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Biomedical subjects

R Guzman

Publications and source records attributed to R Guzman.

At least 73 records · Page 4Linked to original sources
PubMed1987

Effect of adenosine and inosine on ureagenesis in hepatocytes.

Adenosine and inosine produced a dose-dependent stimulation of ureagenesis in isolated rat hepatocytes. Hypoxanthine, xanthine and uric acid were without effect. Half-maximally effective concentrations were 0.08 microM for adenosine and 5 microM for inosine. Activation of ureagenesis by both nucleosides had the following characteristics: (a) it was observed with either glutamine or (NH4)2CO3, provided that glucose was present; (b) it was not detected when glucose was replaced by lactate plus oleate; (c) it was mutually antagonized by glucagon, but not by adrenaline; and (d) it was dependent on Ca2+. We suggest that the action of adenosine and inosine on ureagenesis might be of physiological significance.

Adenosine↗
PubMed1986

Regional cerebral cortical deficits in the immune-deficient nude mouse: a preliminary study.

This report examines the morphology of the cerebral cortex in the nude mouse (nu/nu) compared with the BALB/c(+/+). Our experiments indicated that the thickness of the lateral frontal lobe was significantly reduced (8%; P less than 0.005) in the 4-month-old, female nude mouse compared with a BALB/c mouse of like sex and age. In addition, the left hemisphere was, in general, thinner in the nude than in the BALB/c, with area 18 being significantly thinner (2%; P less than 0.005). Oligodendrocytes were less in area 18 in the nude by 25% (P less than 0.02) in contrast to the BALB/c. In some respects these results extend the work of others. In previously published experiments, we showed that the thickness of the frontal cortex from enriched and impoverished female rats (from 60 to 116 days of age) differed by 2 to 3% (P less than 0.001) and in male rats by 3 to 4% (P less than 0.001). The oligodendrocytes in area 18 in the male differed by 20% (P less than 0.02) in rats exposed to their respective enriched or impoverished environments from 25 to 105 days of age. Thus, we have shown that the frontal cortical morphology as well as the occipital cortical oligodendrocytes were altered in both immune-deficient animals and environmentally stimulated or impoverished animals. Based on these separate sets of data, we will be interested to learn if our environmental conditions can alter the immune system and if so by what mechanism.

Animals↗
PubMed1983

Response of end bud cells from immature rat mammary gland to hormones when cultured in collagen gel.

End buds from 4- to 5-week-old rat mammary glands were isolated and cultured within a rat tail tendon collagen gel matrix. Media containing equine serum or porcine serum and cholera toxin promoted growth, but not the production of casein or thioesterase II, nor did they induce a state of differentiation as assessed by cell ultrastructure. Medium supplemented with only 5% porcine serum, insulin and cholera toxin did not support growth or differentiation. However, when prolactin, estradiol, progesterone and hydrocortisone were added to this medium, growth was stimulated greatly and a differentiated state was induced as assessed by the production of casein and thioesterase and by the appearance of a highly secretory ultrastructure.

Animals↗
PubMed1983

Comparison of the growth of normal and neoplastic mouse mammary cells on plastic, on collagen gels and in collagen gels.

The growth of normal and neoplastic mouse mammary cells was compared in primary cultures on plastic, on rat tail collagen gels and in rat tail collagen gels. Cells on plastic grew for the first few days, then stopped with only a 1- to 3-fold increase in cell number after 2 weeks in culture. Cells grown on or in collagen gels grew continuously over the 2-week culture period with up to 10-fold increase in cell number for cultures on collagen gels and a 20-fold increase for cells embedded in collagen gels. The difference in growth rates between cells grown in collagen gel and those grown on collagen gels was due, in part, to the three-dimensional growth of the colonies in collagen gel their two-dimensional growth on collagen gel. Cells grown on and in collagen gel can produce an electron-dense basal lamina-like structure which is associated with collagen IV and laminin as judged by immunofluorescence. Cells grown on plastic do not form this structure. Cis-OH-proline blocks the production of collagen and inhibits the growth of the cultured cells indicating collagen production to be involved in growth. Rat tail collagen gels are a superior substratum for the growth of mouse mammary cells and this may be related to the cells' ability to form a collagen IV-containing basal lamina-like structure.

