3,816 research outputs found
Parameters of Coseismic Reverse- and Oblique-Slip Surface Ruptures of the 2008 Wenchuan Earthquake, Eastern Tibetan Plateau.
No full textThe Human Cytomegalovirus UL116 Glycoprotein Is a Chaperone to Control gH-Based Complexes Levels on Virions
Full text linkHuman cytomegalovirus (HCMV) relies in large part upon the viral membrane fusion glycoprotein B and two alternative gH/gL complexes, gH/gL/gO (Trimer) and gH/gL/UL128/UL130/UL131A (Pentamer) to enter into cells. The relative amounts of Trimer and Pentamer vary among HCMV strains and contribute to differences in cell tropism. Although the viral ER resident protein UL148 has been shown to interact with gH to facilitate gO incorporation, the mechanisms that favor the assembly and maturation of one complex over another remain poorly understood. HCMV virions also contain an alternative non-disulfide bound heterodimer comprised of gH and UL116 whose function remains unknown. Here, we show that disruption of HCMV gene UL116 causes infectivity defects of ∼10-fold relative to wild-type virus and leads to reduced expression of both gH/gL complexes in virions. Furthermore, gH that is not covalently bound to other viral glycoproteins, which are readily detected in wild-type HCMV virions, become undetectable in the absence of UL116 suggesting that the gH/UL116 complex is abundant in virions. We find evidence that UL116 and UL148 interact during infection indicating that the two proteins might cooperate to regulate the abundance of HCMV gH complexes. Altogether, these results are consistent with a role of UL116 as a chaperone for gH during the assembly and maturation of gH complexes in infected cells
HCMV spread and cell tropism are determined by distinct virus populations.
Full text linkHuman cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells
The human cytomegalovirus protein ul116 interacts with the viral endoplasmic-reticulum-resident glycoprotein ul148 and promotes the incorporation of gH/gL complexes into virions
No full textHeterodimers of glycoproteins H (gH) and L (gL) comprise a basal element of the viral membrane fusion machinery conserved across herpesviruses. In human cytomegalovirus (HCMV), the glycoprotein UL116 assembles onto gH at a position similar to that occupied by gL, forming a heterodimer that is incorporated into virions. Here, we show that UL116 promotes the expression of gH/gL complexes and is required for the efficient production of infectious cell-free virions. UL116-null mutants show a 10-fold defect in production of infectious cell-free virions from infected fibroblasts and epithelial cells. This defect is accompanied by reduced expression of two disulfide-linked gH/gL complexes that play crucial roles in viral entry: the heterotrimer of gH/gL with glycoprotein O (gO) and the pentameric complex of gH/gL with UL128, UL130, and UL131. Kifunensine, a mannosidase inhibitor that interferes with endoplasmic reticulum (ER)-associated degradation (ERAD) of terminally misfolded glycoproteins, restored levels of gH, gL, and gO in UL116-null-infected cells, indicating that constituents of HCMV gH complexes are unstable in the absence of UL116. Further, we find that gH/UL116 complexes are abundant in virions, since a major gH species not covalently linked to other glycoproteins, which has long been observed in the literature, is detected from wild-type but not UL116-null virions. Interestingly, UL116 coimmunoprecipitates with UL148, a viral ER-resident glycoprotein that attenuates ERAD of gO, and we observe elevated levels of UL116 in UL148-null virions. Collectively, our findings argue that UL116 is a chaperone for gH that supports the assembly, maturation, and incorporation of gH/gL complexes into virions. IMPORTANCE HCMV is a betaherpesvirus that causes dangerous opportunistic infections in immunocompromised patients as well as in the immune-naive fetus and preterm infants. The potential of the virus to enter new host cells is governed in large part by two alternative viral glycoprotein H (gH)/glycoprotein L (gL) complexes that play important roles in entry: gH/gL/gO and gH/gL/UL128-131. A recently identified virion gH complex, comprised of gH bound to UL116, adds a new layer of complexity to the mechanisms that contribute to HCMV infectivity. Here, we show that UL116 promotes the expression of gH/gL complexes and that UL116 interacts with the viral ER-resident glycoprotein UL148, a factor that supports the expression of gH/gL/gO. Overall, our results suggest that UL116 is a chaperone for gH. These findings have important implications for understanding HCMV cell tropism as well as for the development of vaccines against the virus
