Showing papers in "Experimental Biology and Medicine in 1993"
TL;DR: In vitro and in vivo studies indicate that insulin is the primary regulator of IGFBP-1 expression in these tissues, and that the primary effect of insulin is rapid inhibition of transcription.
Abstract: Insulin-like growth factor-binding protein (IGFBP)-1 is one of six homologous proteins that specifically bind and modulate the mitogenic and metabolic actions of insulin-like growth factor (IGF)-I and IGF-II. Of the six IGFBP, IGFBP-1 is the only one that displays rapid dynamic regulation in vivo, with serum levels varying 10-fold or more in relation to meals. The complementary cDNA for IGFBP-1 was first reported in 1988. The predicted 234-amino acid sequence has a molecular mass of 25.3 kDa. The N-terminal and C-terminal regions are highly homologous among rat, human, and bovine sequences, and contain 18 conserved cysteines which are postulated to provide a framework for ligand binding. The 65-residue midregion is less homologous and does not contain cysteines, but does include a Pro-Glu-Ser-Thr (PEST) domain that is typical of rapidly metabolized proteins. The gene for IGFBP-1 has been localized to human chromosome region 7p12-p14, where it is contiguous with the gene for IGFBP-3. IGFBP-1 mRNA and protein expression have been identified in human liver and uterine decidua, and in nonhuman kidney. In vitro and in vivo studies indicate that insulin is the primary regulator of IGFBP-1 expression in these tissues, and that the primary effect of insulin is rapid inhibition of transcription. On the other hand, cortisol, glucagon, and cAMP stimulate IGFBP-1 production. Limited data also show a potent stimulatory effect of phorbol esters. A detailed review of IGFBP-1 levels and physiology in vivo and in vitro is presented. The function of IGFBP-1 is not completely defined. However, several studies demonstrate that IGFBP-1 inhibits IGF binding to cell surface receptors and thereby inhibits IGF-mediated mitogenic and cell metabolic actions. Furthermore, IGFBP-1 regulation by insulin and glucoregulatory hormones in vitro and limited in vivo data are consistent with a role for IGFBP-1 in glucose counterregulation.
394 citations
TL;DR: Sleep patterns in rabbits inoculated with E. coli, S. aureus, or C. albicans were classified on the basis of the duration of the period of enhanced sleep to support the hypothesis that dynamic changes in sleep over the course of an infectious disease aid in recuperation.
Abstract: Infectious disease alters sleep patterns in rabbits, but the recuperative value of enhanced sleep during infectious disease has not been experimentally verified. To evaluate the relationship between specific sleep patterns and the clinical response to infectious disease, we classified sleep patterns in rabbits inoculated with E. coli, S. aureus, or C. albicans on the basis of the duration of the period of enhanced sleep. Patterns characterized by a long period of enhanced sleep were associated with a more favorable prognosis and less severe clinical signs than were patterns characterized by relatively short periods of enhanced sleep followed by prolonged sleep suppression. A contrasting analysis of these data indicated that animals that eventually died demonstrated reduced sleep compared to rabbits that survived the infection. These observations are consistent with the hypothesis that dynamic changes in sleep over the course of an infectious disease aid in recuperation.
152 citations
TL;DR: Evidence suggests that both CuZnSOD and MnSOD are important in pulmonary defense against oxygen toxicity, and the role of extracellular SOD in the pulmonary defenseagainst oxygen toxicity is not clear.
Abstract: Three forms of superoxide dismutase (SOD) exist in the lung: CuZnSOD, MnSOD and extracellular SOD. Evidence suggests that both CuZnSOD and MnSOD are important in pulmonary defense against oxygen toxicity. Enhancement of pulmonary levels of CuZnSOD by transgenic overexpression of CuZnSOD, or tracheal insufflation of liposome-encapsulated or polyethylene glycol-conjugated CuZnSOD, protects animals against oxygen toxicity. Likewise, transgenic overexpression of MnSOD, or induction of endogenous MnSOD by endotoxin, tumor necrosis factor, or interleukin 1, also protects animals against oxygen toxicity. The role of extracellular SOD in the pulmonary defense against oxygen toxicity is not clear.
139 citations
TL;DR: Progression in the skin is characterized by genetic changes that result in several distinct changes in the levels of structural proteins, growth factors, and proteases, and the mechanisms involved in progression are being studied in epidermal cell culture.
