in the placenta. These results indicate that IGF-II regulates cell number in the placenta and may play an important role in the differentiation of glycogen cells and the production of glycogen by placental cells.

Animals↗
PubMed1995

Purification and characterization of a DNA strand transferase from broccoli.

A protein with DNA binding, renaturation, and strand-transfer activities has been purified to homogeneity from broccoli (Brassica oleracea var italica). The enzyme, broccoli DNA strand transferase, has a native molecular mass of at least 200 kD and an apparent subunit molecular mass of 95 kD and is isolated as a set of isoforms differing only in charge. All three activities are saturated at very low stoichiometry, one monomer per approximately 1000 nucleotides of single-stranded DNA. Strand transfer is not effected by nuclease activity and reannealing, is only slightly dependent on ATP, and is independent of added Mg2+. Transfer requires homologous single- and double-stranded DNA and at higher enzyme concentrations results in very high molecular mass complexes. As with Escherichia coli RecA, transfer by broccoli DNA strand transferase depends strongly on the presence of 3' homologous ends.

Brassica↗
PubMed1994

A glutathione S-transferase (GST) isozyme from broccoli with significant sequence homology to the mammalian theta-class of GSTs.

A novel glutathione S-transferase (GST) was purified from broccoli (Brassica oleracea var. italica). Partial amino-acid sequencing indicated that the protein shared significant homology with several different plant GSTs from maize, silene, Dianthus, Nicotiana and Triticum, but little homology to yeast (Issatchenkia) GST. One region of the polypeptide near the N-terminal also shared significant homology to a region of rat 5-5, rat 12-12 and human theta-GST (collectively referred to as the theta-GST-class) but little structural homology to the common mammalian cytosolic GSTs (alpha-, mu- or pi-classes). The broccoli GST was retained on a novel membrane based glutathione affinity matrix and displayed activity towards 1-chloro-2,4-dinitro-benzene (CDNB), a general GST substrate, as well as 4-nitrophenethyl bromide, a marker substrate for the theta-class of GSTs. The characteristics of the broccoli GST potentially define it as a member of the theta-class. This is consistent with the view that the theta-class may have arisen prior to the divergence of animals and plants while the mammalian mu-, pi- and alpha-classes evolved after the two kingdoms were established.

Amino Acid Sequence↗
PubMed1994

Expression of growth hormone-binding protein with a hydrophilic carboxyl terminus by the mouse placenta: studies in vivo and in vitro.

GH-binding protein (GHBP) or GH receptor is present in numerous extrahepatic tissues in the rodent. From mid- to late gestation in the mouse, the maternal serum concentration of GHBP increases 30- to 50-fold. We have investigated whether the placenta might synthesize GHBP and potentially contribute to this increase. RNA was isolated from placentas and subjected to Northern analysis using a cDNA probe to the shared region of GHBP and GH receptor-encoding mRNAs. From day 8 to day 18 of gestation, the GHBP-encoding mRNA (1.4 kb) increased 45-fold in quantity. The GH receptor-encoding mRNA (4.2 kb) increased sixfold by day 14 and then remained steady until day 18. These changes which were not co-ordinated parallel reported changes in the steady-state concentrations of 1.4 and 4.2 kb mRNAs in maternal liver, suggesting shared regulatory factors. Extracts of freshly isolated trophoblasts were assayed for GHBP with a radioimmunoassay specific for GHBP with a hydrophilic carboxyl terminus. The cytosolic content of immunoreactive GHBP increased fourfold from mid- to late gestation. Trophoblasts were isolated from placentas and cultured for 2 days on collagen gels in defined medium. Cultured cells were at least 90% viable and secreted mouse placental lactogen-II (mPL-II). Immunocytochemistry was carried out simultaneously on cells cultured from day 7 to day 17 of gestation using a monoclonal antibody (MAb 4.3), which recognizes the hydrophilic C-terminus of GHBP. Cell-localized GHBP was present in trophoblasts cultured for 2 days, but GHBP was not detectable by radioimmunoassay or by immunoprecipitation in concentrated culture media from cultures treated with 100 ng mouse GH/ml or 100 ng mPL-II/ml or from untreated cultures. RNA was isolated from cells cultured in an identical manner to those analysed by immunocytochemistry. Three GH receptor/GHBP mRNA species of 8, 4.2 and 1.4 kb were observed. The quantity of 4.2 and 1.4 kb mRNAs did not change significantly in cultures from day 7 to day 15 of gestation but, in cultures from day 17 of gestation, the amount of 1.4 kb mRNA dropped significantly, while that of the 4.2 kb mRNA remained unchanged. GHBP- and GH receptor-encoding mRNAs are not co-ordinately regulated in vivo or in vitro. Although mPL-II was secreted into the medium by cultured trophoblasts, secretion of GHBP could not be detected. The culture medium may not contain the specific factors required for secretion of placental GHBP, or placental GHBP may not be destined for secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

