bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Dimension for the Development of Fluorescence-Based Assays in Solution: From Physical Principles of FCS Detection to Biological Applications
Authors: Földes-Papp Zeno , Demel Ulrike , Domej Wolfgang , Tilz Gernot P.
Published: July 2017

Ultrasensitive detection methods such as laser-induced fluorescence represent the current state-of-the-art in analytics. Single-molecule detection in solution has received a remarkable amount of attention in the last few years because of its applicability to life sciences. Studies have been performed on the fundamentals of the detection processes themselves and on some biological systems. Fluorescence correlation spectroscopy (FCS) is the link for ultrasensitive multicomponent analysis, showing possibilities for experiments on molecular interactions. Based on the theoretical background of FCS, this article gives full explanation of FCS and an update of highlights in experimental biology and medicine studied by FCS. We focus on a repertoire of diverse immunoglobulin specificities, a ribosome display system, single-molecule DNA sequencing, and a mutant enzyme generated by random mutagenesis of amino acids. We describe the usefulness and the enormous potential of the methodology. Further, this contribution clearly Indicates that FCS is a valuable tool for solution-phase single-molecule (SPSM) experiments In immunobiology and medicine. In experiments with the Goodpasture autoantibody, we worked out conditions for the design of experiments on a complex single molecule in solution. The possibility to use SPSM-FCS as a quantitation methodology opens up other important applications beyond the scope of this article. Original results extending the published studies are presented for the rational foundation of SPSM-FCS. In this original contribution, we deal with experimental systems for biology and medicine where the number of molecules In solution is very small. This article Is mandatory for gaining confidence In the interpretation of experimental SPSM-FCS results on the selfsame, Individual single molecule In solution.
Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers
Authors: Szabó Imre L. , Pai Rama , Jones Michael K. , Ehring George R. , Kawanaka Hirofumi , Tarnawski Andrzej S.
Published: July 2017

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.
Pathophysiological Effects of Nicotine on the Pancreas: An Update
Authors: Chowdhury Parimal , MacLeod Stewart , Udupa Kodetthor B. , Rayford Phillip L.
Published: July 2017

Epidemiological evidence strongly suggests an association between cigarette smoking and pancreatic diseases. It is well recognized that nicotine, a major component in cigarette smoke, is an addictive agent and, therefore, reinforces smoking behavior. The current review update focuses on the genetics of nicotine dependence and its role on the development of pancreatic diseases. The role of smoking and nicotine in pancreatitis and pancreatic cancer development is also discussed. Exposure of laboratory animals to nicotine clearly supports the notion that nicotine can induce pancreatic injury. The mechanism by which nicotine induces such effects is perhaps mediated via signal transduction pathways in the pancreatic acinar cell, leading to enhanced levels of intracellular calcium release, resulting in cytotoxicity and eventual cell death. The induction of pancreatic injury by nicotine may also involve activation and expression of protooncogene, H-ras, which can increase cytosolic calcium via second messenger pathways. Development of pancreatic carcinoma in cigarette smokers as observed in human populations may be the result of activation and mutation of the H-ras gene. A possible pathogenetic mechanism of nicotine in the pancreas activating multiple signal transduction pathways is schematically summarized in Figure 1.
Characterization of Acetylcholinesterase in Caco-2 Cells
Authors: Plageman Lauren R. , Pauletti Giovanni M. , Skau Kenneth A.
Published: July 2017

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-β-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.
Regulatory Effects of Eotaxin, Eotaxin-2, and Eotaxin-3 on Eosinophil Degranulation and Superoxide Anion Generation1
Authors: Badewa A.P. , Hudson C.E. , Heiman A.S.
Published: July 2017

Eosinophilic leukocytes have been implicated as primary effector cells in inflammatory and allergic diseases. When activated by cytokines, human eosinophils secrete and produce a variety of proinflammatory or tissue damaging substances. Although well known for their chemoattractant effects, little is known about the precise contribution of the eosinophil-selective chemokines, eotaxin, eotaxin-2, and eotaxin-3 to the effector functions of eosinophils. This forms the central focus of these investigations for which clone 15-HL-60 human eosinophilic cells were used as the in vitro model. Investigation results suggest that all three subtypes of eotaxin directly stimulate eosinophil superoxide anion generation that is inhibited by neutralizing eotaxin antibody or pretreatment of cells with the receptor antibody anti-CCR3. Pretreatment or co-treatment with each of the eotaxins augmented phorbol myristate-induced superoxide generation. Concentration-dependent degranulation of eosinophil peroxidase was noted for all three chemokines, and potentiation of calcium lonophore-induced degranulation was observed with eotaxin pretreatments. Results of interleukin-5 pretreatment studies suggest that the eotaxin chemokines may act cooperatively to enhance effector functions of eosinophils. Collectively, the present studies have advanced knowledge of the eotaxin family of chemokines to include eosinophil priming and modulation of eosinophil activation and secretion effector functions.
Chromatin Structure and Control of β-Like Globin Gene Switching
Authors: Harju Susanna , McQueen Kellie J. , Peterson Kenneth R.
Published: July 2017