Abstract: Carcinogenesis in mouse skin can be divided into three distinct stages: initiation, promotion, and progression (malignant conversion). Initiation, induced by a single exposure to a genotoxic carcinogen, can result from a mutation in a single critical gene (e.g., rasHa), apparently in only a few epidermal cells. The change is irreversible. Promotion, resulting in the development of numerous benign tumors (papillomas), is accomplished by the repeated application of a nonmutagenic tumor promoter. The effects of single applications of tumor promoters are reversible since papillomas do not develop after insufficient exposure of initiated skin to promoters or when the interval between individual promoter applications is increased sufficiently. The reversibility of promotion suggests an epigenetic mechanism. Promoter treatment provides an environment that allows the selective clonal expansion of foci of initiated cells. The conversion of squamous papillomas to carcinomas (termed progression or malignant conversion) occurs spontaneously at a low frequency. The rate of progression to malignancy can be significantly increased by treatment of papilloma-bearing mice with certain genotoxic agents. These progressor agents or converting agents are likely to act via a second genetic change in papillomas already bearing the initiating mutation. Progression in the skin is characterized by genetic changes that result in several distinct changes in the levels or activity of structural proteins, growth factors, and proteases. The mechanisms involved in progression are being studied in epidermal cell culture. In order to determine the in vivo phenotype of cultured cells, a grafting system was developed in which the cells were transferred from culture to a prepared skin bed in athymic mice. Introduction of an activated v-fos oncogene into initiated cells bearing an activated rasHa gene produced cells with a carcinoma phenotype, i.e., carcinomas formed when the cells were grafted as part of reconstituted skin. Grafted keratinocytes containing the rasHa gene alone produced papillomas; with v-fos alone, normal skin formed when grafted. The rasHa/fos carcinomas showed changes in differentiation markers characteristic of chemically induced carcinomas. A cell culture assay utilizing cells initiated by the introduction of an activated rasHa oncogene was developed to study progression. After exposure of initiated cells to progressor agents under conditions in which the proliferation of the rasHa-initiated cells was suppressed, proliferating foci developed, with a good correlation of activity in the assay with activity in the progression stage in vivo. The cell culture assay provides a quantitative model to study chemically induced neoplastic progression and may be useful to identify potential progressor agents.
122 citations
TL;DR: The transport, tissue concentration, and relative biologic function of tocopherol and tocotrienol appear somewhat disparate and possibly unrelated.
Abstract: Certain aspects of tocopherol and tocotrienol absorption, plasma transport, and tissue distribution were examined in humans and hamsters. Plasma transport differed in that tocopherols were found primarily in low density lipoprotein and high density lipoprotein in association with plasma surface components, whereas tocotrienols disappeared from plasma with chylomicron clearance. In keeping with transport by triglyceride-rich lipoproteins, tocotrienols were deposited in conjunction with triglycerides in the adipose tissue of hamsters. In hamsters, tocopherols were the only tocol readily detected in all tissues, except adipose during tocotrienol supplementation. In fasting humans, the plasma tocotrienol concentration was not significantly increased after tocotrienol supplementation, whereas the platelet concentration of delta-tocotrienol doubled. Furthermore, tocotrienol intake did not appear to modulate the plasma cholesterol concentration in normolipemic hamsters. Thus, the transport, tissue concentration, and relative biologic function of tocopherol and tocotrienol appear somewhat disparate and possibly unrelated.
116 citations
TL;DR: A mathematical model of manganese metabolism in rats fed 54Mn was developed and it was determined that the liver, not the pancreas, was the major source of endogenous gut losses ofManganese.
Abstract: Manganese homeostasis is believed to be maintained by excretion of excess absorbed manganese through the gut, but the extent of endogenous gut losses of manganese has not been quantitated. ...
115 citations
TL;DR: Of the four recognized peptide hormones secreted by the endocrine pancreas, pancreatic polypeptide was the third to be discovered, isolated, and characterized and remains somewhat of an enigma both in name and in function.
Abstract: Historical Background. Of the four recognized peptide hormones secreted by the endocrine pancreas, pancreatic polypeptide (PP) was the third to be discovered, isolated, and characterized. Insulin (1921-22) and glucagon (1923) preceded and somatostatin (1975) followed the isolation of PP in 1968. Despite this sequence of events—and the time interval since its discovery—PP remains somewhat of an enigma both in name and in function. The reluctance to label this polypeptide with a more descriptive title than the lackluster PP it bears stems largely from the fact that while broad actions may be attributed to it, no outstanding or dominating effect has been identified with its actions. Thus, the only characteristic carried by use of the phrase PP is to signify the species of PP one is discussing, such as avian, bovine, rat, porcine, ovine, and human, for example. The avian structure has 15 identities (of a total of 36) with that of bovine PP, and the latter differs by one or two residues from most other...
113 citations
TL;DR: A functional role for the stimulation of tetrahydrobiopterin biosynthesis by cytokines is the formation of a limiting cofactor required for the enzymatic conversion of L-arginine to citrulline and nitric oxide.
Abstract: Biosynthesis of tetrahydrobiopterin starts from guanosine triphosphate by the action of guanosine triphosphate cyclohydrolase I, which yields the first intermediate, 7,8-dihydroneopterin triphosphate. This compound is then converted by subsequent enzymes, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase, to tetrahydrobiopterin, the biologically active metabolite. Cytokines such as gamma-interferon or tumor necrosis factor-alpha strongly stimulate the activity of guanosine triphosphate cyclohydrolase I in murine and human cells, yielding a potentiation of intracellular tetrahydrobiopterin concentrations. In human cells, particularly in human monocytes and macrophages, the low activity of 6-pyruvoyl tetrahydropterin synthase leads to the additional accumulation of neopterin derivatives, which leak from the cells after dephosphorylation and are found increased in body fluids of humans with diseases challenging cell-mediated immunity. A functional role for the stimulation of tetrahydrobiopterin biosynthesis by cytokines is the formation of a limiting cofactor required for the enzymatic conversion of L-arginine to citrulline and nitric oxide.
101 citations
TL;DR: The observations that sub-populations of proliferating oval cells phenotypically similar to early hepatoblasts, and that oval cells originate in or around the ductular structures in the portal area, strongly support the notion that the hepatic stem cell compartment resides in these structures.