High resolution 2-D peptide mapping with subsequent analysis of peptides by microsequencing or lectin binding directly from PVDF membrane blots.

Peptide mapping is a technique that is frequently used to characterize proteins. Typically, the method involves the cleavage of proteins in solution or in a gel with the subsequent separation of the peptide fragments on a 1-D SDS PAGE gel. Electrophoretic peptide maps are often used to compare homologous proteins from related organisms to derive evolutionary relationships. Other applications of peptide mapping include immunoblotting studies of selected proteins. Two-dimensional peptide mapping, a less common technique, has traditionally involved a combination of thin layer or paper chromatography and electrophoresis. Amino acid sequencing of peptides that were separated using this method and then subsequently blotted to PVDF membrane was reported recently. However, the resolution achieved with these methods is far below that which can be achieved with conventional 2-D electrophoresis of proteins in polyacrylamide gels. This report describes an electrophoretic system for the high resolution 2-D separation of peptides in gels with subsequent blotting to a novel cationic PVDF membrane, Immobilon-CD, and microsequencing directly from the 2-D blot. In addition, the high resolution peptide maps can be further analyzed by techniques such as lectin probing to determine post-translational modifications.

Amino Acid Sequence↗
PubMed1992

Purification and properties of DNA topoisomerase I from broccoli.

We have purified a topoisomerase activity from broccoli (Brassica oleracea var. italica) to near homogeneity. The enzyme is an 80 kDa monomer as judged by gel filtration chromatography and SDS gel electrophoresis, though it may represent a proteolytic fragment of a larger protein. The enzyme is capable of removing both negative and positive supercoils in steps of one, does not absolutely require Mg2+, is only very weakly stimulated by NaCl, is inhibited by camptothecin, and cross-reacts with an antibody directed against human DNA topoisomerase I. These properties identify the enzyme as a eukaryotic type I topoisomerase.

Blotting, Western↗
PubMed1992

A mechanically strong matrix for protein electrophoresis with enhanced silver staining properties.

Duracryl is a mechanically strong and elastic acrylamide-based matrix, useful for a wide variety of electrophoretic applications. The matrix is stable as a refrigerated solution for one year. Upon addition of appropriate catalysts, Duracryl forms a polymer-reinforced polyacrylamide gel matrix suitable for electrophoresis. The polymer-reinforced gel is superior to conventional polyacrylamide gels in terms of mechanical strength, elasticity and protein silver staining properties. Protein detection sensitivity by silver staining, as well as the linear response of silver deposition versus protein load, is equivalent to standard acrylamide/N,N'-methylene bisacrylamide gels. Additionally, the silver staining properties of the Duracryl matrix result in proteins appearing as monochromatic shades of grey instead of red, brown and yellow, as is the case of conventional polyacrylamide matrices. Monochromatic shades of grey are more suitable for image analysis and densitometry. The matrix is compatible with standard electroblotting and protein N-terminal sequencing procedures. Low acrylic acid content and conductivity allow incorporation of the matrix into isoelectric focusing gels. The matrix was found not to alter polypeptide migration relative to the standard acrylamide/N,N'-methylene bisacrylamide matrix.