The human β-globin locus is a complex genetic system widely used for analysis of eukaryotic gene expression. The locus consists of five functional β-like globin genes, ε,Gγ,Aγ, δ, and β, arrayed on the chromosome in the order that they are expressed during ontogeny. Globin gene expression is regulated, in part, by the locus control region, which physically consists of five DNasel-hypersensitive sites located 6-22 Kb upstream of the ε-globin gene. During ontogeny two switches occur in β-globin gene expression that reflect the changing oxygen requirements of the fetus. The first switch from embryonic ε- to fetal γ-globin occurs at six weeks of gestation. The second switch from γ- to adult δ- and β-globin occurs shortly after birth. Throughout the locus, cis-acting elements exist that are dynamically bound by trans-acting proteins, including transcription factors, co-activators, repressors, and chromatin modifiers. Discovery of novel erythroid-specific transcription factors and a role for chromatin structure in gene expression have enhanced our understanding of the mechanism of globin gene switching. However, the hierarchy of events regulating gene expression during development, from extracellular signaling to transcriptional activation or repression, is complex. In this review we attempt to unify the current knowledge regarding the interplay of cis-acting elements, transcription factors, and chromatin modifiers into a comprehensive overview of globin gene switching.
Developing a Laboratory Animal Model for Perinatal Endocrine Disruption: The Hamster Chronicles
Authors: Hendry William J. , Sheehan Daniel M. , Khan Shafiq A. , May Jeffrey V.
Published: July 2017

At the biomedical, regulatory, and public level, considerable concern surrounds the concept that inappropriate exposure to endocrine-disrupting chemicals, especially during the prenatal and/or neonatal period, may disrupt normal reproductive tract development and adult function. The intent of this review was to 1. Describe some unique advantages of the hamster for perinatal endocrine disruptor (ED) studies, 2. Summarize the morphological and molecular consequences of exposure to the established perinatal ED, diethylstilbestrol, in the female and male hamster, 3. Present some new, histomorphological insight into the process of neonatal diethylstilbestrol-induced disruption in the hamster uterus, and 4. Introduce recent efforts and future plans to evaluate the potency and mechanism of action of other putative EDs in the hamster experimental system. Taken together, the findings indicate that the hamster represents a unique and sensitive in vivo system to probe the phenomenon of endocrine disruption. The spectrum of candidate endpoints includes developmental toxicity, neoplasia, and more subtle endpoints of reproductive dysfunction.
Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium
Authors: Welt Corrine , Sidis Yisrael , Keutmann Henry , Schneyer Alan
Published: July 2017

It has been 70 years since the name inhibin was used to describe a gonadal factor that negatively regulated pituitary hormone secretion. The majority of this period was required to achieve purification and definitive characterization of inhibin, an event closely followed by identification and characterization of activin and follistatin (FS). In contrast, the last 15–20 years saw a virtual explosion of information regarding the biochemistry, physiology, and biosynthesis of these proteins, as well as identification of activin receptors, and a unique mechanism for FS action—the nearly irreversible binding and neutralization of activin. Many of these discoveries have been previously summarized; therefore, this review will cover the period from the mid 1990s to present, with particular emphasis on emerging themes and recent advances. As the field has matured, recent efforts have focused more on human studies, so the endocrinology of inhibin, activin, and FS in the human is summarized first. Another area receiving significant recent attention is local actions of activin and its regulation by both FS and inhibin. Because activin and FS are produced in many tissues, we chose to focus on a few particular examples with the most extensive experimental support, the pituitary and the developing follicle, although nonreproductive actions of activin and FS are also discussed. At the cellular level, it now seems that activin acts largely as an autocrine and/or paracrine growth factor, similar to other members of the transforming growh factor β superfamily. As we discuss in the next section, its actions are regulated extracellularly by both inhibin and FS. In the final section, intracellular mediators and modulators of activin signaling are reviewed in detail. Many of these are shared with other transforming growh factor β superfamily members as well as unrelated molecules, and in a number of cases, their physiological relevance to activin signal propagation remains to be elucidated. Nevertheless, taken together, recent findings suggest that it may be more appropriate to consider a new paradigm for inhibin, activin, and FS in which activin signaling is regulated extracellularly by both inhibin and FS whereas a number of intracellular proteins act to modulate cellular responses to these activin signals. It is therefore the balance between activin and all of its modulators, rather than the actions of any one component, that determines the final biological outcome. As technology and model systems become more sophisticated in the next few years, it should become possible to test this concept directly to more clearly define the role of activin, inhibin, and FS in reproductive physiology.
Nitric Oxide-Cyclic GMP Signaling Pathway in the Regulation of Rabbit Clitoral Cavernosum Tone1
Authors: Park Jong Kwan , Kim Jung Ui , Lee Soon Oak , Hwang Pyoung Han , Yi Ho Keun , Kim Young Gon , Cho Kyung Woo
Published: July 2017