Abstract: There is increasingly robust experimental evidence in support of the presence of a pluripotent cell compartment in the liver 1, 2, 3, 4, 5, 6, 7. This compartment can under certain conditions function as a stem cell compartment and provide the needed progeny for regeneration of the hepatic parenchyma 8, 9. In the adult rat, specific conditions can be utilized to induce proliferation of a distinct population of small epithelial cells in the ductal structures of the liver 10, 11. These cells, conventionally described as oval cells, are characterized by ovoid nuclei and basophilic cytoplasma 10, and display features of both bile duct cells and fetal hepatocytes 11, 12, 13. There are three experimental systems, two in the rat and one in the mouse, in which it has been conclusively demonstrated that oval cells are capable of differentiation into hepatocytes 8, 11, 14. The developmental potential of oval cells is, however, not restricted to hepatic lineages. Oval cells can differentiate into intestinal-type epi...
100 citations
TL;DR: Data suggest that the rotating-wall vessel affords a new tissue culture model for investigation of growth, regulatory, and differentiation processes within normal tissues.
Abstract: A new low shear stress, low turbulence microcarrier culture system has been developed at NASA's Johnson Space Center that permits large-scale three-dimensional tissue culture. Tissue cultur...
94 citations
TL;DR: It is now clear that the p53 tumor suppressor gene product mediates apoptosis by E1A, and that the E1B gene encodes independent, overlapping functions to disable p53-mediated apoptosis.
Abstract: The human DNA tumor virus adenovirus encodes two transforming genes, the E1A and E1B oncogenes, which cooperate to oncogenically transform primary rodent cells. The E1A products efficiently stimulate cell proliferation, but fail to transform cells due to the induction of programmed cell death (apoptosis). Expression of either the E1B oncogene, or the human bcl-2 proto-oncogene, blocks E1A-associated apoptosis to produce transformation with high frequency. Thus, induction of proliferation by E1A must be coupled to suppression of an intrinsic cell suicide pathway for the efficient transformation of primary cells.The E1B oncogene encodes the unique 19-kDa and 55-kDa proteins, both of which independently suppress apoptosis and greatly enhance transformation by E1A. Suppression of apoptosis by the E1B 19-kDa protein is required not only in transformation of rodent cells with E1A, but also in adenovirus-infected human cells, where it sustains cell viability to maximize virus production. The E1B 19-kDa protein h...
TL;DR: A hypothetical working model explaining the events leading to cardiac failure in the copper-deficient rat heart is presented based on the present body of knowledge, and the pathology is compared with other models of cardiomyopathies.
Abstract: Dietary copper restriction in rats results in cardiomyopathy. In rats fed copper-restricted diets from weaning for 5 to 8 weeks, a concentric hypertrophy is apparent, whereas postweaning copper restriction does produce cardiomyopathy without apparent hypertrophy. Both sets of circumstances appear to affect the integrity of the basal laminae of cardiac myocytes and capillaries. In rats fed copper-restricted diets from weaning, decreases in cytochrome c oxidase are related not only to copper's role as a coenzyme, but also to a marked decrease in the nuclear encoded subunits of the enzyme complex. Decreased levels of the delta-subunit of ATP synthase have been observed. However, such aberrations in mitochondrial enzymes, as well as morphologic alterations, apparently do not affect cardiac levels of ATP. This review suggests mechanisms of cardiac adaptation and initiation factors leading to cardiac hypertrophy. We present a hypothetical working model explaining the events leading to cardiac failure in the copper-deficient rat heart based on the present body of knowledge, and compare the pathology with other models of cardiomyopathies.
TL;DR: The search for liver stem or progenitor cells has a long history, but there is now sufficient evidence to indicate that such cells do exist in adult animals and probably also in humans, and this work has now been extended to encompass work in fetal and adult normal livers as well as acutely injured liver.
Abstract: The search for liver stem or progenitor cells has a long history, but there is now sufficient evidence to indicate that such cells do exist in adult animals and probably also in humans (see Refs. 1-6 for reviews). They constitute a reserve compartment that is activated in situations of severe liver injury in which hepatocytes cannot mount an appropriate proliferative response. Proliferation of cells of this compartment is seen at the early stages of hepatocarcinogenesis induced by many chemicals as well as in noncarcinogenic toxic injury, such as that produced by galactosamine administration. The nonparenchymal epithelial cells that can be detected in these conditions received the name “oval cells” because of their shape. Oval cells are not, however, a homogeneous population, but rather form a cellular compartment that contains cells at various stages of differentiation, either in the hepatocyte or bile duct lineages. A very small proportion of these cells seem capable of serving as progenitors for both l...
TL;DR: Protocols for identifying and isolating antigenically related cell populations present in normal tissues using monoclonal antibodies to oval cell antigens and fluorescence-activated cell sorting are developed and found ones that uniquely identify either hepatic or hemopoietic cells.
