Efficient Parvovirus Replication Requires CRL4Cdt2-Targeted Depletion of p21 to Prevent Its Inhibitory Interaction with PCNA

PubMed Central

Pintel, David J.

2014-01-01

Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA. PMID:24699724

Efficient parvovirus replication requires CRL4Cdt2-targeted depletion of p21 to prevent its inhibitory interaction with PCNA.

PubMed

Adeyemi, Richard O; Fuller, Matthew S; Pintel, David J

2014-04-01

Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

Human CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1

PubMed Central

Zhu, Qianzheng; Wei, Shengcai; Sharma, Nidhi; Wani, Gulzar; He, Jinshan; Wani, Altaf A.

2017-01-01

Acetylated histone H3 lysine 56 (H3K56Ac) diminishes in response to DNA damage but is restored following DNA repair. Here, we report that CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1. We show that H3K56Ac accumulates at DNA damage sites. The restoration of H3K56Ac but not H3K27Ac, H3K18Ac and H3K14Ac depends on CAF-1 function, whereas all these acetylations are mediated by CBP/p300. The CRL4DDB2 components, DDB1, DDB2 and CUL4A, are also required for maintaining the H3K56Ac and H3K9Ac level in chromatin, and for restoring H3K56Ac following induction of DNA photolesions and strand breaks. Depletion of CUL4A decreases the recruitment of CAF-1 p60 and p150 to ultraviolet radiation- and phleomycin-induced DNA damage. Neddylation inhibition renders CRL4DDB2 inactive, decreases H3K56Ac level, diminishes CAF-1 recruitment and prevents H3K56Ac restoration. Mutation in the PIP box of DDB2 compromises its capability to elevate the H3K56Ac level but does not affect XPC ubiquitination. These results demonstrated a function of CRL4DDB2 in differential regulation of histone acetylation in response to DNA damage, suggesting a novel role of CRL4DDB2 in repair-driven chromatin assembly. PMID:29262658

Variation of heat shock protein gene expression in the brain of cold-induced pulmonary hypertensive chickens.

PubMed

Hassanpour, H; Khosravi Alekoohi, Z; Madreseh, S; Bahadoran, S; Nasiri, L

2016-10-01

Quantitative real-time PCR was carried out to evaluate gene expression of heat shock proteins (HSP) (HSP27, HSP56, HSP60, HSP70, HSP90 and ubiquitin) in the brain (hindbrain, midbrain, forebrain) of chickens with cold-induced pulmonary hypertension. The ratio of the right ventricle to the total ventricle (index of pulmonary hypertension in chickens) was increased in the cold-induced pulmonary hypertensive chickens at 42 d of age compared with control. The HSP genes were expressed in the three parts of the brain in the two experimental groups. In the hindbrain of cold-induced pulmonary hypertensive chickens, the relative gene expression of HSP27, HSP60, HSP70 and HSP90 was decreased while gene expression of HSP56 and ubiquitin was increased compared with controls. In the midbrain of cold induced-pulmonary hypertensive chickens, the expression of HSP56, HSP60, HSP70 and ubiquitin genes was increased compared with controls while HSP27 and HSP90 were decreased. In the forebrain of cold induced-pulmonary hypertensive chickens, the expression of HSP56, HSP60, HSP70 and ubiquitin genes was increased while the expression of the HSP27 gene was decreased compared with controls. It is concluded that overexpression of HSPs in the forebrain and midbrain probably delays the pathological process of cold stress whereas diminished expression of HSP genes in the hindbrain may affect the normal function of brain centres in this area to exacerbate pulmonary hypertension.

Persistent HyperCKemia in Athletes

PubMed Central

Brancaccio, Paola; Maffulli, Nicola; Politano, Luisa; Lippi, Giuseppe; Limongelli, Francesco Mario

2011-01-01

Summary We compared the effects of exercise on serum levels of creatin kinase (CK) in athletes with persistent hyperCKemia at rest (CK group) and in healthy athletes (control group). Prospective controlled study. Eighteen male Caucasian athletes with high serum CK levels at rest (CK between 80 and 150 U/L) and 25 male Caucasian athletes with normal serum CK levels at rest (CK between 10 and 80 U/L) Main Outcome Measures Blood samples were collected at rest, 30 minutes, 6 hours, 24 hours, 48 hours and 72 hours after a progressive cycloergometer test to exhaustion. The levels of serum CK and its isoenzymes were measured. In the control group, serum CK values at rest were normal (48.18 ± 14.14 U/L). After exercise, they increased slightly, though they always remained <80 U/L, decreasing to the rest level after 48 hours. The CK group had serum CK levels at rest higher than normal (116.56 ± 33.30 U/L). Serum CK levels were still outwith the normal range after 48 hours (130.11 ± 46.95 U/L) and 72 hours (116.55 ± 24.84 U/L). Serum CK levels were significantly different in both groups both before and after progressive cycloergometer test to exhaustion. In athletes with high serum CK levels at rest, serum CK levels remained elevated and had a different kinetics after exercise when compared with healthy athletes. PMID:23738242

RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes

PubMed Central

Masschaele, Delphine; De Ceuninck, Leentje; Wauman, Joris; Defever, Dieter; Stenner, Frank; Lievens, Sam; Peelman, Frank; Tavernier, Jan

2017-01-01

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARα receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies. PMID:28542518

The nucleolar helicase DDX56 redistributes to West Nile virus assembly sites.

PubMed

Reid, Colleen R; Hobman, Tom C

2017-01-01

Flaviviruses, including the human pathogen, West Nile virus (WNV), are known to co-opt many host factors for their replication and propagation. To this end, we previously reported that the nucleolar DEAD-box RNA helicase, DDX56, is important for production of infectious WNV virions. In this study, we show that WNV infection results in relocalization of DDX56 from nucleoli to virus assembly sites on the endoplasmic reticululm (ER), an observation that is consistent with a role for DDX56 in WNV virion assembly. Super-resolution microscopy revealed that capsid and DDX56 localized to the same subcompartment of the ER, however, unexpectedly, stable interaction between these two proteins was only detected in the nucleus. Together, these data suggest that DDX56 relocalizes to the site of virus assembly during WNV infection and that its interaction with WNV capsid in the cytoplasm may occur transiently during virion morphogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Hematologic and plasma biochemistry reference intervals of healthy adult barn owls (Tyto alba).

PubMed

Szabo, Zoltan; Klein, Akos; Jakab, Csaba

2014-06-01

Hematologic and plasma biochemistry parameters of barn owls (Tyto alba) were studied in collaboration by the Exotic Division of the Faculty of Veterinary Science of the Szent Istvan University and the Eötvös Loránd University, both in Budapest, Hungary. Blood samples were taken from a total of 42 adult barn owls kept in zoos and bird repatriation stations. The following quantitative and qualitative hematologic values were determined: packed cell volume, 46.2 +/- 4%; hemoglobin concentration, 107 +/- 15 g/L; red blood cell count, 3.2 +/- 0.4 x 10(12)/L; white blood cell count, 13.7 +/- 2.7 x 10(9)/L; heterophils, 56.5 +/- 11.5% (7.8 +/- 2 x 10(9)/L); lymphocytes, 40.3 +/- 10.9% (5.5 +/- 1.9 x 10(9)/L); monocytes, 1.8 +/- 2.1% (0.3 +/- 0.3 x 10(9)/ L); eosinophils, 1 +/- 1% (0.1 +/- 0.1 x 10(9)/L); and basophils, 0.6 +/- 0.5% (0.1 +/- 0.1 x 10(9)/L). The following plasma biochemistry values also were determined: aspartate aminotransferase, 272 +/- 43 U/L; L-gamma-glutamyltransferase, 9.5 +/- 4.7 U/L; lipase, 31.7 +/- 11.1 U/L; creatine kinase, 2228 +/- 578 U/L; lactate dehydrogenase, 1702 +/- 475 U/L; alkaline phosphatase, 358 +/- 197 U/L; amylase, 563 +/- 114 U/L; glutamate dehydrogenase, 7.5 +/- 2.5 U/L; total protein, 30.6 +/- 5.3 g/L; uric acid, 428 +/- 102 micromol/L; and bile acids, 43 +/- 18 micromol/L. These results provide reliable reference values for the clinical interpretation of hematologic and plasma biochemistry results for the species.

Modulation of Phototropic Responsiveness in Arabidopsis through Ubiquitination of Phototropin 1 by the CUL3-Ring E3 Ubiquitin Ligase CRL3NPH3[W

PubMed Central

Roberts, Diana; Pedmale, Ullas V.; Morrow, Johanna; Sachdev, Shrikesh; Lechner, Esther; Tang, Xiaobo; Zheng, Ning; Hannink, Mark; Genschik, Pascal; Liscum, Emmanuel

2011-01-01

Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3NPH3. Under low-intensity BL, CRL3NPH3 mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi- and polyubiquitinated by CRL3NPH3, with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters. PMID:21990941

Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-Ring E3 ubiquitin ligase CRL3(NPH3).

PubMed

Roberts, Diana; Pedmale, Ullas V; Morrow, Johanna; Sachdev, Shrikesh; Lechner, Esther; Tang, Xiaobo; Zheng, Ning; Hannink, Mark; Genschik, Pascal; Liscum, Emmanuel

2011-10-01

Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3(NPH3). Under low-intensity BL, CRL3(NPH3) mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi- and polyubiquitinated by CRL3(NPH3), with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.