We investigated the role of nitric oxide (NO)-guanosine 3′,5′-cyclic monophosphate (cGMP) signaling in the regulation of rabbit clitoral cavernosum (CC) tone. Tension measurements, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and NADPH-diaphorase staining were performed in CC. In the precontracted CC strips with phenylephrine (10–5 M), acetylcholine (ACh) relaxed, dependent on dosage. Pretreatment with atropine, Nω nitro-l-arginine-methyl ester (NAME) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), guanylate cyclase inhibitor abolished the ACh-induced relaxations, but tetrodotoxin (TTX) did not. Sodium nitroprusside relaxed the strips in the presence of atropine and NAME, but not in the presence of ODQ. Electrical field stimulation (EFS) relaxed the strips dependent on stimulus strength. Pretreatment with TTX, NAME, or ODQ abolished the EFS-induced relaxation, but atropine did not. l-Arginine partially restored the inhibited response to ACh and EFS. The inducible NO synthase (iNOS) and neuronal NOS (nNOS) mRNAs and iNOS and endothelial NOS (eNOS) proteins were identified in the CC. NADPH-diaphorase staining revealed the positivity on the nerve trunks and fine nerve fibers in the CC. Finally, results demonstrate that the nNOS, ENOS, and the NO-cGMP signaling pathway are involved in the regulation of clitoral tumescence.
Protein Kinase C-Mediated Desmin Phosphorylation is Related to Myofibril Disarray in Cardiomyopathic Hamster Heart1
Authors: Huang Xupei , Li Jian , Foster Dalton , Lemanski Sharon L. , Dube Dipak K. , Zhang Chi , Lemanski Larry F.
Published: July 2017

The cardiomyopathic (CM) Syrian golden hamster (strain UM-X7.1) exhibits a hereditary cardiomyopathy, which causes premature death resulting from congestive heart failure. The CM animals show extensive cardiac myofibril disarray and myocardial calcium overload. The present study has been undertaken to examine the role of desmin phosphorylation in myofibril disarray observed in CM hearts. The data from skinned myofibril protein phosphorylation assays have shown that desmin can be phosphorylated by protein kinase C (PKC). There is no significant difference in the content of desmin between CM and control hamster hearts. However, the desmin from CM hearts has a higher phosphorylation level than that of the normal hearts. Furthermore, we have examined the distribution of desmin and myofibril organization with immunofluorescent microscopy and immunogold electron microscopy in cultured cardiac myocytes after treatment with the PKC-activating phorbol ester, 12-O-tetradecanylphorbol-13-acetate (TPA). When the cultured normal hamster cardiac cells are treated with TPA, desmin filaments are disassembled and the myofibrils become disarrayed. The myofibril disarray closely mimics that observed in untreated CM cultures. These results suggest that disassembly of desmin filaments, which could be caused by PKC-mediated phosphorylation, may be a factor in myofibril disarray in cardiomyopathic cells and that the intermediate filament protein, desmin, plays an Important role in maintaining myofibril alignment in cardiac cells.
Relaxin Stimulates Bronchial Epithelial Cell PKA Activation, Migration, and Ciliary Beating1
Authors: Wyatt T.A. , Sisson J.H. , Forgèt M.A. , Bennett R.G. , Hamel F.G. , Spurzem J.R.
Published: July 2017

Relaxin is an insulin-like serum protein secreted during pregnancy and found in many tissues, including the lung. Relaxin is reported to stimulate epithelial cell proliferation, but the effects of relaxin on airway epithelium are unknown. We tested the hypothesis that relaxin would stimulate the increased migration of bronchial epithelial cells (BEC) in response to wounding. Using monolayers of BEC in a wound-healing model, relaxin augmented wound closure with maximal closure occurring at 12.hr (1 μM). Unlike cytokines, relaxin did not stimulate increased BEC interleukin-8 (IL-8) release. Relaxin caused a significant stimulation of ciliary beat frequency (CBF) in BEC. Because protein kinase (PKA) activation increases CBF and relaxin can elevate intracellular cAMP levels, we measured PKA activity in BEC treated with relaxin. Relaxin increased PKA activity 3–4 fold by approximately 4 hr, with a return to baseline levels by 8–10 hr. Relaxin-stimulated PKA activity differs temporally from the rapid (1 hr) β-adrenergic activation of PKA in BEC. These data suggest that relaxin augments epithelial repair by increasing airway cell migration and CBF via PKA-dependent mechanisms.
Erythropoietin: Physiology and Pharmacology Update
Authors: Fisher James W.
Published: July 2017