Abstract: Oval cells, small cells with oval-shaped nuclei, are induced to proliferate in the livers of animals treated with carcinogens and are thought to be related to liver stem cells and/or committed liver progenitor cell populations. We have developed protocols for identifying and isolating antigenically related cell populations present in normal tissues using monoclonal antibodies to oval cell antigens and fluorescence-activated cell sorting. We have isolated oval cell-antigen-positive (OCAP) cells from embryonic, neonatal, and adult rat livers and have identified culture conditions permitting their growth in culture. The requirements for growth of the OCAP cells included substrata of type IV collagen mixed with laminin, basal medium with complex lipids and low calcium, specific growth factors (most potently, insulin-like growth factor II and granulocyte-macrophage colony-stimulating factor), and co-cultures of embryonic, liver-specific stroma, strongly suggesting paracrine signaling between hepatic and hemopoietic precursor cells. The growing OCAP cultures proved to be uniformly expressing oval cell markers but were nevertheless a mixture of hepatic and hemopoietic precursor cells. To separate the hepatic and hemopoietic subpopulations of OCAP cells, we surveyed known antibodies and found ones that uniquely identify either hepatic or hemopoietic cells. Several of these antibodies were used in panning procedures and fluorescence-activated cell sorting to eliminate contaminant cell populations, particularly hemopoietic and endothelial cells. Using specific flow cytometric parameters, three cellular subpopulations could be isolated separately that were identified by immunochemistry and molecular hybridization assays as probable: (i) committed progenitors to hepatocytes; (ii) committed progenitors to bile ducts; or (iii) a mixed population of hemopoietic cells that contained a small percentage of hepatic blasts that are possibly pluripotent. The hepatic precursor cells have been characterized using immunochemistry, flow cytometry, and molecular hybridization assays. The hepatic blasts are small (7-10 microns) cells with high nuclear to cytoplasmic ratios and with minimal complexity of the cytoplasm. Cultures of the committed progenitors were found to differentiate into cells with recognizable parenchymal cell fates. We discuss our studies in the context of our model of the liver as stem cell and lineage system and suggest that a slow, unidirectional, terminal differentiation process, paralleling more rapid ones in the skin or gut, occurs at all times in the liver and is thought to vary primarily in kinetics during quiescent versus regenerative states.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: The data suggest that taspine promotes early phases of wound healing in a dose-dependent manner with no substantial modification thereafter, and is probably related to its chemotactic properties on fibroblasts and is not mediated by changes in extracellular matrix.
Abstract: Taspine (mol wt 369,000) is an alkaloid extracted from trees of Croton (family Euphorbiaceae) of the western Amazon region that has been used by natives and others as a vulnerary agent. Taspine was purified from tree sap to test its healing properties using different topical concentrations in the paired rat surgical incision model. Wound tensile strength and histology were evaluated. Samples treated with 250 micrograms, but not those treated with 50 micrograms or 10 micrograms, had significant higher values for MBS than paired controls (26%, P < 0.005, and 30%, P < 0.001, by Days 5 and 7, respectively). Taspine did not modify MBS at Day 12. Sample treated with 250 micrograms had significantly greater mononuclear cellular infiltration at Days 5 and 7 but not at Day 12. To better understand the effect of taspine as an enhancer of wound healing, we conducted in vitro studies in cell cultures. Taspine stimulated chemotaxis for fibroblasts. Taspine did not have an effect on specific assays for macrophage chemotaxis, neutrophil activation, fibroblast proliferation, or matrix assembly. Taken together, the data suggest that taspine promotes early phases of wound healing in a dose-dependent manner with no substantial modification thereafter. Its mechanism of action is probably related to its chemotactic properties on fibroblasts and is not mediated by changes in extracellular matrix.
TL;DR: Inclusion of a “minilocus” containing four HS sites linked to a globin gene resulted in higher expression in transplanted mice, but rearrangement of the provirus still occurs, and it is unclear what significance these experiments have with regard to human marrow stem cell transduction.
Abstract: Gene transfer of human globin genes into human pluripotent stem cells via viral vectors may soon be realized. The high level of globin gene expression believed to be required for the treatment of severe hemoglobinopathies necessitated the inclusion of cis-acting sequences (LCR). Retroviral vectors containing the LCR elements are prone to rearrangement, low titer, and poor expression. Inclusion of a "minilocus" containing four HS sites linked to a globin gene resulted in higher expression in transplanted mice, but rearrangement of the provirus still occurs, and it is unclear what significance these experiments have with regard to human marrow stem cell transduction. Recombinant AAV is among the newest of genetic transfer vectors. This once obscure virus possesses unique properties that distinguish it from all other vectors. Its major advantage is the lack of pathogenicity in humans. Wild-type AAV has the unusual ability to selectively integrate into the mammalian genome at a specific region, thus reducing the concern for genomic disruption and insertional mutagenesis. The ability of AAV to carry regulatory elements without interference from the viral template may enable greater control of transferred gene expression. Disadvantages currently include the inferior packaging systems which yield low numbers of recombinant virions which are contaminated with wild-type adenovirus. The small AAV genome that can be packaged (approximately 5 kb) rules out its use for transfer of larger genes. Recombinant AAV viruses do not appear to demonstrate the same site-specific genomic integration as wild-type viruses. Elucidation of the mechanism of site-specific integration should prove useful in the development of safe vectors for gene transfer as well as provide insight into the nature of DNA recombination in humans.
TL;DR: The transcriptional activity of a start−site sequence generally correlates with its similarity to the initiator consensus, suggesting that there is only one type of initiator.
Abstract: RNA polymerase II initiates transcription at specific DNA sequences. Studies using sequence analysis and molecular genetics suggest a simple and universal model of start-site selection by RNA polymerase II. Two consensus sequences occur at fixed positions in promoters from higher eukaryotes and their viruses: the TATA box around -30 and the initiator at the start site of transcription. Both consensus sequences function as positioning elements that control site-specific initiation. As a first step during initiation, the basal transcription factor TFIID binds to the TATA box; regulatory transcription factors can tether TFIID bind to the TATA box; regulatory transcription factors can tether TFIID to promoters without a consensus TATA box. TFIID then directs the assembly of other basal transcription factors and RNA polymerase II into a preinitiation complex. Finally, RNA polymerase II searches for the best match to the initiator consensus about 30 base pairs downstream of the TATA box to select the exact start site. The transcriptional activity of a start-site sequence generally correlates with its similarity to the initiator consensus, suggesting that there is only one type of initiator.