Animals↗
PubMed1982

Identification of the mglA gene product in the beta-methylgalactoside transport system of Escherichia coli using plasmid DNA deletions generated in vitro.

Three genes (mglA, mglB, and mglC) required for active transport of substrate by the methylgalactoside permease were identified in a hybrid ColE1-DNA plasmid isolated from a clone (pLC3-14) of the Clarke-Carbon bank of Escherichia coli genes. A 4.6-kilobase DNA fragment obtained from pLC3-14 was cloned into the plasmid vector pBR322. The presence of the three mgl genes in the resultant plasmid, pMG3, was verified by genetic complementation and biochemical analysis of mgl mutants transformed with pMG3 DNA. Derivatives of pMG3 containing deletions in each mgl gene were constructed; restriction endonuclease mapping and functional analysis of these plasmids allowed us to physically locate the mgl genes within the inserted plasmid DNA and also to identify a heretofore unknown protein component of the transport system. Expression of these plasmids in vivo resulted in the specific synthesis of three major proteins of apparent molecular weight of 19,000, 36,000, and 52,000. The 36,000-dalton protein is the galactose-binding protein previously identified as the mglB product. The 19,000-dalton protein maybe the product of mglD, a regulatory gene mapping outside of the mgl gene cluster. The 52,000-dalton protein is a new permease component which we have identified here as the mglA product based on the observation that pMG6, a plasmid with a 0.6-kilobase mglA deletion, failed to encode for this protein but produced a truncated polypeptide showing a reduction in molecular weight comparable to the extent of the deletion. In bacteria bearing an mglA+, B-, C+ plasmid (Pmg4), the 52,000-dalton protein is located to a large extent (73%) in the membrane fraction.

Autoradiography↗
PubMed1980

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels.

Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture.

Adrenal Cortex Hormones↗
PubMed1980

Sustained growth in primary culture of normal mammary epithelial cells embedded in collagen gels.

Normal mammary epithelial cells from BALB/cfC3H midpregnant mice were freed from stromal cell types by Percoll density gradient centrifugation after collagenase digestion and were then embedded within collagen gels. Sustained growth leading to an increase in cell number was accomplished in response to cholera toxin and high concentrations of horse serum. The extent of growth was found to be dependent on the horse serum concentration, the maximum growth being attained at 50%. A serum concentration of 12.5% horse serum and 2.5% fetal calf serum, along with cholera toxin at 0.01 mug/ml, allowed maintenance but failed to cause any significant increase in cell number during the experimental period of 2 weeks. This same maintenance medium was used to determine the effects of various exogenously added steroids, protein hormones, and organ extracts on the proliferation of mammary epithelial cells in culture. Hormones failed to elicit any proliferative response, but extracts of kidney, brain, uterus, and spleen produced proliferative responses equal to or greater than the response obtained with 50% horse serum and cholera toxin. Kidney extracts prepared from midpregnant mice, virgin mice, and virgin mice given pituitary isografts all showed comparable activities, suggesting that the concentration of stimulatory factor(s) was not influenced by the hormonal status of the donor. Normal mammary epithelial cells that had undergone a 10- to 15-fold increase in cell number over initial values during 2-3 weeks in culture were passaged to secondary gel cultures. Outgrowth similar to those seen in primary culture were seen again in secondary culture. The present system provides a method for sustaining growth in culture of primary mammary epithelial cells from normal tissues.

Animals↗
PubMed1980

Growth factor- and cyclic nucleotide-induced proliferation of normal and malignant mammary epithelial cells in primary culture.

Sustained growth of normal mouse mammary epithelial cells in primary culture, leading to an increase in cell number, in response to growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] or cholera toxin has been achieved by embedding the cells inside collagen cells. Inclusion of agents known to increase the level of cellular cAMP have been found to be favorable for mammary epithelial cell proliferation. Cholera toxin is by far the best of all of the agents tested (prostaglandins E1 and E2, isoproterenol, theophylline, and dibutyryl cAMP). When growth factors (EGF or FGF) are added with cholera toxin, a synergistic effect resulting in a response much greater than with either of them alone is seen. This synergism was best seen in normal mammary epithelial cells from nonpregnant mice. The extent of this synergistic effect was found to be less in normal cells from pregnant mice, suggesting that these cells may be less responsive to EGF during pregnancy. Tumor cells were found to be rather inconsistent in their responses to EGF and cholera toxin, ranging from a minimal response, similar to that of normal cells from pregnant animals, to a maximal response, similar to that of normal cells from nonpregnant animals.