This minireview is an update of a 1997 review on erythropoletin (EPO) in this journal (1). EPO is a 30,400-dalton glycoprotein that regulates red cell production. In the human, EPO is produced by peritubular cells in the kidneys of the adult and in hepatocytes in the fetus. Small amounts of extra-renal EPO are produced by the liver in adult human subjects. EPO binds to an erythroid progenitor cell surface receptor that includes a p66 chain, and, when activated, the p66 protein becomes dimerized. EPO receptor activation induces a JAK2 tyrosine kinase, which leads to tyrosine phosphorylation of the EPO receptor and several proteins. EPO receptor binding leads to intracellular activation of the Ras/mitogen-activated kinase pathway, which is involved with cell proliferation, phosphatidylinositol 3-kinase, and STATS 1, 3, 5A, and 5B transcriptional factors. EPO acts primarily to rescue erythroid cells from apoptosis (programmed cell death) to increase their survival. EPO acts synergistically with several growth factors (SCF, GM-CSF, 1L-3, and IGF-1) to cause maturation and proliferation of erythroid progenitor cells (primarily colony-forming unit-E). Oxygen-dependent regulation of EPO gene expression is postulated to be controlled by a hypoxia-inducible transcription factor (HIF-1α). Hypoxia-inducible EPO production is controlled by a 50-bp hypoxia-inducible enhancer that is approximately 120 bp 3' to the polyadenylation site. Hypoxia signal transduction pathways involve kinases A and C, phospholipase A2, and transcription factors ATF-1 and CREB-1. A model has been proposed for adenosine activation of EPO production that involves protein kinases A and C and the phospholipase A2pathway. Other effects of EPO include a hematocrit-independent, vasoconstriction-dependent hypertension, increased endothelin production, upregulation of tissue renin, change in vascular tissue prostaglandins production, stimulation of angiogenesis, and stimulation of endothelial and vascular smooth muscle cell proliferation. Recombinant human EPO (rHuEPO) is currently being used to treat patients with anemias associated with chronic renal failure, AIDS patients with anemia due to treatment with zidovudine, nonmyeloid malignancies in patients treated with chemotherapeutic agents, perioperative surgical patients, and autologous blood donation. A novel erythropolesis-stimulating factor (NESP, darbepoetin) has been synthesized and when compared with rHuEPO, NESP has a higher carbohydrate content (52% vs 40%), a longer plasma half-life, the amino acid sequence differs from that of native human EPO at five positions, and has been reported to maintain hemoglobin levels just as effectively in patients with chronic renal failure as rHuEPO at less frequent dosing. The use of rHuEPO and darbepoetin to enhance athletic performance is officially banned by most sports-governing bodies because the excessive erythrocytosis can lead to increased thrombogenicity and can cause deep vein, coronary, and cerebral thromboses.
Prolonged Prenatal Hypernatremia Alters Neuroendocrine and Electrolyte Homeostasis in Neonatal Sheep1
Authors: Wang Shengbiao , Chen Jiexiong , Kallichanda Nathash , Azim Arm , Calvario Glenda , Ross Michael G.
Published: July 2017

Arginine vasopressin (AVP) is a neuroendocrine hormone synthesized in the hypothalamus, and is stored and secreted by the posterior pituitary gland in response to stimuli such as plasma hypertonicity and hypotension. The primary physiologic roles of AVP include plasma osmolality and blood pressure regulation. We have previously demonstrated that chronic prenatal plasma hypertonicity alters the AVP regulatory pathway in newborn lambs. The objectives of the present study were to evaluate prolonged effects of antenatal plasma hypertonicity on neonatal plasma osmoregulation. Pregnant ewes at 119 ± 3 days of gestation were water restricted to achieve and maintain hypertonicity until normal-term delivery. After delivery, ewes were provided food and water ad libitum and lambs were allowed maternal nursing. At the age of 28 days, blood samples were obtained for the analysis of plasma osmolality, electrolytes, and AVP levels from study (n = 5) and age-matched control (n = 6) lambs. Subsequently, lambs were euthanized, and the pituitary and hypothalamus were processed for the determination of pituitary AVP content by radioimmunoassay, and AVP gene expression by Northern analysis. In response to water restriction, maternal plasma osmolality significantly increased (306 ± 1.1 to 326 ±1.2 mOsm/kg, P < 0.001). At the age of 28 days, plasma sodium level was higher in study (prenatally dehydrated) than control lambs (144.6 ± 0.4 vs 142.6 ± 0.3, P < 0.05). Study lambs had higher plasma AVP concentrations than the control lambs (4.1 ± 0.4 vs 1.7 ± 0.4 pg/ml, P < 0.05). Similarly, total pituitary AVP content was higher in the in utero hypertonic lambs than in the control lambs (6.5 ± 1.0 vs 2.8 ± 1.2 μg, P < 0.05). However, there was no difference in hypothalamic AVP mRNA levels between the two groups. The present study demonstrates that chronic maternal and fetal plasma hypertonicity has prolonged effects on pituitary and plasma AVP, as well as plasma sodium in neonatal lambs, providing further evidence suggesting prenatal imprinting of osmoregulation through at least 1 month of age.
Cocoa Flavonols and Procyanidins Promote Transforming Growth Factor-β1Homeostasis in Peripheral Blood Mononuclear Cells1
Authors: Mao T.K. , Water J. Van De , Keen C.L. , Schmitz H.H. , Gershwin M.E.
Published: July 2017

Evidence suggests that certain flavan-3-ols and procyanidins (FP) can have a positive influence on cardiovascular health. It has been previously reported that FP isolated from cocoa can potentially modulate the level and production of several signaling molecules associated with immune function and inflammation, including several cytokines and eicosanoids. In the present study, we examined whether FP fractions monomers through decamers modulate secretion of the cytokine transforming growth factor (TGF)-β1from resting human peripheral blood mononuclear cells (PBMC). A total of 13 healthy subjects were studied and grouped according to their baseline production of TGF-β1. When cells from individuals with low baseline levels of TGF-β1(n = 7) were stimulated by individual FP fractions (25 μg/ml), TGF-β1release was enhanced in the range of 15%–66% over baseline (P < 0.05; monomer, dimer, and tetramer). The low-molecular-weight FP fractions (≤pentamer) were more effective at augmenting TGF-β1secretion than their larger counterparts (≥hexamer), with the monomer and dimer inducing the greatest increases (66% and 68%, respectively). In contrast to the above, TGF-β1secretion from high TGF-β1baseline subjects (n = 6) was inhibited by individual FP fractions (P < 0.05; trimer through decamer). The inhibition was most pronounced with trimeric through decameric fractions (28%–42%), and monomers and dimers moderately inhibited TGF-β1release (17% and 23%, respectively). Given the vascular actions associated with TGF-β1, we suggest that in healthy individuals, homeostatic modulation of its production by FP offers an additional mechanism by which FP-rich foods can potentially benefit cardiovascular health.
From Mouse to Man: Redefining the Role of Insulin-Like Growth Factor-I in the Acquisition of Bone Mass
Authors: Yakar Shoshana , Rosen Clifford J.
Published: July 2017