TL;DR: Strong evidence is provided to show that WB-F344 (WB) rat liver epithelial cells are stem-like precursor cells for hepatocytes and that progeny of BAG2-WB cells can be recovered from livers into which they have been transplanted, which may allow the elucidation of alterations in gene expression that accompany their differentiation.
Abstract: From a review of past studies and the report of new studies from our laboratory, this article provides strong evidence to show that WB-F344 (WB) rat liver epithelial cells are stem-like precursor cells for hepatocytes. WB cells are structurally and phenotypically simple epithelial cells that were isolated from the liver of an adult male Fischer 344 rat, under conditions that excluded their origin from hepatocytes in vivo. WB cells express a phenotypic repertory that overlaps, but is distinct from, that of both hepatocytes and bile duct epithelial cells. The complex phenotype of WB cells is compatible with their being embryonic or undifferentiated variants of either hepatocytes or bile duct epithelial cells. When WB cells are tagged genetically with genes for bacterial beta-galactosidase and neomycin resistance (BAG2-WB), they and their progeny can be distinguished from parental WB cells and hepatocytes by the expression of these gene products. Progeny of BAG2-WB cells that were transplanted into the liver parenchyma of syngeneic rats integrated into hepatic plates and acquired the morphological and functional attributes of adjacent host hepatocytes; the progeny of BAG2-WB cells in the liver express albumin, tyrosine aminotransferase, alpha-1-antitrypsin, and transferrin. We also demonstrate that progeny of BAG2-WB cells can be recovered from livers into which they have been transplanted, which may allow the elucidation of alterations in gene expression that accompany their differentiation.
TL;DR: The characteristics of progression—increased chromosomal damage, aneuploidy, growth of AHF in the absence of continued tumor promotion, the presence of foci-in-foci, and an increased incidence of malignant neoplasia—have been used as end points for the demonstration of progressor activity by ENU.
Abstract: Carcinogenesis is a multistage process consisting of the three distinct stages: initiation, promotion, and progression. The initiation-promotion-progression (IPP) protocol models these stages and establishes a method whereby agents that possess a carcinogenic risk can be classified as acting primarily at any one or combination of these stages. In one hepatocarcinogenesis IPP protocol, rats were initiated with 10 mg of diethylnitrosamine/kg body wt at 5 days of age, started on the promoting agent phenobarbital at weaning, subjected to a 70% partial hepatectomy at 6 months, and, at the peak of proliferation, given a putative progressor agent, ethylnitrosourea ([ENU] 100 mg/kg, ip) or hydroxy-urea ([HU] 3 x 150 mg/kg, ip). Administration of the promoting agent was discontinued after the progressor agent was given, and the rats were sacrificed 6 months later. The number and volume fraction of promoter-independent (growth in the absence of the promoting agent) altered hepatic foci (AHF) were then determined by quantitative stereology. The number of such AHF increased with either ENU or HU treatment compared with animals not given a progressor agent. In addition, hepatocytes isolated from animals subjected to an IPP regimen with ENU as the progressor agent exhibited a greater degree of chromosomal breakage and aneuploidy than animals not given a second initiator. A variation of this model, in which the promoting agent was maintained after administration of the progressor agent, was examined. In this IPP model, the number of heterogeneous AHF (foci-in-foci) increased after application of the progressor agent (ENU or HU). An increased incidence of hepatocellular carcinoma was also observed in animals subjected to the IPP protocol when promotion was maintained until sacrifice. Thus, the characteristics of progression--increased chromosomal damage, aneuploidy, growth of AHF in the absence of continued tumor promotion, the presence of foci-in-foci, and an increased incidence of malignant neoplasia--have been used as end points for the demonstration of progressor activity by ENU. In addition, the potential progressor activity of HU and benzene has been demonstrated with the IPP model of rat hepatocarcinogenesis.
TL;DR: The findings indicate that after GalN injury, the liver responds with activation of putative progenitor cells that proliferate and then differentiate through the hepatocyte lineage, whereas the regenerative response after CCl4 administration is primarily through proliferation of preexisting hepatocytes.