Filterability and extracellular polymeric substances of aerobic granules for AGMBR process
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Characterization of MdMADS2, a member of the SQUAMOSA subfamily of genes, in apple
No full textA MADS-box gene, MdMADS2, was isolated from the apple (Malus x domestica Borkh.) var Fuji and its developmental expression pattern was studied during flower development. MdMADS2 shares a high degree of amino acid sequence identity with the SQUAMOSA subfamily of genes. RNA blot analysis showed that MdMADS2 is transcribed through all stages of flower development, and its transcription was seen in the four floral organs. RNA in situ hybridization revealed that the MdMADS2 mRNA is expressed both in the inflorescence meristem and in the floral meristem. The MdMADS2 transcript was detected at all stages of flower development. Protein localization analysis showed that MdMADS2 protein was excluded from the stamen and carpel primordia, in which a considerable MdMADS2 mRNA signal was detected. This indicates that posttanscriptional regulation may be involved in the MdMADS2-mediated control of flower development. Transgenic tobacco expressing the MdMADS2 gene from the cauliflower mosaic virus 35S promoter showed early flowering and shorter bolts, but did not show any homeotic changes in the floral organs. These results suggest that MdMADS2 plays an important role during early stages of flower development.X1179sciescopu
Short term growth hormone (GH) treatment of GH-deficient adults increases body sodium and extracellular water, but not blood pressure
No full textInitiation of GH treatment in adults is frequently complicated by the development of symptomatic fluid retention. To investigate the mechanism and extent of fluid retention that occurs with dosages of GH used in the treatment of GH-deficient adults, we conducted a double blind study in which seven GH-deficient patients (aged 24-74 yr) each received in random order daily sc injections of placebo, a physiological dose of GH (0.04 U/kg, low dose), and a supraphysiological dose of GH (0.08 U/kg, high dose) for 7 days, separated by 21-day washout periods. On the seventh day, measurements were made of serum insulin-like growth factor I, body weight, exchangeable sodium, plasma volume, angiotensinogen, PRA, aldosterone, atrial natriuretic peptide (ANP), and mean 24-h ambulatory heart rate and blood pressure. GH significantly increased mean insulin-like growth factor I levels from 105 ± 11 to 304 ± 45 μg/L during low dose treatment (P = 0.006) and 400 ± 76 μg/L during high dose treatment (P = 0.004). High dose GH caused a 1.2 ± 0.3 kg increase in body weight (P = 0.01) and a 193 ± 65 mmol increase in exchangeable sodium (P = 0.008). Low dose GH had a lesser effect, with no significant increase in body weight, but an increase in exchangeable sodium of 113 ± 37 mmol (P = 0.02). Plasma volume was not significantly affected by GH treatment. Mean supine angiotensinogen levels were significantly higher during both GH treatments compared to placebo (low dose, P = 0.017; high dose, P = 0.028) as were mean supine pRA levels (low dose, P = 0.0002; high dose, P = 0.0025). Supine angiotensin II, aldosterone, and ANP levels were not significantly affected by GH treatment. There was no significant change from placebo in any of the sodium-regulating hormones in the erect posture. The mean 24-h heart rate was significantly higher during low dose (82 ± 2 beats/min; P = 0.0001) and high dose (88 ± 3 beats/min; P = 0.0001) GH treatment than during placebo (67 ± 3 beats/min). However, no significant change in mean 24-h systolic or diastolic blood pressure was observed. In summary, acute GH administration using doses currently employed in treating adults causes a dose-related increase in body weight and body sodium, but no associated increase in blood pressure. We conclude that 1) sodium retention is a physiological effect of GH, but does not cause an acute rise in blood pressure; and 2) the mechanism of sodium and fluid retention is not primarily due to enhanced aldosterone secretion or inhibition of ANP release, but more likely to a direct renal tubular effect
The GH-IGF axis and its potential role in the ovary of zebrafish, Danio rerio.