The insulin-like growth factor system (IGF) has been linked to the process of bone acquisition through epidemiologic analyses of large cohorts and in vitro studies of bone cells. But the exact relationship between expression of IGF-I in bone and skeletal homeostasis or pathologic conditions, such as osteoporosis, remains poorly defined. Recent advances in genomic engineering have resulted in the development of better in vivo models to test the role of IGF-I during development and maintenance of the adult skeleton. It is now established that skeletal expression of IGF-I is critical for differentiative bone cell function. It may also be essential for the full anabolic effects of parathyroid hormone on trabecular bone and for some component of biomineralization. Evidence from conditional mutagenesis studies suggests that serum IGF-I may represent more than a storage depot or permissive factor during the final phase of skeletal acquisition. This work re-examines the original tenets of the “somatomedin hypothesis” in light of these newer mouse models and their remarkable skeletal phenotypes. The implications are far reaching and suggest that newer approaches for manipulating the IGF regulatory system may one day be useful as therapeutic adjuncts for the treatment of osteoporosis.
Discovery of Water-Soluble Anticancer Agents (Edotides) from a Vegetable Found in Benin City, Nigeria
Authors: Izevbigie Ernest B.
Published: July 2017

Cancer claims the lives of more than six million people each year in the world. About 1,268,000 new cancer cases, and 553,400 deaths were reported in the United States in 2001. Current treatment approaches have yielded significant progress in the fight against cancer, but the incidence of developing certain types of cancer continues to rise. This is especially true in the African-American communities. African Americans are about 33% more likely to die of cancer than are whites and more than twice likely to die of cancer as are Asian-Islander, American-Indians, and Hispanics. This increase coupled with the harsh side effects of some of the cancer chemotherapies have led to the search for more natural biological products, especially those derived from plant products, currently known as herbal medicine. There is a need for a continued search for novel natural products that may be used as cancer chemopreventive and/or chemotherapeutic agents. The objective of this study was to evaluate the effect(s) of a novel water-soluble leaf extract of Vernonia amygdalina (VA) on human breast cancer cell DNA synthesis. MCF-7 cell line, considered a suitable model, was used in this study. Treatment of cells with physiologically relevant concentrations of water-soluble VA extract potently inhibited DNA synthesis in a concentration-dependent fashion both in the absence and presence of serum. Fractions of VA extract separated using preparative reverse-phase chromatography also inhibited DNA synthesis (P < 0.005). These results suggest that VA vegetable, if incorporated in the diet, may prevent or delay the on-set of breast cancer.
Regulation of α-Globin mRNA Stability
Authors: Waggoner Shelly A. , Liebhaber Stephen A.
Published: July 2017

mRNA stability is a critical determinant of normal red blood cell development and function. The long half-life of globin mRNA is central to the continued synthesis of globin proteins throughout all stages of erythropoiesis, even as the cells undergo programmed transcriptional arrest during terminal differentiation. Studies of a naturally occurring α-thalassemic mutation that triggers marked destabilization of α-globin mRNA first led investigators to search for a stability determinant in the 3-untranslated region (3′UTR). Analysis of this region identified three cytosine-rich (C-rich) segments that contributed to α-globin mRNA stability when studied in transfected erythroid cells. Subsequently, in vitro studies demonstrated assembly of a sequence-specific ribonucleic-protein (RNP) complex at this site. Mutations in the 3′UTR that blocked formation of this “α-complex” in vitro resulted in a parallel destabilization of α-globin mRNA in transfected cells. Members of the α-globin poly(C)-binding protein (αCP) subfamily of heteronuclear (hn) RNP K homology (KH) domain RNA-binding proteins have been identified as essential protein components of the α-complex. In vitro studies suggested that additional proteins may also contribute to α-complex structure and/or function. Surveys of additional highly stable mRNAs point to a general function for the α-complex in mRNA stabilization. In vitro and in vivo analyses indicated that the α-complex stabilizes α-globin mRNA by two mechanisms: control of 3′-terminal deadenylation and steric protection of an endoribonuclease-sensitive site. Confirmation of these pathways, determination of their relative importance, and generalization of these findings to additional systems await future studies.
Dietary Magnesium Intake Influences Circulating Pro-Inflammatory Neuropeptide Levels and Loss of Myocardial Tolerance to Postischemic Stress
Authors: Kramer Jay H. , Mak I. Tong , Phillips Terry M. , Weglicki William B.
Published: July 2017