Animals↗
PubMed1980

Primary culture of human mammary epithelial cells embedded in collagen gels.

Human mammary epithelial cells were dissociatd from mastectomy tissues. The contaminating fibroblasts were removed by the use of Percoll density-gradient centrifugation, which utilizes the difference in buoyant densities between epithelial cells and fibroblasts. A preparation highly enriched for mammary epithelial cells ws then embedded in collagen gel and cultured in Ham's F12 medium containing 12.5% horse serum, 2.5% fetal calf serum, 0.1 microgram cholera toxin/ml, an extract prepared from human male urine (L microgram protein/ml), and a hormone combination of 10 microgram insulin/ml, 10 microgram human placental lactogen/ml, 1 microgram aldosterone/ml, and 0.5 microgram hydrocortisone/ml. Sustained growth leading to an increase of tenfold to thirtyfold in cell number over the initial value was accomplished in primary culture, and this growth was maintained even after passage to secondary culture. Deletion of either the urine extract or the hormone combination resulted in less than optimal growth. Subsequent studies showed that hydrocortisone alone could replace the hormone combination. In addition, urine extract could be replaced by extracts prepared from human kidneys or brains. The collagen gel system provies a reproducible and consistent method for sustained three-dimensional growth of mammary epithelial cells from human breast tissue in primary as well as passaged cultures.

Aldosterone↗
PubMed1979

Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels.

We have developed a method for embedding cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H]thymidine incorporation during 8 weeks in culture. An increase in DNA content of the cultured cells as a function of time was observed. Mouse mammary tumor cells cultured in the conventional monolayer system failed to show any significant increase in cell number during a culture period of 6 weeks. In addition, in such monolayer systems, cells progressively became detached from the dishes in long-term culture. The mammary epithelial cell origin of the collagen gel cell outgrowths was shown by electron microscopic demonstration of polarized cells containing tight junctions and budding mammary tumor virus particles. In addition, in vivo transplantation of collagen gel outgrowths resulted in the development of mammary adenocarcinoma histologically similar to the donor tumor. Cellular outgrowth patterns resembling those from tumor cells were also seen in similar collagen gel cultures of normal mammary cells from mouse and human and of hyperplastic alveolar nodule cells from mouse. The significance and usefulness of this system in comparison to the conventional monolayer system are discussed.

Animals↗
PubMed1978

Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme.

Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody,

Antibodies↗
PubMed1976

Roles of individual mgl gene products in the beta-methylgalactoside transport system of Escherichia coli K12.

Previous findings showed that galactose-binding protein defective mutants (mgl B-,A+,C+) of Escherichia coli K12 are still capable of growth on methyl-beta-D-galactopyranoside, while mgl A- and mgl C- mutants are not. When assayed by previous methods, none of these mutants exhibited methylgalactoside transport system activity. In this study, we present a modified assay developed for measuring low levels of transport. Using this assay, we found that mgl B-,A+,C+ mutants defective in galactose-binding protein accumulate methyl-beta-D-galactopyranoside up to six times the concentration gradient while mgl A- and mgl C- mutants failed to accumulate this substrate. Similar results were obtained using D-glyceryl-beta-D-galactopyranoside, another substrate of the methylgalactoside transport system. In contrast, all sugars tested which are not substrates of this system were transported equally by all mgl- mutants. The kinetic parameters of transport in mgl B- mutants were compared to those of the isogenic mgl+ strain which accumulates methyl-beta-D-galactopyranoside against a 10,000-fold concentration gradient. The apparent Km of methyl-beta-D-galactopyranoside influx was 1,000 times greater in mgl B- than in mgl+ strains. In contrast, there was no significant difference between these strains in either the Vmax of substrate influx or the rate of substrate exit. D-Galactose competitively inhibited methyl-beta-D-galactopyranoside influx into both mgl B- and mgl+ strains; the Ki of inhibition in mgl B- cells was 2,000-fold greater than that in mgl+ cells.

Biological Transport, Active↗