Full text linkYu, Man Ying Susana.Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.Includes bibliographical references (leaves 103-117).Abstracts in English and Chinese.Abstract (in English) --- p.iAbstract (in Chinese) --- p.ivAcknowledgement --- p.viTable of contents --- p.viiiSymbols and abbreviations --- p.xiiScientific names --- p.xivList of figures --- p.xvChapter Chapter 1 --- General Introduction --- p.1Chapter 1.1 --- Structure of ovarian follicles --- p.1Chapter 1.2 --- Regulation of ovarian follicle development --- p.3Chapter 1.2.1 --- Endocrine regulation --- p.3Chapter 1.2.1.1 --- Gonadotropins- FSH and LH --- p.3Chapter 1.2.1.2 --- Co-gonadotropin- growth hormone --- p.5Chapter 1.2.2. --- Paracrine regulation --- p.6Chapter 1.2.2.1 --- Activin --- p.6Chapter 1.2.2.2 --- Insulin-like growth factor I (IGF-I) --- p.7Chapter 1.3 --- The GH-IGF-I axis --- p.7Chapter 1.3.1 --- The somatomedin hypothesis --- p.8Chapter 1.3.2 --- "Structure and signaling of GH, GHR" --- p.8Chapter 1.3.3 --- Structure and signaling of IGF system --- p.9Chapter 1.3.4 --- Role of GH-IGF system in reproduction --- p.11Chapter 1.3.5 --- GH action in ovarian functions --- p.12Chapter 1.3.6 --- IGF-I action in ovarian functions --- p.13Chapter 1.3.7 --- The mini GH-IGF axis within the ovary --- p.14Chapter 1.4 --- Objectives of present study --- p.14Chapter Chapter 2 --- "Expression Profiles of the GH-IGF System in the Ovary of Zebrafish, Danio rerio" --- p.19Chapter 2.1 --- Introduction --- p.19Chapter 2.2 --- Material and Methods --- p.21Chapter 2.2.1 --- Animals --- p.21Chapter 2.2.2 --- Isolation of tissues and different stages of follicles from the zebrafish --- p.22Chapter 2.2.3 --- Separation of somatic follicle layers and oocytes --- p.22Chapter 2.2.4 --- Primary follicle cell culture --- p.22Chapter 2.2.5 --- Total RNA extraction --- p.23Chapter 2.2.6 --- Reverse transcription --- p.23Chapter 2.2.7 --- "Validation of semi-quantitative RT-PCR assays for GH (gh), GHR (ghr), IGF-I (igf1), IGF-II (igf2), and IGF-I receptor (igf1r)" --- p.24Chapter 2.2.8 --- Data analysis --- p.25Chapter 2.3 --- Results --- p.25Chapter 2.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.25Chapter 2.3.2 --- Spatial expression of GH-IGF in different tissues of zebrafish --- p.26Chapter 2.3.3 --- "Localization of gh, ghr, igf1, igf2 and igf1r within the zebrafish follicle" --- p.26Chapter 2.3.4 --- Temporal expression profiles of GH-IGF system during folliculogenesis --- p.28Chapter 2.4 --- Discussion --- p.28Chapter Chapter 3 --- Regulation of the GH-IGF-I System and Its Cross-talk with the Activin System in the Zebrafish Ovary --- p.43Chapter 3.1 --- Introduction --- p.43Chapter 3.2 --- Material and methods --- p.45Chapter 3.2.1 --- Animals --- p.45Chapter 3.2.2 --- Chemicals and hormones --- p.45Chapter 3.2.3 --- Primary follicle cell culture --- p.45Chapter 3.2.4 --- Preparation of ovarian fragments --- p.45Chapter 3.2.5 --- Total RNA extraction --- p.45Chapter 3.2.6 --- RT-PCR --- p.47Chapter 3.2.7 --- Construction of real-time PCR standards --- p.47Chapter 3.2.8 --- Real-time PCR and semi-quantitative RT-PCR --- p.48Chapter 3.2.9 --- Data analysis --- p.49Chapter 3.3 --- Results --- p.49Chapter 3.3.1 --- "Expression of growth hormone (gh), growth hormone receptors (ghr1 and ghr2\ IGF-I (igf1), IGF-II (igf2), IGF-I receptor (igf1ra and igf1rb), activin subunits (inhba and inhbb) and follistatin (fst) in cultured zebrafish ovarian fragments" --- p.49Chapter 3.3.2 --- "Establishment of real-time RT-PCR for zebrafish inhba, inhbb and