Severe dietary Mg restriction (Mg9, 9% of recommended daily allowance [RDA], plasma Mg = 0.25 m M) induces a proinflammatory neurogenic response in rats (substance P [SP]), and the associated increases in oxidative stress in vivo and cardiac susceptibility to ischemla/reperfusion (I/R) injury were previously shown to be attenuated by SP receptor blockade and antioxidant treatment. The present study assessed if less severe dietary Mg restriction modulates the extent of both the neurogenic/oxidative responses in vivo and I/R injury in vitro. Male Sprague-Dawley rats maintained on Mg40(40% RDA, plasma Mg = 0.6 m M) or Mg100(100% RDA, plasma Mg = 0.8 m M) diets were assessed for plasma SP levels (CHEM-ELISA) during the first 3 weeks and were compared with the Mg9group; red blood cell (RBC) glutathione and plasma malondialdehyde levels were compared at 3 weeks in Mg9, Mg20(plasma Mg = 0.4 m M), Mg40, and Mg100rats; and 40-min global ischemia/30-min reperfuslon hearts from 7-week-old Mg20, Mg40, and Mg100rats were compared with respect to functional recovery (cardiac work, and diastolic, systolic, and developed pressures), tissue LDH release, and free radical production (ESR spectroscopy and α-phenyl- N-tert butyinitrone [PBN; 3 m M] spin trapping). The Mg40diet induced smaller elevations in plasma SP (50% lower) compared with Mg9, but with a nearly identical time course. RBC glutathione and plasma malondialdehyde levels revealed a direct relationship between the severity of oxidative stress and hypomagnesemia. The dominant lipid free radical species detected in all I/R groups was the alkoxyl radical (PBN/alkoxyl: αH= 1.93 G, αN= 13.63 G); however, Mg40and Mg20hearts exhibited 2.7- and 3.9-fold higher alkoxyl levels, 40% and 65% greater LDH release, and lower functional recovery (Mg20< Mg40) compared with Mg100. Our data suggest that varying dietary Mg intake directly influences the magnitude of the neurogenic/oxidative responses in vivo and the resultant myocardial tolerance to I/R Stress.
Leucine-Enkephalin Interrupts Sympathetically Mediated Tachycardia Prejunctionally in the Canine Sinoatrial Node
Authors: Stanfill Amber A. , Jackson Keith , Farias Martin , Barlow Matthew , Deo Shekhar , Johnson Shavsha , Caffrey James L.
Published: July 2017

This study examined the role of leucine-enkephalin (LE) in the sympathetic regulation of the cardiac pacemaker. LE was administered by microdialysis into the interstitium of the canine sinoatrial node during either sympathetic nerve stimulation or norepinephrine infusion. In study one, the right cardiac sympathetic nerves were isolated as they exit the stellate ganglion and were stimulated to produce graded (low, 20–30 bpm; high 40–50 bpm) increases in heart rate (HR). LE (1.5 nmoles/min) was added to the dialysis inflow and the sympathetic stimulations were repeated after 5 and 20 min of LE infusion. After 5 min, LE reduced the tachycardia during sympathetic stimulation at both low (18.2 ± 1.3 bpm to 11.4 ± 1.4 bpm) and high (45 ± 1.5 bpm to 22.8 ± 1.5 bpm) frequency stimulations. The Inhibition was maintained during 20 min of continuous LE exposure with no evidence of opioid desensitization. The δ-opioid antagonist, naltrindole (1.1 nmoles/min), restored only 30% of the sympathetic tachycardia. Nodal δ-receptors are vagolytic and vagal stimulations were included in the protocol as positive controls. LE reduced vagal bradycardia by 50% and naltrindole completely restored the vagal bradycardia. In Study 2, additional opioid antagonists were used to determine if alternative opioid receptors might be implicated in the sympatholytic response. Increasing doses of the K-antagonist, norbinaltorphimine (norBNI), were combined with LE during sympathetic stimulation. NorBNI completely restored the sympathetic tachycardia with an ED50 of 0.01 nmoles/min. A single dose of the μ-antagonist, CTAP (1.0 nmoles/min), failed to alter the sympatholytic effect of LE. Study 3 was conducted to determine if the sympatholytic effect was prejunctional or postjunctional in character. Norepinephrine was added to the dialysis Inflow at a rate (30–45 pmoles/min) sufficient to produce intermediate increases (35.2 ± 1.8 bpm) in HR. LE was then combined with norepinephrine and responses were recorded at 5-min intervals for 20 min. The tachycardia mediated by added norepinephrine was unaltered by LE or LE plus naltrindole. At the same 5-min intervals, LE reduced vagal bradycardia by more than 50%. This vagolytic effect was again completely reversed by naltrindole. Collectively, these observations support the hypothesis that the local nodal sympatholytic effect of LE was mediated by κ-opioid receptors that reduced the effective interstitial concentration of norepinephrine and not the result of a postjunctional interaction between LE and norepinephrine.
Urinary chiro-Inositol and myo-inositol Excretion is Elevated in the Diabetic db/db Mouse and Streptozotocin Diabetic Rat
Authors: Kawa Julianne M. , Przybylski Roman , Taylor Carla G.
Published: July 2017

