bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigallocatechin Gallate Modulates Cytokine Production by Bone Marrow-Derived Dendritic Cells Stimulated with Lipopolysaccharide or Muramyldipeptide, or Infected with Legionella pneumophila
Authors: Rogers James , Perkins Izabella , Olphen Alberto van , Burdash Nicholas , Klein Thomas W. , Friedman Herman
Published: July 2017

The primary polyphenol in green tea extract is the catechin epigallocatechin gallate (EGCG). Various studies have shown significant suppressive effects of catechin on mammalian cells, either tumor or normal cells, including lymphoid cells. Previous studies from this laboratory reported that EGCG has marked suppressive activity on murine macrophages infected with the intracellular bacterium Legionella pneumophila (Lp), an effect mediated by enhanced production of both tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ). In the present study, primary murine bone marrow (BM)-derived dendritic cells (DCs), a phagocytic monocytic cell essential for innate immunity to intracellular microorganisms, such as Lp, were stimulated in vitro with the microbial stimulant lipopolysaccharide (LPS) from gram-negative bacteria, the cell wall component from gram-positive bacteria muramyldipeptide (MDP) or infected with Lp. Production of the T helper cell (Th1)-activating cytokine, interleukin-12 (IL-12) and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), produced mainly by phagocytic cells and important for antimicrobial immunity, was determined in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Treatment of the cells with EGCG inhibited, in a dose-dependent manner, production of IL-12. In contrast, enhanced production of TNF-α occurred in a dose-dependent manner in the DC cultures stimulated with either soluble bacterial product or infected with Lp. Thus, the results of this study show that the EGCG catechin has a marked effect in modulating production of these immunoregulatory cytokines in stimulated DCs, which are important for antimicrobial immunity, especially innate immunity. Further studies are necessary to characterize the physiologic function of the effect of EGCG on TNF-α and IL-12 during Lp infection, and the mechanisms involved.
Ontogeny of Expression of Organic Anion Transporters 1 and 3 in Ovine Fetal and Neonatal Kidney
Authors: Wood Charles E. , Cousins Roderick , Zhang Daying , Keller-Wood Maureen
Published: July 2017

Organic ions are excreted into the urine via the action of organic anion transporters (OATs). In adult kidney, both OAT1 and OAT3, both multispecific transporters, are abundant; OAT1 is a known transporter of para-aminohippurate (PAH) and OAT3 is a known transporter of sulfoconjugated estrogens. The present study was designed to test the hypotheses that the expression of both OAT1 and OAT3 are developmentally regulated and that the expression increases in late gestation. Fetal kidneys were collected at sacrifice of fetal sheep at 80, 100, 120, 130, and 145 days of gestation, as well as 1 day and 1 week after birth (n = 4–5 per group). Renal tissue was separated into cortex and medulla and snap-frozen in liquid nitrogen for later extraction of mRNA. The expression levels of OAT1 and OAT3 were measured using real-time reverse transcriptase polymerase chain reaction (RT-PCR), with specific probes and primers designed in our laboratory. Cellular distribution of protein expression was identified using immunohistochemistry with commercially available antisera. The OAT1 and OAT3 mRNA in renal cortex was increased in the more mature animals. At 145 days of gestation, OAT1 mRNA abundance was increased and remained elevated postnatally. Compared with prenatal ages, OAT3 mRNA was increased postnatally. The expression of both transporters was not significantly changed as a function of development in the renal medulla. The protein expression of OAT1 and OAT3 was identified in tubular epithelium in renal cortex, although the immunoreactivity for OAT1 was greater than for OAT3. We conclude that there is a developmental pattern of expression of both OAT1 and OAT3 in ovine renal cortex, and that the pattern of expression suggests that the function of both transporters is likely to be greater starting in late gestation.
Adult Stem Cells for Cardiac Repair: A Choice Between Skeletal Myoblasts and Bone Marrow Stem Cells
Authors: Ye Lei , Haider Husnain Kh , Sim Eugene K. W.
Published: July 2017

The real promise of a stem cell–based approach for cardiac regeneration and repair lies in the promotion of myogenesis and angiogenesis at the site of the cell graft to achieve both structural and functional benefits. Despite all of the progress and promise in this field, many unanswered questions remain; the answers to these questions will provide the much-needed breakthrough to harness the real benefits of cell therapy for the heart in the clinical perspective. One of the major issues is the choice of donor cell type for transplantation. Multiple cell types with varying potentials have been assessed for their ability to repopulate the infarcted myocardium; however, only the adult stem cells, that is, skeletal myoblasts (SkM) and bone marrow–derived stem cells (BMC), have been translated from the laboratory bench to clinical use. Which of these two cell types will provide the best option for clinical application in heart cell therapy remains arguable. With results pouring in from the long-term follow-ups of previously conducted phase I clinical studies, and with the onset of phase II clinical trials involving larger population of patients, transplantation of stem cells as a sole therapy without an adjunct conventional revascularization procedure will provide a deeper insight into the effectiveness of this approach. The present article discusses the pros and cons of using SkM and BMC individually or in combination for cardiac repair, and critically analyzes the progress made with each cell type.
Soy Protein Isolate Induces CYP3A1 and CYP3A2 in Prepubertal Rats
Authors: Ronis Martin J. , Chen Ying , Badeaux Jamie , Laurenzana Elizabeth , Badger Thomas M.
Published: July 2017