The first genome sequence of a metatherian herpesvirus: Macropodid herpesvirus 1.

PubMed

Vaz, Paola K; Mahony, Timothy J; Hartley, Carol A; Fowler, Elizabeth V; Ficorilli, Nino; Lee, Sang W; Gilkerson, James R; Browning, Glenn F; Devlin, Joanne M

2016-01-22

While many placental herpesvirus genomes have been fully sequenced, the complete genome of a marsupial herpesvirus has not been described. Here we present the first genome sequence of a metatherian herpesvirus, Macropodid herpesvirus 1 (MaHV-1). The MaHV-1 viral genome was sequenced using an Illumina MiSeq sequencer, de novo assembly was performed and the genome was annotated. The MaHV-1 genome was 140 kbp in length and clustered phylogenetically with the primate simplexviruses, sharing 67% nucleotide sequence identity with Human herpesviruses 1 and 2. The MaHV-1 genome contained 66 predicted open reading frames (ORFs) homologous to those in other herpesvirus genomes, but lacked homologues of UL3, UL4, UL56 and glycoprotein J. This is the first alphaherpesvirus genome that has been found to lack the UL3 and UL4 homologues. We identified six novel ORFs and confirmed their transcription by RT-PCR. This is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade. Members of the Simplexvirus genus are remarkably conserved, so the absence of ORFs otherwise retained in eutherian and avian alphaherpesviruses contributes to our understanding of the Alphaherpesvirinae. Further study of metatherian herpesvirus genetics and pathogenesis provides a unique approach to understanding herpesvirus-mammalian interactions.

Diagnostic value of sputum adenosine deaminase (ADA) level in pulmonary tuberculosis.

PubMed

Binesh, Fariba; Jalali, Hadi; Zare, Mohammad Reza; Behravan, Farhad; Tafti, Arefeh Dehghani; Behnaz, Fatemah; Tabatabaee, Mohammad; Shahcheraghi, Seyed Hossein

2016-06-01

Tuberculosis is still a considerable health problem in many countries. Rapid diagnosis of this disease is important, and adenosine deaminase (ADA) has been used as a diagnostic test. The aim of this study was to assess the diagnostic value of ADA in the sputum of patients with pulmonary tuberculosis. The current study included 40 patients with pulmonary tuberculosis (culture positive, smear ±) and 42 patients with non tuberculosis pulmonary diseases (culture negative). ADA was measured on all of the samples. The median value of ADA in non-tuberculosis patients was 2.94 (4.2) U/L and 4.01 (6.54) U/L in tuberculosis patients, but this difference was not statistically significant (p=0.100). The cut-off point of 3.1 U/L had a sensitivity of 61% and a specificity of 53%, the cut-off point of 2.81 U/L had a sensitivity of 64% and a specificity of 50% and the cut-off point of 2.78 U/L had a sensitivity of 65% and a specificity of 48%. The positive predictive values for cut-off points of 3.1, 2.81 and 2.78 U/L were 55.7%, 57.44% and 69.23%, respectively. The negative predictive values for the abovementioned cut-off points were 56.75%, 57.14% and 55.88%, respectively. Our results showed that sputum ADA test is neither specific nor sensitive. Because of its low sensitivity and specificity, determination of sputum ADA for the diagnosis of pulmonary tuberculosis is not recommended.

The E3 ubiquitin-ligase SEVEN IN ABSENTIA like 7 mono-ubiquitinates glyceraldehyde-3-phosphate dehydrogenase 1 isoform in vitro and is required for its nuclear localization in Arabidopsis thaliana.

PubMed

Peralta, Diego A; Araya, Alejandro; Busi, Maria V; Gomez-Casati, Diego F

2016-01-01

The E3 ubiquitin-protein ligases are associated to various processes such as cell cycle control and diverse developmental pathways. Arabidopsis thaliana SEVEN IN ABSENTIA like 7, which has ubiquitin ligase activity, is located in the nucleus and cytosol and is expressed at several stages in almost all plant tissues suggesting an important role in plant functions. However, the mechanism underlying the regulation of this protein is unknown. Since we found that the SEVEN IN ABSENTIA like 7 gene expression is altered in plants with impaired mitochondria, and in plants deficient in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase 1, we decided to study the possible interactions between both proteins as potential partners in plant signaling functions. We found that SEVEN IN ABSENTIA like 7 is able to interact in vitro with glyceraldehyde-3-phosphate dehydrogenase and that the Lys231 residue of the last is essential for this function. Following the interaction, a concomitant increase in the glyceraldehyde-3-phosphate dehydrogenase catalytic activity was observed. However, when SEVEN IN ABSENTIA like 7 was supplemented with E1 and E2 proteins to form a complete E1-E2-E3 modifier complex, we observed the mono-ubiquitination of glyceraldehyde-3-phosphate dehydrogenase 1 at the Lys76 residue and a dramatic decrease of its catalytic activity. Moreover, we found that localization of glyceraldehyde-3-phosphate dehydrogenase 1 in the nucleus is dependent on the expression SEVEN IN ABSENTIA like 7. These observations suggest that the association of both proteins might result in different biological consequences in plants either through affecting the glycolytic flux or via cytoplasm-nucleus relocation. Copyright © 2015 Elsevier Ltd. All rights reserved.

High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays.

PubMed

Masschaele, Delphine; Wauman, Joris; Vandemoortele, Giel; De Sutter, Delphine; De Ceuninck, Leentje; Eyckerman, Sven; Tavernier, Jan

2018-04-06

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-associated cytokine receptors by ubiquitinating and suppressing USP8, which, in turn, destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: affinity purification-mass spectrometry, BioID, and Virotrap. We combined these results with known data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high-confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1.

Electrical and Hydrometeor Structure of Thunderstorms that produce Upward Lightning

NASA Astrophysics Data System (ADS)

dos Santos Souza, J. C.; Albrecht, R. I.; Lang, T. J.; Saba, M. M.; Warner, T. A.; Schumann, C.

2017-12-01

Upward lightning (UL) flashes at tall structures have been reported to be initiated by in-cloud branching of a parent positive cloud-to-ground (CG) or intracloud (IC) lightning during the decaying stages of thunderstorms, and associated with stratiform precipitation. This in-cloud branching of the parent CG lightning into lower layers of the stratiform precipitation, as well as other situational modes of UL triggering, are indicative of a lower charge center. The objective of this study is to determine the hydrometeor characteristics of thunderstorms that produce UL, especially at the lower layers of the stratiform region where the bidirectional leader of the parent CG or IC lightning propagates through. We investigated 17 thunderstorms that produced 56 UL flashes in São Paulo, SP, Brazil and 10 thunderstorms (27 UL) from the UPLIGHTS field experiment in Rapid City, SD, USA. We used polarimetric radar data and 3D lighting mapping or the combination of total (i.e., intracloud and cloud-to-ground) and cloud-to-ground lightning strokes data. The Hydrometeor Identification for the thunderstorms of this study consider the information from polarimetric variables ZH, ZDR, KDP and RHOHV to infer radar echoes into rain (light, medium, heavy), hail, dry snow, wet snow, ice crystals, graupel and rain-hail mixtures. Charge structure is inferred by the 3D very-high-frequency (VHF) Lightning Mapping Array by monitoring lightning propagation closely in time and space and constructing vertical histograms of VHF source density. The results of this research project are important to increase the understanding of the phenomenon, the storm evolution and the predictability of UL.

The host ubiquitin-dependent segregase VCP/p97 is required for the onset of human cytomegalovirus replication

PubMed Central

Lin, Yao-Tang; Grey, Finn

2017-01-01

The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection. PMID:28494016

Exercise responses in patients with chronically high creatine kinase levels.

PubMed

Cooper, Christopher B; Dolezal, Brett A; Neufeld, Eric V; Shieh, Perry; Jenner, John R; Riley, Marshall

2017-08-01

Elevated serum creatine kinase (CK) is often taken to reflect muscle disease, but many individuals have elevated CK without a specific diagnosis. How elevated CK reflects muscle metabolism during exercise is not known. Participants (46 men, 48 women) underwent incremental exercise testing to assess aerobic performance, cardiovascular response, and ventilatory response. Serum lactate, ammonia, and CK were measured at rest, 4 minutes into exercise, and 2 minutes into recovery. High-CK and control subjects demonstrated similar aerobic capacities and cardiovascular responses to incremental exercise. Those with CK ≥ 300 U/L exhibited significantly higher lactate and ammonia levels after maximal exercise, together with increased ventilatory responses, whereas those with CK ≥200 U/L but ≤ 300 U/L did not. We recommend measurement of lactate and ammonia profiles during a maximal incremental exercise protocol to help identify patients who warrant muscle biopsy to rule out myopathy. Muscle Nerve 56: 264-270, 2017. © 2016 Wiley Periodicals, Inc.

Long-term functional and oncological outcomes of patients undergoing sural nerve interposition grafting during robot-assisted laparoscopic radical prostatectomy.