Abstract: Activation of liver progenitor cells was studied in rat liver induced to regenerate after carbon tetrachloride (CCl4) or D-galactosamine (GalN) injury. A change in the concentration of histone-3 mRNA was used as a marker for cell proliferation and the fetal form of alpha-fetoprotein (AFP) mRNA as a marker for fetal hepatoblasts. gamma-Glutamyltranspeptidase (GGT) and glutathione-S-transferase P were used as markers for activation of putative liver progenitor cells. After CCl4 administration, the proliferative response was high but confined primarily to parenchymal cells. No changes in the relative expression of albumin, glutathione-S-transferase P or insulin-like growth factor-II were observed. On the other hand, the level of AFP mRNA was increased modestly and predominantly in the nonparenchymal cell (NPC) fraction. After GalN administration, proliferation of NPC began within 24 hr, primarily in the portal area around the bile ducts. Activated cells were bile "duct-like" in appearance, had scant cytoplasm, and a pale, oval-shaped nucleus. On Day 2, they formed rows and clusters, expanding from the portal zone and invading the parenchyma, as well as proliferating in regions of focal necrosis. On Days 3 and 5, NPC expressing histone-3 mRNA expanded further, forming pseudoducts and islet-like structures (NPC structures) throughout the hepatic lobule. Proliferating NPC were positive for GGT. Some GGT-positive cells on Days 3 and 5 were also positive for fetal AFP mRNA. Expression of fetal AFP mRNA lagged behind that of GGT by 24 hr, was highest on Day 5, and then declined. Expression of albumin mRNA and glucose 6-phosphatase decreased during the first 48 hr after GalN administration and then resumed. These findings indicate that after GalN injury, the liver responds with activation of putative progenitor cells that proliferate and then differentiate through the hepatocyte lineage, whereas the regenerative response after CCl4 administration is primarily through proliferation of preexisting hepatocytes.
TL;DR: The tissue uptake of 67Cu from ceruloplasmin versus albumin and transcuprein, after its intravenous administration to pregnant rats, in the last 4 days of gestation is examined to conclude that Cu destined for the fetus is delivered mainly or exclusively by cerulplasmin.
Abstract: We have examined the tissue uptake of 67Cu from ceruloplasmin versus albumin and transcuprein, after its intravenous administration to pregnant rats, in the last 4 days of gestation. 67Cu infused as in vivo-labeled ceruloplasmin remained on ceruloplasmin in the maternal circulation over the 4- to 6-hr time period examined, as determined by gel chromatography and immunoreactivity. That infused as in vitro-labeled serum was initially on transcuprein and albumin but soon also with new ceruloplasmin. On the basis of percent dose as well as total actual Cu transferred (taking into account the sizes of the two plasma Cu pools), ceruloplasmin was the preferred source of Cu for most tissues. Total uptake of Cu from ceruloplasmin was seven times greater than that from albumin and transcuprein for the placenta, whole fetus, and fetal liver. It was 2- to 6-fold greater for other tissues (except liver and kidney). When synthesis of maternal 67Cu-ceruloplasmin (from 67Cu administered on albumin and transcuprein) was inhibited with cycloheximide, uptake by nonhepatic tissues was reduced markedly. In the fetal circulation, entering 67Cu was initially associated with transcuprein and alpha-fetoprotein (or albumin), but then also appeared with ceruloplasmin. Specific receptors for ceruloplasmin were detected on membranes from the placenta as well as fetal liver; mRNA for ceruloplasmin was detected on the endoplasmic reticulum-bound polyribosomes of placenta/yolk sac, and of fetal and maternal liver. We conclude that Cu destined for the fetus is delivered mainly or exclusively by ceruloplasmin. It may enter via placental receptors, arriving in fetal plasma in ionic form, for later incorporation into fetal ceruloplasmin. The importance of ceruloplasmin as a source of plasma Cu for nonhepatic organs is also confirmed.
TL;DR: The results suggest that the inhibition of the activity of 3β-HSD may be partially responsible for the disruption of testicular steroidogenesis during immobilization stress.
Abstract: We have examined the effect of 3 hr of immobilization stress on plasma luteinizing hormone, testosterone, and corticosterone levels, and on the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in microsomal and mitochondrial fractions of the testis from adult rats. Immobilization for 3 hr increased plasma corticosterone and reduced plasma testosterone concentrations by 57%. Plasma luteinizing hormone levels were lower, although not significantly (P = 0.093) so, in stressed animals. Immobilization (3 hr) reduced the Vmax values of 3 beta-HSD in the mitochondria and in the microsomal fraction of the testis by 40% and 34%, respectively, but had no effect on the Km values of 3 beta-HSD in the two cellular compartments. These results suggest that the inhibition of the activity of 3 beta-HSD may be partially responsible for the disruption of testicular steroidogenesis during immobilization stress.
TL;DR: Under dosing conditions commonly used to assess uterotrophic activity, these “antiestrogens” are complete, albeit less potent, estrogen agonists in the luminal epithelium and, unlike estrogens, induce hypertrophy in the glandular epithelia.
Abstract: Triphenylethylene antiestrogens are considered weak estrogen agonists based on their limited ability to induce estrogen responses, in particular uterine growth. We compared the uterotrophic activity of naturally occurring and synthetic estrogens with that of antiestrogens by quantitating uterine wet weight and hypertrophy in the uterine luminal and glandular epithelium. Immature rats received five daily injections of either an estrogen (17 beta-estradiol [E2], diethylstilbestrol [DES], or ethynyl estradiol [EE]) or an antiestrogen (tamoxifen [TAM], monohydroxytamoxifen [OH-TAM], or clomiphene citrate [CC]) (0.001-100 micrograms/rat/day) subcutaneously in sesame oil and were sacrificed approximately 2 hr after the last injection. Both DES and EE increased uterine weight at doses between 0.01-100 micrograms/rat/day; E2 was about 10-fold less potent. The antiestrogens increased uterine weight only slightly. DES, EE, and the three antiestrogens each increased luminal epithelium hypertrophy to over 3-fold above that in controls. While the potencies of these synthetic compounds differed (DES = EE > OH-TAM > TAM = CC), each hypertrophic response occurred over two log doses, and the response curves displayed identical slopes. E2, however, required a range of four log doses to achieve the same degree of luminal epithelium hypertrophy. The three antiestrogens elicited glandular epithelium hypertrophy up to 2-fold above controls at the same doses that induced luminal epithelium hypertrophy; the order of potency was OH-TAM > TAM = CC. However, the three estrogens increased glandular epithelium hypertrophy only marginally. Thus, under dosing conditions commonly used to assess uterotrophic activity, these "antiestrogens" are complete, albeit less potent, estrogen agonists in the luminal epithelium and, unlike estrogens, induce hypertrophy in the glandular epithelium.