Feeding soy diets has been shown to induce cytochrome P450s in gene family CYP3A in Sprague-Dawley rat liver. We compared expression of CYP3A enzymes on postnatal Day 33 (PND33) rats fed casein or soy protein isolate (SPI+)–based AIN-93G diets continuously from gestational Day 4 through PND33 or the diets were switched on PND15 (n = 3–6 litters) to examine the potential imprinting effects of soy on drug metabolism. In addition rats were fed casein, SPI+, SPI+ stripped of phytochemicals (SPI–), or casein diets supplemented with the soy-associated isoflavones genistein or daidzein from weaning through PND33 to examine the hypothesis that the isoflavones are responsible for CYP3A induction by soy feeding. Feeding SPI either continuously or from weaning induced hepatic CYP3A1 and CYP3A2 mRNA, apoprotein, and CYP3A-dependent testosterone 6β-hydroxylase activity in liver microsomes 2- to 5-fold (P < 0.05). CYP3A mRNA expression was also elevated 2- to 3-fold in the jejunum of SPI-fed rats (P < 0.05). CYP3A was not induced in livers of rats switched to casein from soy at weaning. Induction of CYP3A1 also did not occur in rats fed SPI–, but CYP3A2 mRNA and apoprotein were induced (P < 0.05) in females fed SPI–. Offspring weaned onto genistein-supplemented diets had no elevation of CYP3A mRNAs or apoproteins. Weaning onto daidzein diets increased CYP3A2 mRNA and apoprotein expression in male rats (P < 0.05). These data suggest that early soy consumption may increase the metabolism of a wide variety of CYP3A substrates, but that soy does not imprint the expression of CYP3A enzymes. Effects on CYP3A1 expression appear to be primarily due to phytochemical components of SPI other than isoflavones. In contrast, consumption of soy protein and daidzein appear to be associated with the induction of CYP3A2.
ECRG1, a Novel Candidate of Tumor Suppressor Gene in the Esophageal Carcinoma, Triggers a Senescent Program in NIH3T3 Cells
Authors: Zhao Nianxi , Huang Ge , Guo Liping , Lu Shih-Hsin
Published: July 2017

Esophageal cancer–related gene 1 (ECRG1) is a novel tumor-suppressor gene candidate identified from the human esophagus. Previous studies showed that ECRG1 overexpression could inhibit cell growth and induce G1 cell cycle arrest and p15INK4b expression by interacting with Miz-1 (Myc-interacting zinc finger protein). Such evidence suggests the alterations in ECRG1 may play an important role in tumorigenesis. To further study the biological function of the ECRG1 gene, we transfected ECRG1 into NIH3T3 cells. Expression of ECRG1 in these cells caused senescence-like changes characterized in terms of altered cell morphology, cell cycle arrest at the G1/S phase, and significantly impaired cell proliferation (P < 0.01). Moreover, NIH3T3 cells transfected with ECRG1 stained positive for SA-β-gal staining (pH 6.0), which is a specific marker of cellular senescence. We also studied changes in telomerase activity and the related senescence genes, such as p21 and p16. The results indicated that when ECRG1 induced a senescence-like state, telomerase activity was markedly decreased (P < 0.05), and expression of p21 was distinctly increased, whereas no changes were detected in p16 and telomerase-component RNA levels. These findings suggest that ECRG1 may be of importance in murine ceil senescence, promoting senescence by regulating expression of p21.
Targeting Apoptosis with Dietary Bioactive Agents
Authors: Martin Keith R.
Published: July 2017

Apoptosis, a form of programmed cell death, is a pivotal defense against the occurrence of cancer and is essential to metazoans in maintaining tissue homeostasis. Apoptosis exhibits a distinctive phenotype and involves elimination of potentially deleterious cells. Many diseases have been associated with aberrantly regulated apoptotic cell death, ultimately leading to inhibition of apoptosis and propagation of diseases such as cancer. Elucidation of the critical events associated with carcinogenesis provides the opportunity for dietary intervention to prevent cancer development through induction of apoptosis, Particularly by bioactive agents or functional foods. Diet is a significant environmental factor in the overall cancer process and can exacerbate or interfere with carcinogenesis. Apoptosis occurs primarily through two well-recognized pathways in cells, including the Intrinsic, or mitochondrial-mediated, effector mechanism and the extrinsic, or death receptor-mediated, effector mechanism. In addition to diet's effects on protein expression and function, evidence is also accumulating that a large number of dietary food components can exert effects on the human genome, either directly or indirectly, to modulate gene expression. In fact, many diet-related genes are involved in carcinogenesis as well as apoptosis, and thus are ultimately molecular targets for dietary chemopreventlon. There are multiple steps within pathways in which dietary components can alter gene expression and phenotypes of cells and thus influence cancer outcomes (nutritional transcriptomic effect). Thus, apoptosis is an emerging therapeutic target of bioactive agents of diet. In this review, the process of apoptosis is discussed and the potential mechanistic interaction of bioactive agents, as components of functional foods, is explored within the context of apoptosis.
Gastrointestinal Inflammation: A Central Component of Mucosal Defense and Repair
Authors: Martin Gary R. , Wallace John L.
Published: July 2017