Acrylic Resins↗
PubMed1991

Effect of various detergents on protein migration in the second dimension of two-dimensional gels.

Two-dimensional gel electrophoresis (2D)1 is a powerful technique used to separate complex protein mixtures. The technique involves the separation of proteins by charge in the first dimension and by molecular weight in the second dimension. The effect of substituting various detergents for sodium dodecyl sulfate (SDS) in the second dimension (PAGE) was investigated. Individual C-10 through C-14 alkyl sulfates, C-11 through C-14 alkyl sulfonates, sodium N-lauroyl-N-methyl-taurine, N-lauroylsarcosine, sodium laurate, or benzyldimethyl-n-hexadecylammonium chloride were substituted for SDS in equilibration buffer, gel buffer, and upper running buffer. The cationic benzyldimethyl-n-hexadecylammonium chloride system was run with reversed polarity. Dramatic effects on protein migration from human mesothelial cell extracts were observed when different detergents were utilized. The C-12 (SDS) through C-14 alkyl sulfates and sulfonates resulted in anomalous migration of the simple epithelial keratins. Unlike SDS, the C-10 and C-11 alkyl sulfates and C-11 sulfonate resulted in gels in which the keratins were separated accurately with respect to their gene sequence-determined molecular weights. However, with these shorter chain alkyl sulfates and sulfonate, resolution was compromised, especially with respect to the high-molecular-weight polypeptides. The C-12 alkyl sulfate (SDS) and alkyl sulfonate provided the best resolution of polypeptides. Mixtures of C-11 sulfate and SDS resulted in gels with better sequence molecular weight estimates and high resolution. In addition, trace amounts of sodium tetradecyl sulfate/sodium heptadecyl sulfate in commercial SDS preparations had an effect on polypeptide resolution.

Adipose Tissue↗
PubMed1991

Tris-tricine and Tris-borate buffer systems provide better estimates of human mesothelial cell intermediate filament protein molecular weights than the standard Tris-glycine system.

Human mesothelial cells contain a number of well defined intermediate filament proteins (IFs) that have been completely sequenced including vimentin and the cytokeratins (K7, K8, K18, and K19). The electrophoretic migration of these IFs was monitored as a function of second dimension gel buffer composition using various systems including Tris-glycine (pH 8.3 or 9.2), Tris-glycine with 20% methanol, Tris-borate, Tris-tricine, and sodium phosphate. All of the second dimension buffer chemistries yielded patterns of sufficient resolution to identify the major cytoskeletal proteins but differed in the relative mobilities of the IFs. Using gene sequence calculated molecular weight data, the major cytoskeletal polypeptides of human mesothelial cells were ranked from highest molecular weight to lowest molecular weight. This rank order of sequence calculated molecular weights was then compared to the rank order determined form the actual migration of the polypeptides in the different gel systems. With the Tris-tricine and the Tris-borate gel systems as well as gene sequence data, KS = vimentin greater than beta-tubulin = K7 greater than K18 greater than K19 greater than actin. With the pH 8.3 and 9.2 Tris-glycine systems, as well as the sodium phosphate gel system, the rank order of the polypeptides did not correspond to gene sequence data. Adding 20% methanol to the Tris-glycine system resulted in IF migration that more closely corresponded to the gene sequence derived data. Migration position of the IFs depended upon the temperature of the second dimension separation as well. In mesothelial cells, the migration of a total of 15-25% of the polypeptides was influenced by differing buffer systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Boric Acids↗
PubMed1991

Pituitary-placental interaction: hypophysectomy modulates the secretion of mouse placental lactogen-I.