PubMed

Zorn, Kevin C; Bernstein, Andrew J; Gofrit, Ofer N; Shikanov, Sergey A; Mikhail, Albert A; Song, David H; Zagaja, Gregory P; Shalhav, Arieh L

2008-05-01

For men with high-volume or high-grade prostate cancer, wide excision of the ipsilateral neurovascular bundle is commonly performed. The concept of nerve reconstruction is intriguing as a feasible approach to preserve sexual function (SF). We sought to evaluate the functional, pathologic, and oncologic outcomes of men who underwent robot-assisted sural-nerve graft (SNG) interposition. Between February 2003 and May 2007, 1175 consecutive men underwent robot-assisted laparoscopic radical prostatectomy (RLRP). Database analysis identified 27 men who had SNG: 4 bilateral (BL) and 23 unilateral (UL). SF was prospectively evaluated preoperatively and at 1, 3, 6, 12, and 24 months postoperatively using validated questionnaires. Positive surgical margins (PSMs), biochemical recurrence (BCR), and potency were evaluated. Compared with RLRP patients without SNG, patients with SNG were younger (57.2 v 61.8 years, P=0.02), had a higher Gleason score (P=0.02), and had a higher clinical and pathologic stage (P<0.001 for both). Mean surgical time was significantly longer (349 v 195 min, P<0.001) in patients with SNG. With a mean follow-up of 26.1 months, 11 (47.8%) patients with UL-SNG and zero men with BL-SNG regained potency. No significant difference in SF was observed between UL nerve sparing and no SNG (56%) compared with UL nerve sparing with UL-SNG (P=0.44). Rates of return-to-baseline SF (RTB-SF) at 6, 12, and 24 months were 11%, 36% and 45% for UL-SNG, respectively, which were also comparable to UL nerve sparing only (P>0.05). No patient (0%) in the BL-SNG group ever achieved RTB-SF status at any time point. PSMs were observed in 37% (10/27) of all patients. BCR occurred in nine patients (33.3%), seven of whom had PSM (78%); treatment failure occurred within 6 months of surgery, necessitating androgen deprivation therapy. Despite optimism regarding SNG, long-term functional outcomes have been disappointing, particularly for BL nerve interposition. UL-SNG functional outcomes do not appear to improve outcomes when compared with men with UL nerve preservation. With the greater risk of PSM and BCR in patients who are considered candidates for SNG, newer treatment modalities are needed to cure their disease while preserving SF.

53BP1 is a reader of the DNA damage-induced H2A Lys15 ubiquitin mark

PubMed Central

Fradet-Turcotte, Amélie; Canny, Marella D.; Escribano-Díaz, Cristina; Orthwein, Alexandre; Leung, Charles C.Y.; Huang, Hao; Landry, Marie-Claude; Kitevski-LeBlanc, Julianne; Noordermeer, Sylvie M.; Sicheri, Frank; Durocher, Daniel

2014-01-01

53BP1 (TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains enigmatic since its relocalization involves recognition of H4 Lys20 (H4K20) methylation by its Tudor domain. Here we elucidate how 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mono-nucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitylated on Lys15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a C-terminal extension, termed the ubiquitylation-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone “code” produced by DSB signaling. PMID:23760478

Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5.

PubMed

Becuwe, Michel; Léon, Sébastien

2014-11-07

After endocytosis, membrane proteins can recycle to the cell membrane or be degraded in lysosomes. Cargo ubiquitylation favors their lysosomal targeting and can be regulated by external signals, but the mechanism is ill-defined. Here, we studied the post-endocytic trafficking of Jen1, a yeast monocarboxylate transporter, using microfluidics-assisted live-cell imaging. We show that the ubiquitin ligase Rsp5 and the glucose-regulated arrestin-related trafficking adaptors (ART) protein Rod1, involved in the glucose-induced internalization of Jen1, are also required for the post-endocytic sorting of Jen1 to the yeast lysosome. This new step takes place at the trans-Golgi network (TGN), where Rod1 localizes dynamically upon triggering endocytosis. Indeed, transporter trafficking to the TGN after internalization is required for their degradation. Glucose removal promotes Rod1 relocalization to the cytosol and Jen1 deubiquitylation, allowing transporter recycling when the signal is only transient. Therefore, nutrient availability regulates transporter fate through the localization of the ART/Rsp5 ubiquitylation complex at the TGN.

75 FR 56160 - BNSF Railway Company and Union Pacific Railroad Company-Joint Relocation Project Exemption-in...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-15

... line beginning south of Sun Valley Boulevard (near UP milepost 56.50) that crosses Salt Creek and..., 2006). UP also leases: (1) The BNSF line near Sun Valley Boulevard (beginning near UP milepost 56.50) to the beginning point of the wye at BNSF milepost 0.62 (referred to as the Sun Valley Segment); and...

Comparison of cost-benefit analysis of nitrogen dioxide control in Tokyo, Japan with those in other countries and cities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voorhees, A.S.; Araki, S.; Sakai, R.

1999-07-01

To evaluate the economic effectiveness of past NO{sub 2} controls in Tokyo, the authors compared the results of their cost-benefit analysis (CBA) of these controls with other investigations. The authors carried out a CBA of NO{sub 2} controls in Tokyo using Freeman's benefit methodology and EPA and Dixon et al. cost methodologies and they compared their assumptions and results to work done by other researchers for other countries and cities, which were collected from the literature. The authors assumed 2 to 3 days duration per incidence of respiratory illness. Kenkel suggested 4.1 days and Dixon et al. assumed 2 weeks. They estimated avoided incidence per person in adults as 2.6 (upper limit UL 2.7; lower limit LL 2.4) and in children as 0.33 (UL 0.35; LL 0.30). Ostro estimated 0.20 for respiratory symptoms in adults from NO{sub 2} exposure, 5.2 for respiratory symptoms and 0.078 for asthma attacks in adults from particulates. The authors estimated work loss days (WLDs) per person for workers as 4.7 (UL 5.0; LL 4.4) and for working mothers as 0.61 (UL 0.66; LL 0.56). Shin et al.'s per-person estimates included 4.5 WLDs in Bangkok, 3.7 in Beijing, 2.3 in Shanghai, and 1.1 in Kuala Lumpur. They estimated the cost effectiveness of NO{sub 2} control in Tokyo to bemore » $1,400/ton (UL $1,500; LL $1,300) for motor vehicles, $21,000/ton (UL $23,000; LL $$19,000) for all NO{sub x} sources, and $$91,000/ton (UL $98,000; LL $84,000) for stationary point sources. This compares to $240 to $$1,500/ton in West Virginia for all NO{sub x} sources, $$2,700/ton in northern Virginia from motor vehicles, $5,600/ton from motor vehicles in Virginia, and $17,000 to $26,000/ton from all NO{sub x} sources in the Chesapeake River Watershed. Herein, the benefits in Tokyo exceeded the costs by a ratio of approximately 6 to 1 (UL 7:1; LL 5:1).« less

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) III: Endogenous Inhibition of Angiotensin Converting Enzyme (ACE) Provides Protection against Cardiovascular Diseases

PubMed Central

Fagyas, Miklós; Úri, Katalin; Siket, Ivetta M.; Daragó, Andrea; Boczán, Judit; Bányai, Emese; Édes, István; Papp, Zoltán; Tóth, Attila

2014-01-01

ACE inhibitor drugs decrease mortality by up to one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors. Here we investigated the clinical significance of this potential endogenous ACE inhibition. ACE concentration and activity was measured in patient's serum samples (n = 151). ACE concentration was found to be in a wide range (47–288 ng/mL). ACE activity decreased with the increasing concentration of the serum albumin (HSA): ACE activity was 56±1 U/L in the presence of 2.4±0.3 mg/mL HSA, compared to 39±1 U/L in the presence of 12±1 mg/mL HSA (values are mean±SEM). Effects of the differences in ACE concentration were suppressed in human sera: patients with ACE DD genotype exhibited a 64% higher serum ACE concentration (range, 74–288 ng/mL, median, 155.2 ng/mL, n = 52) compared to patients with II genotype (range, 47–194 ng/mL, median, 94.5 ng/mL, n = 28) while the difference in ACE activities was only 32% (range, 27.3–59.8 U/L, median, 43.11 U/L, and range 15.6–55.4 U/L, median, 32.74 U/L, respectively) in the presence of 12±1 mg/mL HSA. No correlations were found between serum ACE concentration (or genotype) and cardiovascular diseases, in accordance with the proposed suppressed physiological ACE activities by HSA (concentration in the sera of these patients: 48.5±0.5 mg/mL) or other endogenous inhibitors. Main implications are that (1) physiological ACE activity can be stabilized at a low level by endogenous ACE inhibitors, such as HSA; (2) angiotensin II elimination may have a significant role in angiotensin II related pathologies. PMID:24690767

Serum Gamma-Glutamyl-Transferase Independently Predicts Outcome After Transarterial Chemoembolization of Hepatocellular Carcinoma: External Validation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guiu, Boris, E-mail: boris.guiu@chu-dijon.fr; Deschamps, Frederic; Boulin, Mathieu