The mucosal layer of the gastrointestinal (GI) tract is able to resist digestion by the endogenous substances that we secrete to digest foodstuffs. So-called “mucosal defense” is multifactorial and can be modulated by a wide range of substances, many of which are classically regarded as inflammatory mediators. Damage to the GI mucosa, and its subsequent repair, are also modulated by various inflammatory mediators. In this article, we provide a review of some of the key Inflammatory mediators that modulate GI mucosal defense, Injury, and repair. Among the mediators discussed are nitric oxide, polyamines, the elcosanolds (prostaglandins and II-poxlns), protease-activated receptors, and cytokines. Many of these endogenous factors, or the enzymes involved in their synthesis, are considered potential therapeutic targets for the treatment of diseases of the digestive tract that are characterized by Inflammation and ulceration.
Metallothionein and Liver Cell Regeneration
Authors: Cherian M. George , Kang Y. James
Published: July 2017

Hepatocytes in adults are in a nonproliferative state but they have high capacity to regenerate within few hours after an Injury. After partial hepatectomy or chemical injury, hepatocytes undergo a synchronized multistep process consisting of prlming/initiation, proliferation, and termination. These distinct steps are essential for restoring the structure and functions of liver. The mechanisms involved in each of these steps of regeneration are well documented from various laboratories and are described in several reviews. We briefly describe these steps and the Involvement of various cytokines and growth factors for cell regeneration in this short review. Liver cell regeneration may also involve stem cell proliferation. The regenerating cells require large amounts of zinc within a short time, and this requirement is met by induction of a zinc and copper binding protein, metallothionein (MT), during the priming step, soon after an injury. There are several reports on the transfer of zinc from MT to various metalloenzymes and transcription factors. Genetically modified mouse models have been used to study the Involvement of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in cell regeneration. The use of an MT-knockout mouse has enabled us to Investigate the specific role of MT in liver regeneration after partial hepatectomy, chemical injury, and fibrosis. Several studies have suggested a defective liver regeneration after an Injury in MT-knockout mice. There is cumulative evidence that indicates an essential role for MT in liver cell regeneration.
Store-Operated Ca2+ Channel in Renal Microcirculation and Glomeruli
Authors: Ma Rong , Du Juan , Sours Sherry , Ding Min
Published: July 2017

Store-operated Ca2+ channel (SOC) is defined as a channel that opens in response to depletion of the internal Ca2+ stores. During the last decade, many investigators have made a great effort to identify and characterize SOC, and to evaluate its physiologic function and pathophysiologic relevance in a variety of cell lines, primary cultures, and native tissues. To date, accumulating evidence has demonstrated that SOC is an essential Ca2+ entry mechanism in vascular smooth-muscle cells of renal microvasculature and glomerular mesangial cells, both of which tightly control glomerular hemodynamics and filtration. Store-operated Ca2+, combined with other types of Ca2+ entry channels, constitutes a profile of Ca2+ changes in response to physiologic vasoconstrictors and, thereby, regulates renal microcirculation and mesangial function. In addition, SOC is associated with altered Ca2+ signaling occurring in diseased kidneys, such as diabetic nephropathy. Although the gating mechanism and molecular identity of SOC are still enigmatic and may be cell-type and tissue specific, data from several independent groups suggest that protein kinase C plays an important role in SOC activation and that certain isoforms of canonical transient receptor potential (TRPC) proteins are candidates of SOC in renal mlcrovessels and mesangial cells.
Cardiac Cytochrome-c Oxidase Deficiency Occurs During Late Postnatal Development in Progeny of Copper-Deficient Rats
Authors: Johnson W. Thomas , Brown-Borg Holly M.
Published: July 2017

Although cytochrome-c oxidase (CCO) is a copper-dependent enzyme, the effect of maternal copper deficiency on the expression of CCO activity during postnatal development of the neonatal rat heart has not been investigated extensively. Here, we show that CCO activity in heart mitochondria isolated from neonates of copper-deficient dams did not exhibit significant reductions until postnatal days (PND) 15 and 21. In addition, Immunoblot analysis indicated that the CCO subunit (Cox-1) was reduced on postnatal Days 10 and 21, and that Cox-4 was reduced on PND 21 in heart mitochondria of the neonates from copper-deficient dams. These findings indicate that the impairment of CCO activity in neonatal heart by maternal copper deficiency occurs late in the postnatal heart development. Furthermore, the concurrent reductions in Cox-1 and Cox-4 suggest that the impaired CCO activity reflects a CCO deficiency in heart mitochondria. CCO activity and Cox-1 in heart mitochondria were not fully restored by 6 weeks of postweaning copper repletion in the pups of copper-deficient dams. This indicates that prolonged maternal intake of moderately low dietary copper produces CCO deficiency in cardiac mitochondria of neonates during late postnatal heart development, after terminal differentiation of cardiomyocytes occurs. The resistance of CCO deficiency to repair by dietary copper supplementation may be related to the relatively slow turnover of the affected mitochondria in the terminally differentiated heart.
Cyclooxygenase Products Stimulate Carbon Monoxide Production by Piglet Cerebral Microvessels
Authors: Kanu Alie , Gilpin David , Fedinec Alexander L. , Leffler Charles W.
Published: July 2017