Inositol phosphoglycan molecules containing either d-chiro-inositol or myo-inositol have been isolated from various mammalian tissues and are putative mediators of insulin action. Urinary excretion of inositols appears to be altered in diabetes mellitus; however, the relationships with different types of diabetes are unclear. The objective of this study was to determine the urinary excretion of chiro and myo-inosltol in diabetic animal models, including streptozotocin (STZ) rats, db/db mice, and fa/fa Zucker rats. In STZ rats (type 1 diabetes), 12-hr urinary excretion of chiro-inositol was elevated 336-fold and myo-inositol excretion was elevated 47-fold compared with their nondiabetic counterparts. When corrected for creatinine, chiro-inositol excretion was 259-fold higher and myo-inosltol excretion was 36-fold higher in STZ rats than in normal rats. The same pattern was observed in db/db mice (type 2 diabetes), where 12-hr urinary chiro-inositol excretion was elevated 247-fold compared with normal mice. When corrected for creatinine, chiro-inositol excretion was 2455-fold higher and urinary myo-inositol excretion was elevated 8.5-fold in db/db mice compared with normal mice. The fa/fa Zucker rats (impaired glucose tolerance) had a pattern of urinary inositol excretion that was similar to the nondiabetic animals (lean Zucker rats, C57BL/6 mice, and Sprague-Dawley rats). In summary, urinary chiro-inositol and myo-inosltol excretion was elevated in animal models of type 1 and type 2 diabetes mellitus, concomitant with hyperglycemia and glucosuria.
Androgen Receptors Containing Expanded Polyglutamine Tracts Exhibit Progressive Toxicity when Stably Expressed in the Neuroblastoma Cell Line, SH-SY 5Y
Authors: Avila D.M. , Allman D.R. , Gallo J.-M. , McPhaul M.J.
Published: July 2017

The pathogenesis of X-linked spinal and bulbar muscular atrophy (SBMA) has been traced to an expansion of repeated glutamine (Gin) residues within the amino terminus of the human androgen receptor (AR). To examine the mechanisms by which these expanded repeat ARs (Exp-ARs) are toxic to neurons, we have established and characterized a cell culture model by stably transfecting SH-SY 5Y neuroblastoma cells with cDNAs containing either normal AR (81 series; 23 Gins) or Exp-AR (902 series; 56 Gins). At a low passage number, no differences in cell morphology, growth properties, or susceptibility to toxic insults were observed between clones expressing normal AR or Exp-AR. Initially, both types of cultures were found to express similar levels of specific hormone binding in monoiayer binding assays. Immunohistochemical studies demonstrated the vast majority of both the normal AR and Exp-AR were localized to the nucleus in the absence and presence of androgen. As the 902 series of clones were propagated, the Exp-AR content in the cells appeared to decline progressively. However, this decrease actually reflects a gradual disappearance of the Exp-AR cell population. No such selection occurred during the propagation of cells expressing the normal AR. This selection against cells expressing physiological levels of Exp-AR occurs in the absence of intracellular aggregates and suggests that mechanisms other than those Involving the formation of aggregates underlie the observed toxicity of Exp-ARs.
The Role of CSF-1 in Normal Physiology of Mammary Gland and Breast Cancer: An Update
Authors: Sapi Eva
Published: July 2017

Colony stimulating factor (CSF-1) and its receptor (CSF-1R, product of c-fms proto-oncogene) were initially implicated as essential for normal monocyte development as well as for trophoblastic implantation. However, studies have demonstrated that CSF-1 and CSF-1R have additional roles in mammary gland development during pregnancy and lactation. This apparent role for CSF-1/CSF-1R in normal mammary gland development is very intriguing because this receptor/ligand pair has also been found to be important in the biology of breast cancer in which abnormal expression of CSF-1 and its receptor correlates with tumor cell invasiveness and adverse clinical prognosis. Recent findings also implicate tumor-produced CSF-1 in promotion of bone metastasis in breast cancer, and a certain membrane-associated form of CSF-1 appears to induce immunity against tumors. This review aims to summarize recent findings on the role of CSF-1 and its receptor in normal and neoplastic mammary development that may elucidate potential relationships of growth factor–induced biological changes in the breast during pregnancy and tumor progression.
The Asymmetry of Existence: Do We Owe Our Existence to Cold Dark Matter and the Weak Force?
Authors: Borchers Andrea T. , Davis Paul A. , Gershwin M. Eric
Published: July 2017

A common theme throughout biology is homochirality, including its origin and especially implications. Homochirality has also intrigued scientists because of the hypothesis that life, as it currently exists, could not have occurred without it. In this review, we discuss several hypotheses regarding homochirality and their linkage to processes that range from subatomic in scale to processes that help define the structure of the universe. More importantly, this exploration begins with the knowledge that humans inhabit the universe in which there is an excess of normal matter over antimatter. It is a universe characterized by homochirality but is nonetheless contained in what is most easily described as a 3+1 dimensional spacetime wherein most laws of physics are invariant under spacetime transformations. This restriction on spacetime poses significant constraints on the processes that can be invoked to explain homochirality. However, in dealing with such restraints, including the total mass contained in the universe, the concepts of cold dark matter and dark energy can be incorporated into cosmological models with resultant behaviors and predictions very much in accord with the findings of the cosmic background surveys. Indeed, the introduction of cold dark matter and dark energy to solve problems relating to the mass found in the universe may provide a means for generating the needed asymmetry to allow homochirality to arise.
The Focal Segmental Glomerulosclerosis Permeability Factor: Biochemical Characteristics and Biological Effects
Authors: Sharma Mukut , Sharma Ram , McCarthy Ellen T. , Savin Virginia J.
Published: July 2017