Purpose: An Asian study showed that gamma glutamyl transpeptidase (GGT) can predict survival after transarterial chemoembolization (TACE) of hepatocellular carcinoma (HCC). This study was designed to validate in a European population this biomarker as an independent predictor of outcome after TACE of HCC and to determine a threshold value for clinical use. Methods: In 88 consecutive patients treated by TACE for HCC, the optimal threshold for GGT serum level was determined by a ROC analysis. Endpoints were time-to-treatment failure (TTTF) and overall survival (OS). All multivariate models were internally validated using bootstrapping (90 replications). Results: Median follow-up lasted 373 days,more » and median overall survival was 748 days. The optimal threshold for GGT was 165 U/L (sensitivity: 89.3%; specificity: 56.7%; area under the ROC curve: 0.7515). Median TTTF was shorter when GGT was {>=}165 U/L (281 days vs. 850 days; P < 0.001). GGT {>=}165 U/L (hazard ratio (HR) = 2.06; P = 0.02), WHO PS of 2 (HR = 5.4; P = 0.002), and tumor size (HR = 1.12; P = 0.014) were independently associated with shorter TTTF. Median OS was shorter when GGT was {>=}165 U/L (508 days vs. not reached; P < 0.001). GGT {>=} 165 U/L (HR = 3.05; P = 0.029), WHO PS of 2 (HR = 12.95; P < 0.001), alfa-fetoprotein (HR = 2.9; P = 0.01), and tumor size (HR = 1.096; P = 0.013) were independently associated with shorter OS. The results were confirmed by bootstrapping. Conclusions: Our results provide in a European population the external validation of GGT as an independent predictor of outcome after TACE of HCC. A serum level of GGT {>=} 165 U/L is independently associated with both shorter TTTF and OS.« less

Effect of Upper Limb Rehabilitation Compared to No Upper Limb Rehabilitation in Lung Transplant Recipients: A Randomized Controlled Trial.

PubMed

Fuller, Louise M; El-Ansary, Doa; Button, Brenda M; Corbett, Monique; Snell, Greg; Marasco, Silvana; Holland, Anne E

2017-10-16

To investigate the effect of a supervised upper limb (UL) program (SULP) compared to no supervised UL program (NULP) after lung transplantation (LTx). Randomized controlled trial. Physiotherapy gym. Participants (N=80; mean age, 56±11y; 37 [46%] men) were recruited after LTx. All participants underwent lower limb strength thrice weekly and endurance training. Participants randomized to SULP completed progressive UL strength training program using handheld weights and adjustable pulley equipment. Overall bodily pain was rated on the visual analog scale. Shoulder flexion and abduction muscle strength were measured on a hand held dynamometer. Health related quality of life was measured with Medical Outcomes Study 36-item Short Form health Survey and the Quick Dash. Measurements were made at baseline, 6 weeks, 12 weeks, and 6 months by blinded assessors. After 6 weeks of training, participants in the SULP (n=41) had less overall bodily pain on the visual analog scale than did participants in the NULP (n=36) (mean VAS bodily pain score, 2.1±1.3cm vs 3.8±1.7cm; P<.001) as well as greater UL strength than did participants in the NULP (mean peak force, 8.4±4.0Nm vs 6.7±2.8Nm; P=.037). At 12 weeks, participants in the SULP better quality of life related to bodily pain (76±17 vs 66±26; P=.05), but at 6 months there were no differences between the groups in any outcome measures. No serious adverse events were reported. UL rehabilitation results in short-term improvements in pain and muscle strength after LTx, but no longer-term effects were evident. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Kinetics of transcription of infectious laryngotracheitis virus genes.

PubMed

Mahmoudian, Alireza; Markham, Philip F; Noormohammadi, Amir H; Browning, Glenn F

2012-03-01

The kinetics of expression of only a few genes of infectious laryngotracheitis virus (ILTV) have been determined, using northern blot analysis. We used quantitative reverse transcriptase PCR to examine the kinetics of expression of 74 ILTV genes in LMH cells. ICP4 was the only gene fully expressed in the presence of cycloheximide, and thus classified as immediate-early. The genes most highly expressed early in infection, and thus classified as early, included UL1 (gL), UL2, UL3, UL4, UL5, UL6, UL7, UL8, UL13, UL14, UL19, UL20, UL23 (TK), UL25, UL28, UL29, UL31, UL33, UL34, UL38, UL39, UL40, UL42, UL43, UL44 (gC), UL47, UL48 (α-TIF), UL49, UL54 (ICP27), US3 and US10. ORF A, ORF B, ORF C, ORF E, sORF 4/3, UL[-1], UL0, UL3.5, UL9, UL10 (gM), UL11, UL15a, UL15b, UL18, UL22 (gH), UL24, UL26, UL30, UL32, UL36, UL45, UL49.5 (gN), UL52, US2, US4 (gG), US5 (gJ) and US9 were most highly expressed late in infection and were thus considered late genes. Several genes, including ORF D, UL12, UL17, UL21, UL27 (gB), UL35, UL37, UL41, UL46, UL50, UL51, UL53 (gK), US8 (gE), US6 (gD) and US7 (gI), had features of both early and late genes and were classified as early/late. Our findings suggest transcription from most of ILTV genes is leaky or subject to more complex patterns of regulation than those classically described for herpesviruses. This is the first study examining global expression of ILTV genes and the data provide a basis for future investigations of the pathogenesis of infection with ILTV. Copyright © 2011 Elsevier Ltd. All rights reserved.

Variation in glycogen concentrations within mantle and foot tissue in Amblema plicata plicata: Implications for tissue biopsy sampling

USGS Publications Warehouse

Naimo, T.J.; Monroe, E.M.

1999-01-01

With the development of techniques to non-lethally biopsy tissue from unionids, a new method is available to measure changes in biochemical, contaminant, and genetic constituents in this imperiled faunal group. However, before its widespread application, information on the variability of biochemical components within and among tissues needs to be evaluated. We measured glycogen concentrations in foot and mantle tissue in Amblema plicata plicata (Say, 1817) to determine if glycogen was evenly distributed within and between tissues and to determine which tissue might be more responsive to the stress associated with relocating mussels. Glycogen was measured in two groups of mussels: those sampled from their native environment (undisturbed mussels) and quickly frozen for analysis and those relocated into an artificial pond (relocated mussels) for 24 months before analysis. In both undisturbed and relocated mussels, glycogen concentrations were evenly distributed within foot, but not within mantle tissue. In mantle tissue, concentrations of glycogen varied about 2-fold among sections. In addition, glycogen varied significantly between tissues in undisturbed mussels, but not in relocated mussels. Twenty-four months after relocation, glycogen concentrations had declined by 80% in mantle tissue and by 56% in foot tissue relative to the undisturbed mussels. These data indicate that representative biopsy samples can be obtained from foot tissue, but not mantle tissue. We hypothesize that mantle tissue could be more responsive to the stress of relocation due to its high metabolic activity associated with shell formation.

Data forwarding mechanism for supporting real-time services during relocations in UMTS systems

NASA Astrophysics Data System (ADS)

Cai, Wei; Liao, Xianglong; Zheng, Liang; Liu, Zehong

2004-04-01

To minimize the interruption during the handovers or relocations invoked by subscribers moving is a very critical factor to enhance the performance of the UMTS systems. We know that the 2G systems have been optimized to minimize the interruption of speech during handovers by two main technologies: one is the bi-casting for the DL traffic and the other is the fast radio resynchronization by the UE for the UL traffic. In the UMTS systems, we have also implemented lossless relocations for non real-time services with high reliability by data buffering in the source RNC and target RNC for the UE. However, the UMTS systems support four QoS classes traffic flow: conversational class, streaming class, interactive class and background class. The main distinguishing factor between these QoS classes is how delay sensitive the traffic is: Conversational and Streaming classes are mainly used to carry real-time traffic flows, like video telephony, interactive and background classes are mainly used by traditional Internet applications like WWW, E-mail and FTP. It"s essential to provide the solutions for supporting real-time services to meet the requirement for QoS in UMTS systems. Apparently, the Data buffering mechanism is not adapted to real-time services because of it"s delay may exceed the basic requirement for real-time services. Under this background, the paper discussed two data forwarding solutions for real-time services from the PS domain in the UMTS systems: packet duplication and Core Network bi-casting. The former mechanism does not require any new procedures, messages nor information elements. The later mechanism requires that the GGSN or SGSN is able to bi-cast the DL traffic to the target RNC according to the relocations involving two SGSNs or just involving one SGSN. It also implicitly shows that we need change procedures at the nodes SGSN, GGSN and RNC which are involved in the relocation procedure based on existing procedures that we have already designed if adopt the later solution. In a detail way, the paper analyzed the characteristic for these two solutions respectively, concentrated on the packet flows and the message flows in those nodes involved in relocations. Additionally, also gave out the impact on present transport technologies in the wireless communication systems. However we shall minimize the impact of evolution of transport mechanism and utilize the resource efficiently according to the general requirements for QoS in UMTS systems.