Products of arachidonic acid (AA) metabolism by cyclooxygenase (Cox) are important in regulation of neonatal cerebral circulation. The brain and cerebral microvessels also express heme oxygenase (HO) that metabolizes heme to carbon monoxide (CO), biliverdin, and iron. The purpose of this study in newborn pig cerebral microvessels was to address the hypothesis that Cox products affect HO activity and HO products affect Cox activity. AA (2.0–20 u.μM) increased prostaglandin E2 (PGE2) measured by radioimmunoassay (RIA) and also CO measured by gas chromatography/mass spectrometry (GC/MS). Further, 10-4 M indomethacin, which inhibited Cox, reduced both AA and heme-induced CO production. Conversely, neither exogenous 2 × 106 M heme, which markedly increased CO production, nor the inhibitor of HO, chromium mesoporphyrin, altered PGE2 synthesis. Because AA metabolism by Cox generates both prostanoids and superoxides, we determined the effects of the predominant prostanoid and superoxide on CO production. Although PGE2 caused a small increase in CO production, xanthine oxidase plus hypoxanthine, which produces superoxide, strongly stimulated the production of CO by cerebral microvessels. This increase was mildly attenuated by catalase. These data suggest that Cox-catalyzed AA metabolites, most likely superoxide and/or a subsequent reactive oxygen species, increase cerebrovascular CO production. This increase seems to be caused, at least in part, by the elevation of HO-2 catalytic activity. Conversely, Cox activity is not affected by HO-catalyzed heme metabolites. These data suggest that some cerebrovascular functions attributable to Cox activity could be mediated by CO.
Redox State and O2•- Production in Neutrophils of Crohn's Disease Patients
Authors: Biagioni Chiara , Favilli Fabio , Catarzi Serena , Marcucci Tommaso , Fazi Marilena , Tonelli Francesco , Vincenzini Maria T. , Iantomasi Teresa
Published: July 2017

The aim of this in vitro study was to evaluate the intracellular redox state and respiratory burst (RB) in neutrophils of patients with Crohn's disease (CD). The intracellular redox state and RB in neutrophils was assessed by the superoxide anion (O2•-) production induced in these cells after stimulation by various factors related to the molecular mechanisms that, if altered, may be responsible for an abnormal immune response. This can, in part, cause the onset of inflammation and tissue damage seen in CD. This study demonstrated a decreased glutathione/glutathione disulfide (GSH/GSSG) ratio index of an increased oxidative state in CD patient neutrophils. Moreover, our findings showed a decrease in tumor necrosis factor (TNF-α)- or phorbol 12-myristate 13-acetate (PMA)–induced O2•- production in CD patient neutrophils adherent to fibronectin as compared with controls. A decreased adhesion was also demonstrated. For this reason, the involvement of altered mechanisms of protein kinase C (PKC) and β-integrin activation in CD patient neutrophils is suggested. These data also showed that the harmful effects of TNF-α cannot be caused by excessive reactive oxygen species (ROS) production Induced by neutrophils. Decreased cell viability after a prolonged time of adhesion (20 hrs) was also measured in CD patient neutrophils. The findings of this study demonstrate, for the first time, that granulocyte-macrophage colony–stimulating factor (GM-CSF), a compound recently used in CD therapy, is able to activate the RB for a prolonged time both in control and CD patient neutrophils. Increased viability of CD patient neutrophils caused by GM-CSF stimulation was also observed. In conclusion, our results indicate that decreased O2•- production and adhesion, caused, in part, by an anomalous response to TNF-α, together with low GSH level and low cell viability, may be responsible for the defective neutrophil function found in CD patients. This can contribute to the chronic inflammation and relapses that characterize this pathology. A possible role of GM-CSF in inducing O2•- production and in restoring the defensive role of neutrophils in CD patients is suggested.
Effects of an S84E Mutation of Bovine Growth Hormone in Transgenic Mice
Authors: Schenck E. J. H. , Brooks C. L.
Published: July 2017

The ability of mutant bovine growth hormones (bGH) to serve as either agonist or antagonist has been demonstrated in transgenic mice. We have prepared two transgenic strains of FVB/N mice, one expressing wild-type bGH and a second with a glutamic acid mutation at serine 84 in helix 2. Comparison of their phenotypes to those of nontransgenic littermates indicates that wild-type bGH induces a previously described phenotype for hyper-somatotrophic mice. In contrast, the replacement of the side chain hydroxyl at serine 84 with acetic acid produced a phenotype that expressed bGH at appreciable concentrations, but failed to elicit the phenotype observed with either an agonist or an antagonist of bGH. These results indicate that serine 84 is crucial for the activity of bGH despite this site being distal to the receptor binding surfaces.
Investigations of a Human Embryonic Globin Gene Silencing Element Using YAC Transgenic Mice
Authors: Navas Patrick A. , Li Qiliang , Peterson Kenneth R. , Stamatoyannopoulos George
Published: July 2017

A silencing element has been previously located upstream of the human ε-globin gene promoter using transient assays and transgenic mice carrying plasmid constructs in which the element has been deleted or its transcriptional motifs have been mutated. To investigate whether this element functions in the context of the whole β-globin locus, we analyzed ε-globin gene expression in transgenic mice carrying a deletion of the silencing element in the context of a 213-kilobase human β-globin yeast artificial chromosome (β-YAC). ε-Globin gene expression was measured during embryonic and fetal development and in adult mice. ε-mRNA levels in embryonic cells in Day 12 blood were as high as those measured in wild-type β-YAC controls, indicating that the deletion does not affect ε gene promoter function. ε-Globin gene expression was confined to the embryonic cells, indicating that deletion of this silencing element did not affect ε-globin developmental expression in the context of the β-YAC. These results suggest that in the context of the whole β-globin locus, other proximal and upstream ε gene promoter elements as well as competition by the downstream globin genes contribute to the silencing of the ε-globin gene in the cells of definitive erythropoiesis.
Nerve Growth Factor Increases in Pancreatic β Cells After Streptozotocin-Induced Damage in Rats
Authors: Larrieta Maria Elena , Vital Paz , Mendoza-Rodríguez Adriana , Cerbón Marco , Hiriart Marcia
Published: July 2017