Focal segmental glomerulosclerosis (FSGS) is characterized by steroid resistant nephrotic syndrome and progression to end-stage renal disease. Proteinuria in certain patients with FSGS may be caused by a circulating factor (FSGS permeability factor [FSPF]). The current report documents the biochemical characteristics and the biological and molecular effects of 70% ammonium sulfate supernatant of plasma from patients with recurrence of FSGS after transplantation (FSGS 70% supernatant). FS permeability activity, defined as the capacity of plasma from patients with FSGS to increase albumin permeability (Palb) of isolated glomeruli, was assessed in vitro. Permeability activity was not affected by lyophilization. FSPF bound strongly to matrices containing Mono-Q anion exchanger or protein A. It eluted from matrix-bound Cibacron blue F3GA over a wide range of salt concentrations, indicating a potential binding with other proteins, such as albumin. FSPF caused a maximal increase in Palb within 2 mins of incubation in vitro. Cellular proteins isolated from glomeruli with increased Palb showed decreased tyrosine phosphorylation of focal adhesion kinase, paxillin, and other proteins. Tyrosine phosphatase inhibition prevented the increase in Palb. Intravenous administration of as little as 3 mg protein in FSGS 70% supernatant increased Palb, while 9 mg or more were required to produce proteinuria. We conclude that FSPF is a low-molecular-weight protein, carries an anionic charge, and binds to protein A. Effects of FSPF on the glomerular permeability barrier are rapid and dose dependent and involve signaling through altered phosphorylation of cellular proteins. Identification of these biochemical and biological characteristics may be used to design strategies for removing FSPF from circulation and for purification and identification of this factor.
Growth Hormone Secretion: Molecular and Cellular Mechanisms and In Vivo Approaches
Authors: Anderson Lloyd L. , Jeftinija Srdija , Scanes Colin G.
Published: July 2017

Growth hormone (GH) release is under the direct control of hypothalamic releasing hormones, some being also produced peripherally. The role of these hypothalamic factors has been understood by in vitro studies together with such in vivo approaches as stalk sectioning. Secretion of GH is stimulated by GH-releasing hormone (GHRH) and ghrelin (acting via the GH secretagogue [GHS] receptor [GHSR]), and inhibited by somatostatin (SRIF). Other peptides/proteins influence GH secretion, at least in some species. The cellular mechanism by which the releasing hormones affect GH secretion from the somatotrope requires specific signal transduction systems (cAMP and/or calcium influx and/or mobilization of intracellular calcium) and/or tyrosine kinase(s) and/or nitric oxide (NO)/cGMP. At the subcellular level, GH release (at least in response to GHS) is accomplished by the following. The GH-containing secretory granules are moved close to the cell surface. There is then transient fusion of the secretory granules with the fusion pores in the multiple secretory pits in the somatotrope cell surface.
Hormone Regulation of Choline Uptake and Incorporation in Mouse Mammary Gland Explants
Authors: Rillema James A.
Published: July 2017

Choline is a nutrient in milk that is essential for the nourishment and growth of newborns. In rat milk, choline is present in concentrations that are more than an order of magnitude higher than in maternal serum. Using cultured mammary tissues taken from 12–14-day pregnant mice, the effects of the three primary lactogenic hormonesȓprolactin (PRL), insulin (I), and cortisol (H)—on choline uptake and incorporation into lipids were determined. By itself or in the presence of H and/or PRL, I was the only hormone that increased the accumulation of choline in aqueous tissue fractions. In contrast, PRL, when present with I plus H, was the only hormone that stimulated the incorporation of choline into the lipid fraction of tissues. Choline uptake was found to be sodium and time dependent; maximum distribution ratios >18 were achieved after a 6-hr uptake time. In kinetic studies the apparent Km for choline uptake was calculated to be ~2.7 mM, whereas the Vmax was 7.4 mM intracellular water per 30 mins. These results suggest the existence of a sodium-dependent active transporter for choline in the mouse mammary gland that is specifically stimulated by I. PRL, in contrast, only stimulates the incorporation of choline into lipids.
Screening for and Treatment of Polycystic Ovary Syndrome in Teenagers
Authors: Salmi Darren J. , Zisser Howard C. , Jovanovic Lois
Published: July 2017

Polycystic ovary syndrome (PCOS) usually arises during puberty and is marked by hyperinsulinemia and hyperandrogenism. Adolescents with PCOS are at an increased risk of developing health problems later on in life such as type 2 diabetes, cardiovascular disease, and infertility. Furthermore, the physical signs of PCOS can be detrimental to a teenage girl's self-image. Early diagnosis and treatment of PCOS in adolescents are essential in ensuring adulthood health and restoring self-esteem. Treatments for an adolescent with PCOS include diet and exercise, metformin, and oral contraceptive pills. Each of these options has been shown to be effective in improving certain aspects of PCOS, and probably the best treatment plan involves some combination of them.