The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

PubMed

Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

2016-10-15

Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Evaluation of an anal sac adenocarcinoma tumor in a Spitz dog

PubMed Central

Javanbakht, Javad; Tavassoli, Abbas; Sabbagh, Atefeh; Hassan, Mehdy Aghamohammmad; Samakkhah, Shohreh Alian; Shafiee, Radmehr; Lakzian, Ali; Ghalee, Vahideh Rahmani; Gharebagh, Sonia Shoja

2013-01-01

A 9-year-old emasculated male Spitz with tenesmus and constipation had a subcutaneous mass at the left ventral aspect of the anus with history of polyuria and polydipsia. A complete blood cell count, serum biochemistry panel, and urinalysis (cystocentesis sample) were evaluated. Abnormalities in the serum biochemistry panel included a mildly elevated serum cholesterol concentration (7.28 mmol/L; reference interval, 2.70–5.94 mmol/L), increased serum alkaline phosphatase activity (184 U/L; reference interval, 9–90 U/L), alanine transaminase (122 U/L; reference interval, 5–60 U/L) activity and aspartate aminotransferase (80 U/L; reference interval, 5–55 U/L) activity, severe increased total calcium concentration (16.3 mg/dL; reference interval, 8.2–12.4 mg/dL or 9.3–11.4 mg/dL), and decreased total calcium concentration (3.4 mg/dL, reference interval, 2.5–5.6mg/dL). Furthermore, testing revealed an increased intact parathyroid hormone concentration (38.6 pmol/L; reference interval, 3–17 pmol/L). On cytologic and histopathologic examinations, various types of cells were observed. Most of the cells were oval to polygonal and had elliptical or elongate nuclei and a moderate amount of pale to basophilic cytoplasm. The remaining cells had round to oval nuclei and pale to basophilic cytoplasm. Cells of both types were loosely adhered to each other and were arranged in rosette-like structures. Both neoplastic cell types had fine homogenous chromatin and either a small indistinct nucleolus or no visible nucleolus. Mild anisokaryosis and anisocytosis were observed. Histologically, the mass consists of glandular structures formed by cuboidal cells admixed with bundles of spindle cells. Based on location and histologic features, the final diagnosis was adenocarcinoma of the apocrine gland of the anal sac, which should be included as a cytologic differential diagnosis when spindle cells and typical epithelial cells are observed in masses in the region of the anal sac of dogs. PMID:23570021

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

PubMed

Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

2016-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

[Legionnaire's pneumonia with rhabdomyolysis and acute renal failure. A case report].

PubMed

Sposato, Bruno; Mariotta, Salvatore; Ricci, Alberto; Lucantoni, Gabriele; Schmid, Giovanni

2003-09-01

Legionella pneumophyla is the agent responsible of Legionnaire's disease. It appears as a severe pneumonia and often requires admission in Intensive Care Unit. In literature, renal failure is reported to occur in 15 percent of Legionnaire disease and this event induce a mortality over 50% of these cases. The authors describe a case of Legionnaire's pneumonia with respiratory failure, rhabdomyolysis and acute renal failure. Patient was a female, 61 yrs old, admitted to our hospital because of fever (38 degrees-38.5 degrees C), severe respiratory failure (pH = 7.49, PaCO2 = 23.1 mmHg, PaO2 = 56.7 mmHg), oliguria (< 200 ml/24 h); chest x-rays and computed tomography (TC) showed a pneumonia at right lower lobe. Among other things, in blood analysis was noted the following values: BUN = 47 mg/dl, creatinine = 2.1 mg/dl, Na+ = 133 mmol/L, Cl- = 97 mmol/L, Ca+ = 7.2 mg/dl, K+ = 5.8 mmol/L, AST = 213 U/L, ALT = 45 U/L, LDH = 1817 U/L, CPK = 16738 U/L, CPK-MB = 229 U/L, myoglobin > 4300 ng/ml., leucocyte count = 17,500/mmc (N = 92%, L = 3%, M = 5%), positive anti Legionella IgG and IgM (IgG > 1:64, IgM > 1:96), evidence of Legionella soluble antigen in the urine analysis. Therapy with clarytromicyne (500 mg b.i.d i.v.) and rifampicin (600 mg/die i.v.) was begun; computed tomography showed after six days an improvement of pulmonary lesion but, in the following days, health status and blood analysis got worse. Patient went on antibiotics and underwent haemotherapy (Hb: 8 gr/dl), haemodialysis because of acute renal failure but healthy status worse furthermore and she died on 18th days after admission. This case point out rhabdomyolysis with acute renal failure is suggestive for Legionnaire's disease and is associated with high rate of mortality.

[Prevalence of anti-CMV antibodies in blood donors in the Sfax region (value in blood transfusion)].

PubMed

Gargouri, J; Elleuch, H; Karray, H; Rekik, H; Hammami, A

2000-01-01

Detection of anti-CMV antibodies was carried out in sera of healthy blood donors, divided into groups of 20 according to age and sex. Sera were tested for anti-CMV by an ELISA test (Enzygnost anti-CMV/IgG-Behring). Among 280 sera, 272 were positive for IgG to CMV (97.14%). The prevalence of those antibodies was high in all age stratum (95-100%) but was higher in women than in men (98.57% versus 95.71%). The titre of IgG to CMV was superior to 12 Ul/ml in 56.43% of CMV positive donors. So, the leucocyte removal is the only alternative for the prevention of post-transfusional CMV infection. The high percentage of donors with anti-CMV antibodies level more than to 12 Ul/ml allow to consider the use of plasmapheresis for preparing specific immunoglobulins to CMV.

Staphylococcal superantigen-specific IgE antibodies: degree of sensitization and association with severity of asthma.

PubMed

Elabras, José; Mello, Fernanda Carvalho de Queiroz; Lupi, Omar; Bica, Blanca Elena Rios Gomes; Papi, José Angelo de Souza; França, Alfeu Tavares

2016-01-01

To determine the presence of staphylococcal superantigen-specific IgE antibodies and degree of IgE-mediated sensitization, as well as whether or not those are associated with the severity of asthma in adult patients. This was a cross-sectional study involving outpatients with asthma under treatment at a tertiary care university hospital in the city of Rio de Janeiro, Brazil. Consecutive patients were divided into two groups according to the severity of asthma based on the Global Initiative for Asthma criteria: mild asthma (MA), comprising patients with mild intermittent or persistent asthma; and moderate or severe asthma (MSA). We determined the serum levels of staphylococcal toxin-specific IgE antibodies, comparing the results and performing a statistical analysis. The study included 142 patients: 72 in the MA group (median age = 46 years; 59 females) and 70 in the MSA group (median age = 56 years; 60 females). In the sample as a whole, 62 patients (43.7%) presented positive results for staphylococcal toxin-specific IgE antibodies: staphylococcal enterotoxin A (SEA), in 29 (20.4%); SEB, in 35 (24.6%); SEC, in 33 (23.2%); and toxic shock syndrome toxin (TSST), in 45 (31.7%). The mean serum levels of IgE antibodies to SEA, SEB, SEC, and TSST were 0.96 U/L, 1.09 U/L, 1.21 U/L, and 1.18 U/L, respectively. There were no statistically significant differences between the two groups in terms of the qualitative or quantitative results. Serum IgE antibodies to SEA, SEB, SEC, and TSST were detected in 43.7% of the patients in our sample. However, neither the qualitative nor quantitative results showed a statistically significant association with the clinical severity of asthma. Determinar a presença de anticorpos IgE específicos para superantígenos estafilocócicos e o grau de sensibilização mediada por esses, assim como se esses estão associados à gravidade da asma em pacientes adultos. Estudo transversal incluindo asmáticos adultos em acompanhamento ambulatorial em um hospital universitário terciário no Rio de Janeiro (RJ). Os pacientes foram alocados consecutivamente em dois grupos de gravidade da asma segundo critérios da Global Initiative for Asthma: asma leve (AL), com asmáticos leves intermitentes ou persistentes, e asma moderada ou grave (AMG). Foram determinados os níveis séricos de anticorpos IgE antitoxinas estafilocócicas, e os resultados foram comparados por análise estatística. Foram incluídos 142 pacientes no estudo: 72 no grupo AL (mediana de idade = 46 anos; 59 do sexo feminino) e 70 do grupo AMG (mediana de idade = 56 anos; 60 do sexo feminino). Na amostra geral, 62 pacientes (43,7%) apresentaram resultados positivos para dosagens de anticorpos IgE antitoxinas estafilocócicas: enterotoxina (TX) A, em 29 (20,4%); TXB, em 35 (24,6%); TXC, em 33 (23,2%); e toxic shock syndrome toxin (TSST), em 45 (31,7%). As médias das dosagens séricas de anticorpos IgE específicos anti-TXA, TXB, TXC e TSST foram, respectivamente, de 0,96 U/l, 1,09 U/l, 1,21 U/l, e 1,18 U/l. Não houve diferença estatisticamente significativa dos resultados qualitativos ou quantitativos entre os grupos. A presença de anticorpos IgE séricos anti-TXA, TXB, TXC e TSST, foi detectada em 43,7% nessa amostra de pacientes, mas não houve associação estatisticamente significativa entre seus resultados qualitativos ou quantitativos e gravidade clínica da asma.

UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

PubMed Central

Reynolds, Ashley E.; Ryckman, Brent J.; Baines, Joel D.; Zhou, Yuping; Liang, Li; Roller, Richard J.

2001-01-01

The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane. PMID:11507225

Identification of interaction domains within the UL37 tegument protein of herpes simplex virus type 1.