We investigated short-term in vivo and in vitro effects of streptozotocin (STZ) on pancreatic β cells. Male Wistar rats were treated with 75 mg/kg STZ, and, after 4 hrs blood glucose and insulin were measured and islet cells were isolated, cultured for 16 hrs, and challenged with 5.6 and 15.6 mM glucose. Treated rats showed hyperglycemia (14 mM) and a 70% decrease in serum insulin levels as compared with controls. Although insulin secretion by isolated β cells from STZ-treated rats was reduced by more than 80%, in both glucose concentrations, nerve growth factor (NGF) secretion by the same cells increased 10-fold. Moreover, NGF messenger RNA (mRNA) expression increased by 30% as compared with controls. Similar results were obtained in an in vitro model of islet cells, in which cells were exposed directly to STZ for 1, 2, and 4 hrs and then challenged for 3 hrs with the same glucose concentrations. Our data strongly suggest that an early increase in NGF production and secretion by β cells could be an endogenous protective response to maintain cell survival and that diabetes mellitus may occur when this mechanism is surpassed.
Involvement of Protein Kinases on the Upregulation of Endothelin Receptors in Rat Basilar and Mesenteric Arteries
Authors: Jamali Roya , Edvinsson Lars
Published: July 2017

EndothelinB (ETB) receptors are upregulated in experimental stroke or after 24 hrs of organ culture. This upregulation is manifested both as stronger contraction and as an increase in ETB receptor messenger RNA (mRNA) levels. The present study was designed to evaluate the importance of protein kinases (c-Jun N-terminal kinase [JNK], protein kinase C [PKC], and extracellular signal-regulated kinase [ERK1/2]) in ETB receptor upregulation after organ culture. Rat basilar and mesenteric arteries were incubated for 24 hrs in Dulbecco's modified Eagle's medium (DMEM) with or without the PKC inhibitor, RO-31–7549; the ERK1/2 inhibitor, SB386023; or the JNK inhibitor, SP600125, added 3, 6, or 12 hrs after initiation of incubation. Subsequently, vessel segments were mounted in myographs and the contractile responses to ET-1 and sarafotoxin 6c were studied. The ETB and ETA receptor mRNA levels were determined with a real-time polymerase chain reaction (PCR). The cellular localization and protein level of ETB receptors were evaluated by immunohistochemistry. The PKC and ERK1/2 inhibitors attenuated the contraction induced by S6c in the basilar arteries more than in the mesenteric arteries. The efficiency of the inhibitors was proportional to the incubation time. Real-time PCR showed a decrease in the ETB receptor mRNA levels in arteries treated with PKC or ERK inhibitors. The JNK inhibitor had a significant inhibitory effect on ETB receptor upregulation in the basilar arteries. Immunohistochemistry revealed that the ETB receptor upregulation occured in the smooth-muscle cells and that it had the same pattern as in the quantitative PCR. Our results show that the PKC, ERK1/2, and JNK are more important for the upregulation of contractile ETB receptors in cerebral arteries compared with mesenteric arteries. ERK1/2 seems to be more important for the ETB receptor upregulation, as compared with PKC and JNK. The evaluation of the time dependency suggests that the phenomenon can be reversed even after its initiation.
Effects of Scutellaria baicalensis Georgi on Macrophage-Hepatocyte Interaction Through Cytokines Related to Growth Control of Murine Hepatocytes
Authors: Wang Jir-You , Chuang Han-Ning , Chiu Jen-Hwey , Fu Shu-Ling , Tsai Tung-Hu , Tsou Ann-Ping , Hu Cheng-Po , Chi Chin-Wen , Yeh Sheau-Farn , Lui Wing-Yiu , Wu Chew-Wun , Chou Chen-Kung
Published: July 2017

The aim of this study is to elucidate the effects of Scutellaria baicalensis Georgi (SbG) extract and its constituents on macrophage-hepatocyte interaction in primary cultures. By using trans-well primary Kupffer cell culture or conditioned medium (CM) from murine macrophage RAW264.7 cell line (RAW cells), effects of SbG on hepatocyte growth were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and trypan blue exclusion assay. Cytokine production, antibody-neutralization studies, and molecular mechanisms of transforming growth factor (TGF)-β1 gene expression were elucidated on SbG-treated RAW264.7 cells. In addition, recombinant human TGF-β1 (r-human TGF-β1) was added to elucidate the mechanisms of SbG effects on cultured hepatocytes. Immunohistochemistry using anti-NF-κB antibody was used to determine the possible signal transduction pathways in primary hepatocyte culture. The results showed that SbG stimulated the proliferation of cultured hepatocytes, possibly through NF-κB, but not of Toll-like receptor 4 activation; whereas SbG-RAW-CM and SbG in trans-well significantly suppressed the proliferation of hepatocytes. Antibody-neutralization studies revealed that TGF-β1 was the main antimitotic cytokine in SbG-treated RAW cells CM. The growth stimulation effect of SbG on cultured hepatocytes was inhibited by exogenous administration of r-human TGF-β1. Furthermore, SbG induced NF-κB translocation into the nuclei of cultured cells. In the RAW264.7 line, SbG and baicalin stimulated TGF-β1 gene expression via NF-κB and protein kinase C activation. We conclude that SbG stimulates hepatocyte growth via activation of the NF-κB pathway and induces TGF-β1 gene expression through the Kupffer cell–hepatocyte interaction, which subsequently results in the inhibition of SbG-stimulated hepatocyte growth.
Effect of Nitric Oxide Synthase Inhibition on Proteinuria in Glomerular Immune Injury
Authors: Datta Prasun K. , Sharma Mukut , Duann Pu , Lianos Elias A.
Published: July 2017