TL;DR: It is shown that dietary or in vitro supplementation with Se results in a significant upregulation of the expression of both the p55 and p70/75 IL-2 binding sites on the surface of concanavalin A-stimulated lymphocytes from C57BL/6J mice.
Abstract: Selenium (Se) is an essential nutritional factor that was shown previously by us to alter the kinetics of expression of high affinity (p55/p75) interleukin 2 receptors (IL-2R). This study shows that dietary (2 ppm for 8 weeks) or in vitro (1 x 10(-7) M) supplementation with Se (as sodium selenite) results in a significant upregulation of the expression of both the p55 and p70/75 IL-2 binding sites on the surface of concanavalin A-stimulated lymphocytes from C57BL/6J mice. This resulted in the formation of significantly higher numbers of high affinity IL-2R/cell with preservation of the normal ratio of high affinity to total IL-2 binding sites/cell. The high affinity IL-2R on cells from Se-supplemented animals functioned normally in terms of ligand binding and kinetics of IL-2 internalization, but their greater numbers/cell resulted in the internalization of significantly larger amounts of IL-2/cell. As Se supplementation results in an earlier expression of greater numbers of high affinity IL-2R, the presence of Se in the cell environment can result in an accelerated clonal expansion of activated lymphocytes.
TL;DR: Results from longitudinal training studies have been inconsistent because of experimental design, i.e., inadequate type, duration, and intensity of exercise intervention, lipid measurements made across the menstrual cycle, and studies carried out in women with high baseline HOL-C.
Abstract: This review summarizes the cross-sectional and training studies (acute and chronic) that have examined the relationship between exercise and plasma lipid and lipoproteins in women. Because women experience major fluctuations in reproductive hormones throughout the life cycle, the effects of the endogenous sex steroid status on the association between exercise and plasma lipoproteins also are addressed. In general, cross-sectional studies report a positive association between exercise and high density lipoprotein-cholesterol (HDL-C) in both pre- and postmenopausal women. Women on hormone replacement therapy who report exercising have higher HDL-C than sedentary women on hormone replacement therapy. Results from longitudinal training studies have been inconsistent because of experimental design, i.e., inadequate type, duration, and intensity of exercise intervention, lipid measurements made across the menstrual cycle, and studies carried out in women with high baseline HDL-C. Since lipids vary approximately 10-25% throughout the menstrual cycle, menstrual phase should be controlled when determining lipid changes after an exercise intervention. In approximately half of the intervention studies, an increase in HDL-C was demonstrated; the magnitude of the response that can be expected is approximately 10%. The responsiveness of pre- versus postmenopausal women to an exercise intervention is unknown. Studies are needed to clarify the interactive effects of exercise and sex hormones on plasma lipoproteins in women of all ages. This information will be useful in developing intervention programs to reduce the risk of coronary heart disease in women.
TL;DR: Although a glucocorticoid receptor-mediated antiproliferative effect is evident, PRL has the capacity to protect the cell against glucocORTicoid-receptor-mediated induction of apoptosis, which is indicative of programmed cell death or apoptosis.
Abstract: The interaction between the immunosuppressive effects of glucocorticoids and the mitogenic effects of prolactin (PRL) were examined in Nb2 lymphoma cells, a pre-T cell line. The synthetic glucocorticoid, dexamethasone (Dex), caused a concentration-dependent (6.25-200 nM) inhibition of basal and ovine PRL (oPRL)-stimulated Nb2 cell proliferation. Although Dex was antiproliferative, the steroid had no effect on cell viability in the presence of PRL. However, when PRL was omitted from the medium, Dex increased the proportion of dead Nb2 cells by 24 hr in a concentration (25-200 nM)-dependent fashion without affecting total cell number. The antiproliferative and cytolytic effects of Dex were mimicked by other corticosteroids (cortisol, corticosterone, aldosterone, and deoxycorticosterone) in the expected order of glucocorticoid potency, but not by other steroids (17-beta-estradiol, progesterone, testosterone, 5-alpha-dihydrotestosterone, and dehydroepiandrosterone) or triiodothyronine. In addition, the antiproliferative and cytolytic effects of glucocorticoids were antagonized by the glucocorticoid receptor antagonist RU 486. Since corticosteroid-induced cytolysis was apparent only in the absence of mitogen, the anticytolytic effects of oPRL were tested. In the presence of Dex (100 nM), oPRL (25-1600 pg/ml) caused a concentration-dependent inhibition of cytolysis without changing cell number. Other lactogenic hormones (human growth hormone, human placental lactogen, rat PRL), but not trophic nonlactogenic hormones (rat growth hormone, human chorionic gonadotropin, ACTH), also inhibited Dex (100 nM)-induced cytolysis. Agarose gel electrophoresis of DNA extracted from Nb2 cells revealed that within 12 hr, 100 nM Dex induced DNA fragmentation, indicative of programmed cell death or apoptosis. Coincubation of cells with Dex and oPRL (1 ng/ml) inhibited Dex-induced fragmentation of Nb2 cell genomic DNA. These studies reveal a complex interaction between glucocorticoids and PRL in Nb2 cells. Although a glucocorticoid receptor-mediated antiproliferative effect is evident, PRL (at concentrations that usually stimulate cell proliferation) has the capacity to protect the cell against glucocorticoid-receptor-mediated induction of apoptosis.