PubMed

Bucks, Michelle A; Murphy, Michael A; O'Regan, Kevin J; Courtney, Richard J

2011-07-20

Herpes simplex virus type 1 (HSV-1) UL37 is a 1123 amino acid tegument protein that self-associates and binds to the tegument protein UL36 (VP1/2). Studies were undertaken to identify regions of UL37 involved in these protein-protein interactions. Coimmunoprecipitation assays showed that residues within the carboxy-terminal half of UL37, amino acids 568-1123, are important for interaction with UL36. Coimmunoprecipitation assays also revealed that amino acids 1-300 and 568-1123 of UL37 are capable of self-association. UL37 appears to self-associate only under conditions when UL36 is not present or is present in low amounts, suggesting UL36 and UL37 may compete for binding. Transfection-infection experiments were performed to identify domains of UL37 that complement the UL37 deletion virus, K∆UL37. The carboxy-terminal region of UL37 (residues 568-1123) partially rescues the K∆UL37 infection. These results suggest the C-terminus of UL37 may contribute to its essential functional role within the virus-infected cell. Copyright © 2011 Elsevier Inc. All rights reserved.

Longitudinal impact of frequent geographic relocation from adolescence to adulthood on psychosocial stress and vital exhaustion at ages 32 and 42 years: the Amsterdam growth and health longitudinal study.

PubMed

Lin, Kuan-Chia; Twisk, J W R; Huang, Hui-Chuan

2012-01-01

We assessed mobility in different life stages over a 29-year period from adolescence through adulthood and its correlation with psychosocial stress and vital exhaustion at ages 32 and 42 years. Data were derived from the Amsterdam Growth and Health Longitudinal Study, an observational longitudinal study of 420 boys and girls from age 13 to 42 years. Measurements included cumulative frequency of geographic relocation (CFGR), psychosocial stress (measured by a Dutch scale of experienced stress, VOEG-13), vital exhaustion (measured by the Maastricht Questionnaire, MQ), demographics, socioeconomic status, and other background characteristics. From 1976 to 2006, total CFGR was 3.56 ± 1.89 (range 0-13). Frequent geographic relocation during 2 life stages (age 22-32 years and 33-42 years) was significantly interrelated; however, this was not evident at age 13 to 21 years, which suggests a unique exposure to relocation during adolescence and youth. After adjusting for anticipated confounders, higher cumulative frequencies of residential changes during adolescence and youth were markedly associated with psychosocial stress and vital exhaustion at ages 32 and 42 years. Frequent geographic relocation during adolescence and youth was an indicator of psychosocial stress and vital exhaustion in the transition to middle adulthood. Further consideration of the pathways in this web of causation may aid in stress prevention and minimize negative consequences.

Human Cytomegalovirus UL50 and UL53 Recruit Viral Protein Kinase UL97, Not Protein Kinase C, for Disruption of Nuclear Lamina and Nuclear Egress in Infected Cells

PubMed Central

Sharma, Mayuri; Kamil, Jeremy P.; Coughlin, Margaret; Reim, Natalia I.

2014-01-01

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them. PMID:24155370

Human cytomegalovirus UL50 and UL53 recruit viral protein kinase UL97, not protein kinase C, for disruption of nuclear lamina and nuclear egress in infected cells.

PubMed

Sharma, Mayuri; Kamil, Jeremy P; Coughlin, Margaret; Reim, Natalia I; Coen, Donald M

2014-01-01

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.

Forster's tern chick survival in response to a managed relocation of predatory California gulls

USGS Publications Warehouse

Ackerman, Joshua T.; Herzog, Mark P.; Hartman, C. Alex; Herring, Garth

2014-01-01

Gull populations can severely limit the productivity of waterbirds. Relocating gull colonies may reduce their effects on nearby breeding waterbirds, but there are few examples of this management strategy. We examined gull predation and survival of Forster's tern (Sterna forsteri) chicks before (2010) and after (2011) the managed relocation of the largest California gull (Larus californicus) colony (24,000 adults) in San Francisco Bay, California. Overall, survival of radio-marked Forster's tern chicks from hatching to fledging was 0.22 ± 0.03 (mean ± SE), and daily survival rates increased with age. Gulls were the predominant predator of tern chicks, potentially causing 54% of chick deaths. Prior to the gull colony relocation, 56% of radio-marked and 20% of banded tern chicks from the nearest tern colony were recovered dead in the gull colony, compared to only 15% of radio-marked and 4% of banded chicks recovered dead from all other tern colonies. The managed relocation of the gull colony substantially increased tern chick survival (by 900%) in the nearby (3.8 km) reference tern colony (0.29 ± 0.10 in 2010 and 0.25 ± 0.09 in 2011). Among 19 tern nesting islands, fledging success was higher when gull abundance was lower at nearby colonies and when gull colonies were farther from the tern colony. Our results indicate that the managed relocation of gull colonies away from preferred nesting areas of sensitive waterbirds can improve local reproductive success, but this conservation strategy may shift gull predation pressure to other areas or species.

[Magnesium isoglycyrrhizinate prevention of chemotherapy-induced liver damage during initial treatment of patients with gastrointestinal tumors].

PubMed

Yan, Yulan; Mo, Yongsen; Zhang, Dongmei

2015-03-01

To investigate the preventive effect of magnesium isoglycyrrhizinate against acute drug-induced liver damage from initial chemotherapy treatment in patients with gastrointestinal cancer. A total of 216 cases with early stage gastric cancer and indications for systemic chemotherapy that had been diagnosed with gastrointestinal malignant tumors by pathology in our hospital were enrolled for study during the period of January 2011 to June 2013.Using a prospective randomized controlled study design,differences were assessed between groups treated with glycyrrhizic acid magnesium (experimental group; n=114) or glutathione (control group; n=102) and the FOLFOX regimen (n=104) or the XELOX regimen (n=112).Patients in the FOLFOX group received intravenous infusion of L-OHP (85 mg/m²) at day 1,followed by a bolus injection of 5-FU (400 mg/m²) at days 1-2 and continuous intravenous infusion of 5-FU (600 mg/m²) for 22 h at days 1-2,with one cycle comprising 2 weeks. Patients in the XELOX group received intravenous infusion of L-OHP (130 mg/m²) at day 1, followed by capecitabine (1 000 mg/m²) oral twice a day at days 1-14,with one cycle comprising 3 weeks.In the first cycle of chemotherapy,serum was extracted from the patients at 1 day before chemotherapy and 1 week after chemotherapy.An automated biochemistry analyzer was used to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and alkaline phosphatase (ALP). Differences between groups were statistically analyzed by the t-test and x² test. Among the total 216 cases treated with chemotherapy,40 showed hepatic biochemical abnormalities (12 cases in the experimental group, 28 cases in the control group), and the effect of prevention was significantly different between the two groups (10.53% vs. 27.25%; x² =10.219, P less than 0.005).The acute and subacute hepatic toxicity reaction degrees for the experimental and the control groups were: 0:94.78% vs. 88.2%; 1:5.3% vs. 11.8% (x² =6.99, P < 0.01). One week after chemotherapy, the liver biochemical indexes in the experimental group (ALT:35.93 ± 8.33 U/L; AST:24.84 ±2.91 U/L; TBil:13.29 ± 5.89 mumol/L; ALP:125.1 ± 53.61 U/L) were statically different from those in the control group (all P < 0.05). The liver biochemical indexes before and after chemotherapy were also significantly different between the experimental group (ALT:13.18t3.23 U/L; AST:5.39 ± 2.57 U/L; TBil:2.79 ± 0.23 mumol/L; ALP:52.08 ± 4.83 U/L) and the control group (all P < 0.05).One week after chemotherapy in the experimental group, the groups treated with the FOLFOX regimen or the XELOX regimen showed no statistical differences in the liver biochemical indexes.One week after chemotherapy in the control group, though, the groups treated with the FOLFOX regimen showed significantly lower AST (26.24 ± 3.50 U/L vs. 29.80 ± 6.57 U/L, t=-2.431, P < 0.05),but the residual liver biochemical indexes were not significantly different.In the experimental group, the FOLFOX group showed significantly lower ALP (53.44 ± 2.47 U/L vs. 56.58 ± 6.70 U/L, t =-2.201, P < 0.05), AST (6.48 ± 3.15U/L vs. 9.88 ± 4.57 U/L, t =-5.223, P < 0.05), but the residual liver biochemical index was not significantly different. Magnesium isoglycyrrhizinate is an effective drug for the prevention of drug-induced liver damage after initial chemotherapy in patients with early stage gastrointestinal cancer.

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism

PubMed Central

Leitch, Andrea; Higgs, Martin R.; Bicknell, Louise S.; Yigit, Gökhan; Blackford, Andrew N.; Zlatanou, Anastasia; Mackenzie, Karen J.; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A. M.; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H.; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B.; Nürnberg, Peter; Jackson, Stephen P.; Hurles, Matthew E.; Wollnik, Bernd; Stewart, Grant S.; Jackson, Andrew P.

2015-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism/Seckel syndrome. We establish that TRAIP relocalizes to sites of DNA damage where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to UV irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a novel component of the DNA damage response to replication-blocking DNA lesions. PMID:26595769

Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

PubMed

Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi

2016-01-01

Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects. Copyright © 2016 Elsevier B.V. All rights reserved.