In glomerular immune injury, the inducible isoform of nitric oxide synthase (iNOS) becomes a major catalyst of NO production. Although iNOS-catalyzed NO production is sustained and can be cytotoxic, iNOS inhibition exacerbates the magnitude of proteinuria that accompanies immune injury. To investigate putative mechanisms of this effect, we assessed changes in glomerular permeability to albumin by using the following two approaches: (i) an in vivo rat model of glomerular immune injury induced by antibody against the glomerular basement membrane (GBM), in which urine albumin excretion was measured under conditions of iNOS Inhibition, and (ii) an ex vivo model of isolated rat glomeruli, in which changes in glomerular capillary permeability to albumin were assessed under conditions of NOS inhibition.

In rats with anti-GBM antibody–induced glomerular injury, there was an increase in urine albumin excretion. Treatment with two structurally dissimilar iNOS inhibitors at doses sufficient to decrease urine nitrate and/or nitrite exacerbated proteinuria. In these animals, urine excretion of the isoprostane 8-iso-PGF2α (marker of oxidative stress) was increased. In isolated glomeruli incubated with the NOS inhibitor L-NMMA, the permeability to albumin increased. This effect was reversed by the NO donor DETA NONOate and by the superoxide dismutase mimetic Tempol.

We conclude that NOS-catalyzed NO production is an important mechanism in regulating glomerular permeability to protein. This mechanism involves control of the bioavailability of superoxide.
Expression of Cytosolic NADP+-Dependent Isocitrate Dehydrogenase in Bovine Mammary Epithelium: Modulation by Regulators of Differentiation and Metabolic Effectors
Authors: Liu Wenjing , Capuco Anthony V. , Romagnolo Donato F.
Published: July 2017

The cytosolic NADP+-dependent isocitrate dehydrogenase (IDH1) catalyzes the conversion of isocitrate to α-ketoglutarate in the cytosol, and generates NADPH as a primary source of reducing equivalents for de novo fatty acid synthesis in bovine mammary gland. The enzymatic activity of IDH1 increases dramatically in early lactation in bovine mammary tissue. We hypothesized that the expression of IDH1 in bovine is modulated by regulators of mammary epithelial differentiation. To test this hypothesis, we first examined the changes in IDH1 expression in late pregnancy (20 days) and at various stages (14, 90, 120, and 240 days) of lactation in bovine mammary tissue. IDH1 mRNA levels increased by 2.3-fold after parturition compared to late pregnancy and remained elevated thereafter. Next, we examined the effects of extracellular matrix and lactogenic hormones on the expression of IDH1 in cultured BME-UV bovine mammary epithelial cells. We found that expression of IDH1 mRNA increased in parallel with β-casein expression induced by extracellular matrix. Fetal calf serum and insulin repressed, whereas prolactin stimulated the expression of IDH1 mRNA in a dose-dependent fashion. The inhibitory effects of insulin on IDH1 mRNA levels were antagonized by cotreatment with prolactin. In contrast, treatment with prolactin in the presence of extracellular matrix further increased IDH1 mRNA and protein accumulation. Prolactin-induced IDH1 expression was inhibited by the mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126, and Janus tyrosine kinase 2 (Jak2) inhibitor AG490, suggesting that both MAPK and Jak2 contribute to regulation of IDH1 expression by prolactin. Finally, we report that treatment of BME-UV cells with α-ketoglutarate and palmitic acid reduced IDH1 transcript levels. Taken together, our data suggest that the expression of IDH1 in bovine mammary epithelium is modulated by regulators of differentiation including extracellular matrix and lactogenic hormones as well as metabolic effectors.
Retinoic Acid Exerts Dual Regulatory Actions on the Expression and Nuclear Localization of Interferon Regulatory Factor-1
Authors: Luo Xin M. , Ross A. Catharine
Published: July 2017

Interferon regulatory factor-1 (IRF-1), a transcription factor and tumor suppressor involved in cell growth regulation and immune responses, has been shown to be induced by all-trans retinoic acid (ATRA). However, the factors controlling the cellular location and activity of IRF-1 are not well understood. In this study, we examined the expression of IRF-1 and its nuclear localization, DNA-binding activity, and target gene expression in human mammary epithelial MCF10A cells, a model of breast epithelial cell differentiation and carcinogenesis. Following initial treatment with ATRA, IRF-1 mRNA and protein were induced within 2 hrs, reached a peak (>30-fold induction) at 8 hrs, and declined afterwards. IRF-1 protein was predominantly cytoplasmic during this treatment. Although a second dose of ATRA or Am580 (a related retinoid selective for retinoic acid receptor-α [RARα]), given 16 hrs after the first dose, restimulated IRF-1 mRNA and protein levels to a similar level to that obtained by the first dose, IRF-1 was predominantly concentrated in the nucleus after restimulation. ATRA and Am580 also increased nuclear RARα, whereas retinoid X receptor-α (RXRa)—a dimerization partner for RARα, was localized to the nucleus upon second exposure to ATRA. However, ATRA and Am580 did not regulate the expression or activation of signal transducer and activator of transcription-1 (STAT-1), a transcription factor capable of inducing the expression of IRF-1, indicating an STAT-1–independent mechanism of regulation by ATRA and Am580. The increase in nuclear IRF-1 after retinoid restimulation was accompanied by enhanced binding to an IRF-E DNA response element, and elevated expression of an IRF-1 target gene, 2′,5′-oligoadenylate synthetase-2. The dual effect of retinoids in increasing IRF-1 mRNA and protein and in augmenting the nuclear localization of IRF-1 protein may be essential for maximizing the tumor suppressor activity and the immunosurveillance functions of IRF-1 in breast epithelial cells.
Cyanide Detoxification by the Cobalamin Precursor Cobinamide
Authors: Broderick Kate E. , Potluri Prasanth , Zhuang Shunhui , Scheffler Immo E. , Sharma Vijay S. , Pilz Renate B. , Boss Gerry R.
Published: July 2017