bactin" --- p.50Chapter 3.3.3 --- GH regulation of activin expression in cultured zebrafish follicle cells --- p.50Chapter 3.3.4 --- GH regulation of IGF-I in cultured zebrafish follicle cells --- p.51Chapter 3.3.5 --- IGF-I regulation of activin expression in cultured zebrafish follicle cells --- p.51Chapter 3.3.6 --- Activin regulation of IGF system --- p.52Chapter 3.4 --- Discussion --- p.52Chapter Chapter 4 --- Production of recombinant zebrafish growth hormone --- p.69Chapter 4.1 --- Introduction --- p.69Chapter 4.2 --- Material and Methods --- p.71Chapter 4.2.1 --- Animals --- p.71Chapter 4.2.2 --- Construction of expression plasmids pPIC9K/zfGH --- p.71Chapter 4.2.3 --- Production of recombinant zebrafish GH using Pichia pastoris --- p.73Chapter 4.2.4 --- SDS-PAGE and silver staining --- p.74Chapter 4.2.5 --- Purification --- p.74Chapter 4.2.6 --- Primary follicle cell culture --- p.75Chapter 4.2.7 --- Zebrafish hepatic cell culture --- p.76Chapter 4.2.8 --- RNA extraction and RT-PCR --- p.76Chapter 4.2.9 --- Real-time PCR --- p.77Chapter 4.2.10 --- Cell culture and transient transfection --- p.78Chapter 4.2.11 --- Luciferase reporter gene assay --- p.78Chapter 4.2.12 --- Data analysis --- p.79Chapter 4.3 --- Results --- p.79Chapter 4.3.1 --- Production of recombinant zebrafish GH --- p.79Chapter 4.3.2 --- Effect of recombiant zfGH on the expression of activin β Aand βB in cultured zebrafish follicle cells --- p.80Chapter 4.3.3 --- Effect of zfGH on the expression of igf1 in cultured zebrafish hepatic cells --- p.80Chapter 4.3.4 --- Luciferase reporter gene assay --- p.81Chapter 4.4 --- Discussion --- p.81Chapter Chapter 5 --- General Discussion --- p.94Chapter 5.1 --- Overview --- p.94Chapter 5.2 --- Major achievements of the present study --- p.95Chapter 5.2.1 --- Demonstration of a local mini-GH-IGF-I axis within the zebrafish ovary --- p.96Chapter 5.2.2 --- Differential expression profiles of the GH-IGF system during folliculogenesis --- p.96Chapter 5.2.3 --- The inter-relationship of GH-IGF and activin-follistatin systems --- p.96Chapter 5.2.4 --- Production of recombinant zebrafish GH --- p.97Chapter 5.3 --- Future prospects --- p.97References --- p.102Symbols and AbbreviationsSymbolsα Alphaβ BetaAbbreviations20β-HSD 20β-hydroxysteroid dehydrogenasebp Base paircAMP Cyclic adenosine monophosphatecDNA Complementary cDNACHO Chinese hamster ovary"DHP 17α, 20β-dihydroxy-4-prenane-3 -one"DNA Deoxyribonucleic acidEGF Epidermal growth facto
Activities of Amphioxus GH-Like Protein in Osmoregulation: Insight into Origin of Vertebrate GH Family
Full text linkGH is known to play an important role in both growth promotion and osmoregulation in vertebrates. We have shown that amphioxus possesses a single GH-like hormone (GHl) gene encoding a functional protein capable of promoting growth. However, if GHl can mediate osmoregulation remains open. Here, we demonstrated clearly that GHl increased not only the survival rate of amphioxus but also the muscle moisture under high salinity. Moreover, GHl induced the expression of both the ion transporter Na+-K+-ATPase (NKA) and Na+-K+-2Cl− cotransporter (NKCC) in the gill as well as the mediator of GH action IGFl in the hepatic caecum, indicating that GHl fulfills this osmoregulatory activity through the same mechanisms of vertebrate GH. These results together suggest that the osmoregulatory activities of GH had emerged in the basal chordate amphioxus. We also proposed a new model depicting the origin of pituitary hormone family in vertebrates
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