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) I: Endogenous Angiotensin Converting Enzyme (ACE) Inhibition

PubMed Central

Fagyas, Miklós; Úri, Katalin; Siket, Ivetta M.; Daragó, Andrea; Boczán, Judit; Bányai, Emese; Édes, István; Papp, Zoltán; Tóth, Attila

2014-01-01

Angiotensin-converting enzyme (ACE) inhibitors represent the fifth most often prescribed drugs. ACE inhibitors decrease 5-year mortality by approximately one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors, which endogenous inhibitory effects are much less characterized than that for the clinically administered ACE inhibitors. Here we aimed to investigate this endogenous ACE inhibition in human sera. It was hypothesized that ACE activity is masked by an endogenous inhibitor, which dissociates from the ACE when its concentration decreases upon dilution. ACE activity was measured by FAPGG hydrolysis first. The specific (dilution corrected) enzyme activities significantly increased by dilution of human serum samples (23.2±0.7 U/L at 4-fold dilution, 51.4±0.3 U/L at 32-fold dilution, n = 3, p = 0.001), suggesting the presence of an endogenous inhibitor. In accordance, specific enzyme activities did not changed by dilution when purified renal ACE was used, where no endogenous inhibitor was present (655±145 U/L, 605±42 U/L, n = 3, p = 0.715, respectively). FAPGG conversion strongly correlated with angiotensin I conversion suggesting that this feature is not related to the artificial substrate. Serum samples were ultra-filtered to separate ACE (MW: 180 kDa) and the hypothesized inhibitor. Filtering through 50 kDa filters was without effect, while filtering through 100 kDa filters eliminated the inhibiting factor (ACE activity after <100 kDa filtering: 56.4±2.4 U/L, n = 4, control: 26.4±0.7 U/L, n = 4, p<0.001). Lineweaver-Burk plot indicated non-competitive inhibition of ACE by this endogenous factor. The endogenous inhibitor had higher potency on the C-terminal active site than N-terminal active site of ACE. Finally, this endogenous ACE inhibition was also present in mouse, donkey, goat, bovine sera besides men (increasing of specific ACE activity from 4-fold to 32-fold dilution: 2.8-fold, 1.7-fold, 1.5-fold, 1.8-fold, 2.6-fold, respectively). We report here the existence of an evolutionary conserved mechanism suppressing circulating ACE activity, in vivo, similarly to ACE inhibitory drugs. PMID:24691160

Differential effects of hGH and IGF-I on body proportions.

PubMed

Laron, Zvi; Silbergeld, Aviva; Kauli, Rivka

2012-07-01

The differential growth effects of hGH and IGF-I on the upper/lower (U/L) body segment in relation to height (Ht) were analyzed in 15 patients with isolated Growth hormone deficiency (IGHD,:7M, 8F) mean age 5.0 +/- 3.2 (SD) years treated with hGH; 21 patients with multiple pituitary hormone deficiency including growth hormone (MPHD: 14M, 7F) aged 10.0 +/- 3.8, treated with hGH; 9 patients with Laron Syndrome (LS) (4M,5F) aged 6.9 +/- 5.6 years treated with IGF-I; 9 boys with intrauterine growth retardation (IUGR) aged 6.3 +/- 1.25 years treated by hGH; and 22 boys with idiopathic short stature (ISS) aged 8.0 +/- 1.55 years treated by hGH. The dose of hGH was 33 microg/kg/day, that of IGF-I 180-200 microg/kg/day. the U/L body segment ratio in IGHD patients decreased from 2.3 +/- 0.7 to 1.1 +/- 0.7 (p <0.001), and the Ht SDS increased from -4.9 +/- 1.3 to 2.3 +/- 1 (p < 0.001) following treatment. In MPHD patients the U/L body segment decreased from 1.1 +/- 1.1 to -0.6 +/- 1.0 (p < 0.001), and the Ht SDS increased from -3.3 +/- 1.4 to -2.5 +/- 1.0 (p < 0.009). In the LS group the U/L body segment ratio did not change with IGF-I treatment but Ht improved from -6.1 +/- 1.3 to -4.6 +/- 1.2 (p < 0.001), The differential growth response of the children with IUGR and with ISS resembled that of the children with LS. hGH and IGF-I act differentially on the spine and limbs.

Myxedema coma associated with combination aripiprazole and sertraline therapy.

PubMed

Church, Chelsea O; Callen, Erin C

2009-12-01

To describe a case of myxedema coma (MC) associated with combination aripiprazole and sertraline therapy. A 41-year-old male presented to the emergency department with confusion, right-sided numbness and tingling, slurred speech, dizziness, and facial edema. His blood pressure was 160/113 mm Hg, with a pulse of 56 beats/min and temperature of 35.4 degrees C. Initial abnormal laboratory values included creatine kinase (CK) 439 U/L; serum creatinine 1.6 mg/dL; aspartate aminotransferase 85 U/L; and alanine aminotransferase 35 U/L. Repeat cardiac markers revealed an elevated CK level of 3573 U/L with a CK-MB of 24 ng/mL. Thyroid function tests showed thyroid-stimulating hormone 126.4 microIU/mL and free thyroxine 0.29 ng/dL. Home medications of unknown duration were sertraline 200 mg and aripiprazole 20 mg daily. He was admitted to the intensive care unit and initially treated with intravenous levothyroxine and dexamethasone. By hospital day 4, the patient was clinically stable and discharged to home. Myxedema coma, the most significant form of hypothyroidism (HT), is a rare but potentially fatal condition. The known precipitating causes of MC were ruled out in this patient, which left his home medications as the likely cause. Cases of HT caused by certain atypical antipsychotics and antidepressants are found in the literature, but none was reported with aripiprazole therapy. There are also no reported cases of sertraline or aripiprazole inducing MC. Use of the Naranjo probability scale indicates that the combination of aripiprazole and sertraline was a probable inducer of MC in this patient. Due to the widespread use of psychotropic medications, clinicians should be reminded of the rare, yet life-threatening, occurrence of MC when treating patients, especially with combination therapies such as sertraline and aripiprazole.

Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs.

PubMed

Alvisi, Gualtiero; Ripalti, Alessandro; Ngankeu, Apollinaire; Giannandrea, Maila; Caraffi, Stefano G; Dias, Manisha M; Jans, David A

2006-10-01

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.

The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme

PubMed Central

Wang, Yi-Ping; Du, Wen-Juan; Huang, Li-Ping; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

2016-01-01

Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354–370 and that K354, R355, and K367 are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system. PMID:26913023

Alternate promoter selection within a human cytomegalovirus immediate-early and early transcription unit (UL119-115) defines true late transcripts containing open reading frames for putative viral glycoproteins.

PubMed Central

Leatham, M P; Witte, P R; Stinski, M F

1991-01-01

The human cytomegalovirus open reading frames (ORFs) UL119 through UL115 (UL119-115) are located downstream of the immediate-early 1 and 2 transcription units. The promoter upstream of UL119 is active at all times after infection and drives the synthesis of a spliced 3.1-kb mRNA. The viral mRNA initiates in UL119, contains UL119-117 and UL116, and terminates just downstream of UL115. True late transcripts that are detected only after viral DNA synthesis originate from this transcription unit. True late mRNAs of 2.1 kb, containing ORFs UL116 and UL115, and 1.2 kb, containing ORF UL115 only, are synthesized. The true late viral mRNAs are 3' coterminal with the 3.1-kb mRNA. This transcription unit is an example of late promoters nested within an immediate-early-early transcription unit. The gene products of UL119-117, UL116, and UL115 are predicted to be glycoproteins. Efficient expression of the downstream ORFs at late times after infection may be related to alternate promoter usage and downstream cap site selection. Images PMID:1717716

Inhibition of Human Cytomegalovirus DNA Polymerase by C-Terminal Peptides from the UL54 Subunit

PubMed Central

Loregian, Arianna; Rigatti, Roberto; Murphy, Mary; Schievano, Elisabetta; Palu, Giorgio; Marsden, Howard S.

2003-01-01

In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 1242 at the very C terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially α-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction. PMID:12857903

Biodegradation of Ethyl Carbamate and Urea with Lysinibacillus sphaericus MT33 in Chinese Liquor Fermentation.

PubMed

Cui, Kaixiang; Wu, Qun; Xu, Yan

2018-02-14

It is important to reduce the concentration of ethyl carbamate (EC) in fermented foods. However, controlling the formation of EC and its precursor urea is difficult in spontaneous food fermentation because urea is a natural product of nitrogen metabolism. Biodegradation is a better solution to reduce the concentration of EC. This study aimed to reduce the concentration of EC in Chinese liquor via an indigenous strain Lysinibacillus sphaericus MT33. This strain produced urethanase (940 U/L) and urease (1580 U/L) and degraded 76.52% of EC and 56.48% of urea. After inoculation in liquor fermentation, the maximal relative abundance of Lysinibacillus increased from 0.02% to 8.46%, the final EC and urea contents decreased by 41.77% and 28.15%. Moreover, the concentration of EC decreased by 63.32% in liquor. The negative correlation between abundance of Lysinibacillus and contents of EC and urea indicated the effect of L. sphaericus on EC and urea degradation.

Production of novel cell-associated tannase from newly isolated Serratia ficaria DTC.