Cyanide is a highly toxic agent that inhibits mitochondrial cytochrome-c oxidase, thereby depleting cellular ATP. it contributes to smoke inhalation deaths in fires and could be used as a weapon of mass destruction. Cobalamin (vitamin B12) binds cyanide with a relatively high affinity and is used in Europe to treat smoke inhalation victims. Cobinamide, the penultimate compound in cobalamin biosynthesis, binds cyanide with about 1010 greater affinity than cobalamin, and we found It was several-fold more effective than cobalamin in (i) reversing cyanide inhibition of oxidative phosphorylation in mammalian cells; (ii) rescuing mammalian cells and Drosophila melanogaster from cyanide toxicity; and (iii) reducing cyanide inhibition of Drosophila Malpighian tubule secretion. Cobinamide could be delivered by oral ingestion, inhalation, or injection to Drosophila, and it was as effective when administered up to 5 mins post-cyanide exposure as when given preexposure. We conclude that cobinamide is an effective cyanide detoxifying agent that has potential use as a cyanide antidote, both in smoke inhalation victims and in persons exposed to cyanide used as a weapon of mass destruction.
A Tale of Two Speeds: Challenges for Research Universities
Authors: Feng Da Hsuan
Published: July 2017

This manuscript represents a presentation to the Annual Conference of the Association of Anatomy, Cell Biology and Neurobiology Chairpersons. The author is responding to the question, “How can university administrations nurture both interdisciplinary and inter-institutional collaboration and cooperation?” The presentation makes the point that university administrators must be cognizant of the changing dynamics of interdisciplinary and inter-institutional collaboration and cooperation due to the speed of the Internet and the speed of travel today. Those institutional leaders, and their universities, that recognize that the world has become flat will flourish, while those locked in a silo mentality will perish.
Complainant Issues in Research Misconduct: The Office of Research Integrity Experience
Authors: Pascal Chris B.
Published: July 2017

This paper discusses the experiences of the Office of Research Integrity (ORI) with issues involving complainants who make allegations of research misconduct. The paper describes the legal framework for complainant issues, the various roles of the complainant as the allegation of misconduct proceeds through the steps of investigation and resolution, how allegations of retaliation against the complainant are handled, the responsibilities of ORI and of the research institution where the alleged misconduct occurred, and ORI's experience with several cases of alleged retaliation. In each of these areas, the paper attempts to provide guidance to prospective complainants, research institutions, and other interested persons on effective ways to approach the various problems and concerns that arise, while maintaining a balance between the needs of the complainant, the accused, the research institution handling the allegation, and ORI.
Expression and Properties of Recombinant Ovine Uterine Serpin
Authors: Tekin Şaban , Padua Maria B. , Brad Amber M. , Rhodes Michelle L. , Hansen Peter J.
Published: July 2017

Ovine uterine serpin (OvUS) is produced in the uterus of sheep under the influence of progesterone. It weakly inhibits pepsin and reduces proliferation of lymphocytes, tumor cell lines, and preimplantation embryos. When purified from uterine fluid, the concentration required for its antiproliferative effect in vitro is ~0.25–1 mg/ml. Here we show that recombinant (r) OvUS is a more potent regulator of cell proliferation than native (n) OvUS purified from uterine fluid. To produce rOvUS, RNA was extracted from endometrium from a pregnant ewe and cDNA was amplified by reverse transcription-polymerase chain reaction using gene-specific primers. The purified OvUS cDNA was inserted into the ampicillin-resistant plasmid vector pcDNA3.1/V5-His-TOPO. The plasmid was introduced into the TOP10 Escherichia coli strain, purified, and used for transfection of Freestyle 293-F cells. Digestion of rOvUS with protein N-glycosidase F confirmed that rOvUS was N-glycosylated. Both rOvUS and nOvUS inhibited proliferation of phytohemagglutin-activated sheep lymphocytes and the P388D1 mouse lymphoma and PC-3 prostate cell lines. Inhibition was greater for rOvUS than for nOvUS, and concentrations as low as 15 μg/ml rOvUS were effective at reducing lymphocyte proliferation. Addition of rOvUS to fertilized bovine embryos reduced the cleavage rate and the percentage of embryos that became blastocysts. Native OvUS did not affect cleavage rate and had a smaller effect on development to the blastocyst stage. Experiments demonstrate that OvUS is a more potent inhibitor of cell proliferation and embryonic development than previously believed and add credence to the putative role for the protein in regulating cell proliferation.
Influence of Osmolytes on In Vivo Glucose Monitoring Using Optical Coherence Tomography
Authors: Sapozhnikova Veronika V. , Prough Donald , Kuranov Roman V. , Cicenaite Inga , Esenaliev Rinat O.
Published: July 2017