To determine whether the pituitary gland influences the concentration of mouse placental lactogen-I (mPL-I) in maternal serum, pregnant Swiss Webster mice were hypophysectomized or sham-operated on day 9 of gestation. Blood was collected on days 10-13 and 18, and the mPL-I concentration of the serum was measured by RIA. The serum mPL-I concentration of hypophysectomized mice was significantly higher than that of sham-operated and intact mice on days 10-13. There was no difference in mPL-I concentration on day 18 of pregnancy among the groups. Similar elevations in serum mPL-I concentration were observed when hypophysectomy was performed on day 10 of pregnancy. Steady state levels of placental mPL-I messenger RNA (mRNA) were analyzed by Northern hybridization. No differences were observed in the amount of mPL-I mRNA among hypophysectomized, sham-operated, and intact mice. These results demonstrate that the pituitary gland exerts inhibitory control over the maternal serum mPL-I concentration. This control does not appear to be affected at the level of steady state amounts of mPL-I mRNA.

Animals↗
PubMed1990

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.

Autoradiography↗
PubMed1989

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra.

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.

Acetylene↗
PubMed1985

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.

Actinomycetales↗
PubMedDate not supplied

Reproducibility of polypeptide spot positions in two-dimensional gels run using carrier ampholytes in the isoelectric focusing dimension.

The reproducibility of complex protein patterns in two-dimensional (2-D) gels run with carrier ampholytes in the first dimension has been investigated. Two different laboratories collaborated in the study and 18 or 19 gels were run in each laboratory for comparison. The electrophoresis chemicals, running devices, and samples were standardized in both labs. The resulting 37 gels were scanned with a charge-coupled device (CCD) camera and spots were located, counted, quantified, and matched using a commercially available image analysis system. Subsequently, the reproducibility of spot position was determined. To perform the statistical analysis, the test gels were initially each matched to a master reference gel. Next, three sets of 12 gels (the image analysis software database could analyze only 12 gels at a time) were analyzed and the isoelectric point (pI) and molecular weight (M(r)) positional variation of all the spots that matched across the gels in each set was determined. The resulting statistical analysis indicates very high reproducibility of the carrier ampholyte technique.

Buffers↗
PubMedDate not supplied

Ontogenetic differences in the expression of olfactory-conditioned aversions resulting from a state of acute alcohol intoxication in the rat.

Three experiments examined the effects of acute alcohol intoxication on olfactory conditioning in infant (16 days old) and adult rats (60-65 days old). In both age groups, alcohol administration (2 g/kg, IG) prior to conditioning procedures failed to affect subsequent expression of methyl-conditioned aversions in a two-way odor locational test. Nevertheless, whenever this same alcohol dose was administered prior to testing procedures, the expression of the olfactory conditioning was substantially impaired in infants but unaffected in adults. The factorial design allowed rejection of the possibility that ontogenetic differences in alcohol-induced state-dependent retention might explain this developmental difference (Experiment 1). A subsequent experiment (Exp. 2a) revealed major age related differences in terms of peak blood alcohol levels (BALs) that resulted from the 2 g/kg alcohol dose previously employed. As revealed by gas chromatography, infants exhibited significantly higher BALs than adults at time of conditioning and/or testing. Therefore in Experiment 2b, the alcohol dose administered in adults was increased until BALs were comparable to those attained in infants given 2 g/kg. This was accomplished with a 2.5 g/kg alcohol dose, employed in Experiment 2c to evaluate responsiveness of adults in conditioning circumstances identical to those of Experiment 1. Yet even with this dose the adults' expression of the conditioned olfactory aversion was unaffected by the alcohol at the time of testing. Experiment 3 analyzed the effects of alcohol intoxication on infantile and adult motor responses elicited by methyl and a novel odor (lemon), which might conceivably have affected performance in the two-way odor locational tests used to assess olfactory conditioning. The results failed to support the possibility that the previously observed ontogenetic differences in the expression of olfactory-conditioned aversions were due to alcohol's effect on sensorimotor responsiveness. In accordance with prior studies, the present results seem to indicate age-related sensitivity to alcohol's effects on cognitive processes.

Aging↗