TL;DR: Infants born to monkeys fed low zinc diets are characterized by evidence of DNA damage shortly after birth; this damage may be due to an increased rate of oxidative damage and/or a reduction in the rate of DNA repair.
Abstract: Severe zinc deficiency in rodent models has been shown to influence the frequency of single-strand breaks in DNA isolated from liver. In the current study, we investigated whether DNA isolated from infant monkeys born to mothers fed zinc-restricted diets would be characterized by higher than normal levels of DNA damage. DNA was isolated from 30-day-old infants born to dams fed low zinc (2 or 4 micrograms Zn/g) or control zinc (50 micrograms Zn/g) diets. The amount of single-strand breaks in liver DNA was significantly higher in the low zinc group than in controls; consistent with the above, there was a trend for higher steady state levels of liver 8-hydroxy-2'-deoxyguanosine in the low zinc group. While evidence for DNA damage in the low zinc group was obtained, the activities of several antioxidant enzymes were similar between the low zinc and control groups. In summary, infants born to monkeys fed low zinc diets are characterized by evidence of DNA damage shortly after birth; this damage may be due to an increased rate of oxidative damage and/or a reduction in the rate of DNA repair.
TL;DR: In an attempt to measure plasma thiobarbituric acid-reactive substances (TBARS) during extracorporeal membrane oxygenation, a form of sustained cardiopulmonary bypass, it became apparent that the absorbance signal at the 532-nm wavelength was composed not only of the peak absorbance of TBARS, but also of interfering substances from heme pigments and bilirubin.
Abstract: The 2-thiobarbituric acid reaction with malondialdehyde has been used to assess lipid peroxidation in a variety of biologic systems. However, in an attempt to measure plasma thiobarbituric acid-reactive substances (TBARS) during extracorporeal membrane oxygenation, a form of sustained cardiopulmonary bypass, it became apparent that the absorbance signal at the 532-nm wavelength was composed not only of the peak absorbance of TBARS, but also of interfering substances from heme pigments and bilirubin. A method of subtracting interfering substances was developed and applied to normal human plasma. The method was tested by adding varying amounts of red blood cell hemolysate, bilirubin, and 1,1,3,3-tetramethoxypropane (TMP) standard to plasma and determining TBARS in the resulting mixture. In addition, varying the amount of added desferoxamine was investigated to determine the effects of iron chelation on the assay. This was important because the different samples would have varying amounts of free iron from hemoglobin to catalyze the reaction. It was found that the following equation could be used in this system to determine that amount of 532-nm absorption due to TBARS: MDA532 = 1.22[(A532) - (0.56)(A510) + (0.44)(A560)]. Regression analysis revealed an 86.6% recovery of the TMP spike. Analysis of variance showed that the variability in the model could be explained mainly by the additive increments of TMP spike (94.6%).
TL;DR: Future studies of this least understood and most complex of gluconeogenic enzymes seem to us poised at the edge of a golden era.
Abstract: Areas which we believe merit immediate intensive study include (i) isolation and characterization, chemically and functionally, of all of the individual components of the system; (ii) characterization of possible multiple forms of transport components, as we have done with T2 (24, 25, 36); (iii) optimistically, the reassembly of these components into a functional unit within synthetic biomembranes; (iv) characterization of the impact of components of biomembranes upon function of the various components; (v) cloning of all of the individual proteins of the system; (vi) characterization of mechanisms regulating the biosynthesis of the individual proteins at the transcriptional and translational levels; (vii) demonstration of possible auxiliary functions of translocases other than exclusively with the glucose 6-phosphatase system; (viii) characterization of possible physical associations of some components of the system; (ix) characterization of the glucose 6-phosphatase system from nonhepatic sources; (x) c...
TL;DR: It is concluded that neonatal hormonal imprinting has a significant influence on the incidence of diabetes in the NOD mouse.
Abstract: The nonobese diabetic (NOD) mouse is a model of Type I (insulin-dependent) diabetes. It develops autoimmune pancreatic beta-cell lesions characterized by lymphocytic infiltration and beta-cell destruction. The incidences of diabetes for male and female NOD mice in our colony were 24% and 73%, respectively. In this study, we investigated the effect of neonatal manipulation of the sex hormone profile on the incidence of diabetes in male and female NOD mice. One day after birth, male mice were castrated and female mice were either ovariectomized, given testosterone, or ovariectomized and given testosterone. The mice were maintained for 140 days and blood samples were collected biweekly starting at 42 days old. Diabetes was determined by three consecutive blood glucose levels > 200 mg/dl. Neonatal gonadectomy increased the incidence of diabetes in males but decreased it in females. Females treated with testosterone also had a decreased incidence of diabetes, whereas ovariectomy plus testosterone increased the incidence to 100%. Castration decreased the body weight in males and increased body weight in females. Testosterone treatment with or without ovariectomy also increased body weight. From these studies, we concluded that neonatal hormonal imprinting has a significant influence on the incidence of diabetes in the NOD mouse.