Diabetes mellitus and its complications are the third leading cause of death in the world, exceeded only by cardiovascular disease and cancer. Tighter monitoring and control of blood glucose could minimize complications associated with diabetes. Recently, optical coherence tomography (OCT) for noninvasive glucose monitoring was proposed and tested in vivo. The aim of this work was to investigate the influence of changes in blood glucose concentration ([glu]) and sodium concentration ([Na+]) on the OCT signal. We also investigated the influence of other important analytes on the sensitivity of glucose monitoring with OCT. The experiments were carried out in anesthetized female pigs. The OCT images were acquired continuously from skin, while [glu] and [Na+] were experimentally varied within their physiological ranges. Correlations of the OCT signal slope with [glu] and [Na+] were studied at different tissue depths. The tissue area probed with OCT was marked and cut for histological examination. The correlation of blood [glu] and [Na+] with the OCT signal slope was observed in separate tissue layers. On average, equimolar changes in [glu] produced 2.26 ± 1.15 greater alterations of the OCT signal slope than changes in [Na+]. Variation of concentrations of other analytes did not Influence the OCT signal slope. The influence of [Na+] on relative changes in the OCT signal slope was generally less than [glu]-induced changes. OCT is a promising method for noninvasive glucose monitoring because of its ability to track the influence of changing [glu] on individual tissue layers.
Physiologic and Genomic Analyses of Nutrition-Ethanol Interactions During Gestation: Implications for Fetal Ethanol Toxicity
Authors: Shankar Kartik , Hidestrand Mats , Liu Xiaoli , Xiao Ruin , Skinner Charles M. , Simmen Frank A. , Badger Thomas M. , Ronis Martin J. J.
Published: July 2017

Nutrition-ethanol (EtOH) interactions during gestation remain unclear primarily due to the lack of appropriate rodent models. In the present report we utilize total enteral nutrition (TEN) to specifically understand the roles of nutrition and caloric intake in EtOH-induced fetal toxicity. Time-impregnated rats were intragastrically fed either control or diets containing EtOH (8–14 g/kg/day) at a recommended caloric intake for pregnant rats or rats 30% undernourished, from gestation day (GD) 6–20. Decreased fetal weight and litter size (P < 0.05) and increased full litter resorptions (33% vs. 0%), were observed in undernourished dams compared to adequately fed rats given the same dose of EtOH, while undernutrition alone did not produce any fetal toxicity. Undernutrition led to impairment of EtOH metabolism, increased blood EtOH concentrations (160%), and decreased maternal hepatic ADH1 mRNA, protein, and activity. Microarray analyses of maternal hepatic gene expression on GD15 revealed that 369 genes were altered by EtOH in the presence of undernutrition, as compared to only 37 genes by EtOH per se (±2-fold, P < 0.05). Hierarchical clustering and gene ontology analysis revealed that stress and external stimulus responses, transcriptional regulation, cellular homeostasis, and protein metabolism were affected uniquely in the EtOH-under-nutrition group, but not by EtOH alone. Microarray data were confirmed using real-time RT-PCR. Undernourished EtOH-fed animals had 2-fold lower IGF-1 mRNA and 10-fold lower serum IGF-1 protein levels compared to undernourished controls (P < 0.0005). Examination of maternal GH signaling via STAT5a and -5b revealed significant reduction in both gene and protein expression produced by both EtOH and undernutrition. However, despite significantly elevated fetal BECs, fetal IGF-1 mRNA and protein were not affected by EtOH or EtOH-undernutrition combinations. Our data suggest that undernutrition potentiates the fetal toxicity of EtOH in part by disrupting maternal GH-IGF-1, signaling thereby decreasing maternal uterine capacity and placental growth.
In Situ Cooling in a Lung Hilar Clamping Model of Ischemia-Reperfusion Injury
Authors: Koletsis Efstratios , Chatzimichalis Antonios , Apostolakis Efstratios , Kokkinis Kiriakos , Fotopoulos Vasilios , Melachrinou Maria , Chorti Maria , Crockett James , Marinos Evangelos , Bellenis Ion , Dougenis Dimitrios
Published: July 2017

Experimental models for studying transplantation have up to now been unable to isolate reperfusion injury with minimal surgical manipulation and without the interference of graft rejection. Six pigs were subjected to left hilum preparation only (control group), and eight pigs were subjected to left hilum preparation plus in situ cooling ischemia and reperfusion of the lung (experimental group). The hilum was dissected free from other tissues in both groups. Lung preservation was achieved by antegrade flush perfusion via the left pulmonary artery. Pulmonary veins were clamped at the left atrium and a vent was created. The left main bronchus was clamped. Lung temperature was maintained at 4°–8°C, while core temperature was kept at 38°C. After 3 hrs of cold ischemia the clamps were removed and the lung was reperfused. Elevated pulmonary vascular resistance and local and systemic aspects of ischemia-reperfusion syndrome were consistently reproduced. This large-animal model of in situ unilateral lung cold ischemia with warm reperfusion proved to be very reliable in reproducing all aspects of ischemia-reperfusion injury. It excludes the interference of rejection and extensive surgical manipulation. We therefore propose its use in experimental studies investigating pharmaceutical or cooling modifications affecting lung ischemia-reperfusion outcomes.