PubMed

Belur, Prasanna D; Gopal, Mugeraya; Nirmala, K R; Basavaraj, N

2010-04-01

Five strains of tannic acid degrading bacteria were isolated and identified by phenotypic characterization. All the five isolates showed cell-associated activity, where as only three showed extracellular activity. Serratia ficaria DTC showing highest cell-associated activity (0.29 U/l) was selected for further shake flask studies. Tannase synthesis was growth associated and reached the peak in the late stationary phase of growth. Organic nitrogen sources enhanced the tannase production. Peak tannase production of 0.56 U/l was recorded in the medium having the initial pH of 6. The pH and temperature optima of the enzyme were found to be 8.9 and 35 degrees , respectively. This is the first report of cell-associated activity in case of bacterial tannase. Cell-associated tannase of Serratia ficaria DTC could be industrially important from the perspective of its activity at broad temperature and pH range, its unusually high activity at pH 8.9.

The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

PubMed

Biswas, N; Weller, S K

2001-05-18

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been thought.

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2010-03-02

... Water-Type Fire Extinguishers UL 711 Rating and Fire Testing of Fire Extinguishers UL 796 Printed-Wiring... Equipment UL 1053 Ground-Fault Sensing and Relaying Equipment UL 1054 Special-Use Switches UL 1058... Halon 1211 Recovery/Recharge Equipment UL 2111 Overheating Protection for Motors III. Temporary...

The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seongman; Chul Ahn, Byung; O'Callaghan, Dennis J.

2012-10-25

The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealedmore » that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.« less

Structural basis for sorting mechanism of p62 in selective autophagy.

PubMed

Ichimura, Yoshinobu; Kumanomidou, Taichi; Sou, Yu-shin; Mizushima, Tsunehiro; Ezaki, Junji; Ueno, Takashi; Kominami, Eiki; Yamane, Takashi; Tanaka, Keiji; Komatsu, Masaaki

2008-08-15

Impairment of autophagic degradation of the ubiquitin- and LC3-binding protein "p62" leads to the formation of cytoplasmic inclusion bodies. However, little is known about the sorting mechanism of p62 to autophagic degradation. Here we identified a motif of murine p62 consisting of 11 amino acids (Ser334-Ser344) containing conserved acidic and hydrophobic residues across species, as an LC3 recognition sequence (LRS). The crystal structure of the LC3-LRS complex at 1.56 angstroms resolution revealed interaction of Trp340 and Leu343 of p62 with different hydrophobic pockets on the ubiquitin fold of LC3. In vivo analyses demonstrated that p62 mutants lacking LC3 binding ability accumulated without entrapping into autophagosomes in the cytoplasm and subsequently formed ubiquitin-positive inclusion bodies as in autophagy-deficient cells. These results demonstrate that the intracellular level of p62 is tightly regulated by autophagy through the direct interaction of LC3 with p62 and reveal that selective turnover of p62 via autophagy controls inclusion body formation.

MK-2206, an AKT Inhibitor, Promotes Caspase-Independent Cell Death and Inhibits Leiomyoma Growth

PubMed Central

Sefton, Elizabeth C.; Qiang, Wenan; Serna, Vanida; Kurita, Takeshi; Wei, Jian-Jun; Chakravarti, Debabrata

2013-01-01

Uterine leiomyomas (ULs), benign tumors of the myometrium, are the number one indication for hysterectomies in the United States due to a lack of an effective alternative therapy. ULs show activation of the pro-survival AKT pathway compared with normal myometrium; however, substantial data directly linking AKT to UL cell survival are lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable target for inhibiting UL growth. We used the investigational AKT inhibitor MK-2206, currently in phase II trials, on cultured primary human UL and myometrial cells, immortalized leiomyoma cells, and in leiomyoma grafts grown under the kidney capsule in mice. MK-2206 inhibited AKT and PRAS40 phosphorylation but did not regulate serum- and glucocorticoid-induced kinase and ERK1/2, demonstrating its specificity for AKT. MK-2206 reduced UL cell viability and decreased UL tumor volumes. UL cells exhibited disruption of mitochondrial structures and underwent cell death that was independent of caspases. Additionally, mammalian target of rapamycin and p70S6K phosphorylation were reduced, indicating that mammalian target of rapamycin complex 1 signaling was compromised by AKT inhibition in UL cells. MK-2206 also induced autophagy in UL cells. Pretreatment of primary UL cells with 3-methyladenine enhanced MK-2206-mediated UL cell death, whereas knockdown of ATG5 and/or ATG7 did not significantly influence UL cell viability in the presence of MK-2206. Our data provide molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option to consider for the treatment of ULs. PMID:24002033

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

PubMed Central

Chen, Yan; Carrington-Lawrence, Stacy D.; Bai, Ping; Weller, Sandra K.

2005-01-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface. PMID:15994803

Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

PubMed

Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

2005-07-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

PubMed

Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

2018-05-01

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

Interaction and interdependent packaging of tegument protein UL11 and glycoprotein e of herpes simplex virus.

PubMed

Han, Jun; Chadha, Pooja; Meckes, David G; Baird, Nicholas L; Wills, John W

2011-09-01

The UL11 tegument protein of herpes simplex virus plays a critical role in the secondary envelopment; however, the mechanistic details remain elusive. Here, we report a new function of UL11 in the budding process in which it directs efficient acquisition of glycoprotein E (gE) via a direct interaction. In vitro binding assays showed that the interaction required only the first 28, membrane-proximal residues of the cytoplasmic tail of gE, and the C-terminal 26 residues of UL11. A second, weaker binding site was also found in the N-terminal half of UL11. The significance of the gE-UL11 interaction was subsequently investigated with viral deletion mutants. In the absence of the gE tail, virion packaging of UL11, but not other tegument proteins such as VP22 and VP16, was reduced by at least 80%. Reciprocally, wild-type gE packaging was also drastically reduced by about 87% in the absence of UL11, and this defect could be rescued in trans by expressing U(L)11 at the U(L)35 locus. Surprisingly, a mutant that lacks the C-terminal gE-binding site of UL11 packaged nearly normal amounts of gE despite its strong interaction with the gE tail in vitro, indicating that the interaction with the UL11 N terminus may be important. Mutagenesis studies of the UL11 N terminus revealed that the association of UL11 with membrane was not required for this function. In contrast, the UL11 acidic cluster motif was found to be critical for gE packaging and was not replaceable with foreign acidic clusters. Together, these results highlight an important role of UL11 in the acquisition of glycoprotein-enriched lipid bilayers, and the findings may also have important implications for the role of UL11 in gE-mediated cell-to-cell spread.

Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting

PubMed Central

Müller, Oliver; Ivanova, Lyudmila; Bialy, Dagmara; Pohlmann, Anja; Binz, Anne; Hegemann, Maike; Viejo-Borbolla, Abel; Rosenhahn, Bodo; Bauerfeind, Rudolf; Sodeik, Beate

2017-01-01

Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells. PMID:29284065

The Chromatin Remodeling Factor SMARCB1 Forms a Complex with Human Cytomegalovirus Proteins UL114 and UL44

PubMed Central

Ranneberg-Nilsen, Toril; Rollag, Halvor; Slettebakk, Ragnhild; Backe, Paul Hoff; Olsen, Øyvind; Luna, Luisa; Bjørås, Magnar

2012-01-01

Background Human cytomegalovirus (HCMV) uracil DNA glycosylase, UL114, is required for efficient viral DNA replication. Presumably, UL114 functions as a structural partner to other factors of the DNA-replication machinery and not as a DNA repair protein. UL114 binds UL44 (HCMV processivity factor) and UL54 (HCMV-DNA-polymerase). In the present study we have searched for cellular partners of UL114. Methodology/Principal Findings In a yeast two-hybrid screen SMARCB1, a factor of the SWI/SNF chromatin remodeling complex, was found to be an interacting partner of UL114. This interaction was confirmed in vitro by co-immunoprecipitation and pull-down. Immunofluorescence microscopy revealed that SMARCB1 along with BRG-1, BAF170 and BAF155, which are the core SWI/SNF components required for efficient chromatin remodeling, were present in virus replication foci 24–48 hours post infection (hpi). Furthermore a direct interaction was also demonstrated for SMARCB1 and UL44. Conclusions/Significance The core SWI/SNF factors required for efficient chromatin remodeling are present in the HCMV replication foci throughout infection. The proteins UL44 and UL114 interact with SMARCB1 and may participate in the recruitment of the SWI/SNF complex to the chromatinized virus DNA. Thus, the presence of the SWI/SNF chromatin remodeling complex in replication foci and its association with UL114 and with UL44 might imply its involvement in different DNA transactions. PMID:22479537

Involvement of UL24 in herpes-simplex-virus-1-induced dispersal of nucleolin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lymberopoulos, Maria H.; Pearson, Angela

2007-07-05

UL24 of herpes simplex virus 1 is important for efficient viral replication, but its function is unknown. We generated a recombinant virus, vHA-UL24, encoding UL24 with an N-terminal hemagglutinin tag. By indirect immunofluorescence at 9 h post-infection (hpi), we detected HA-UL24 in nuclear foci and in cytoplasmic speckles. HA-UL24 partially co-localized with nucleolin, but not with ICP8 or coilin, markers for nucleoli, viral replication compartments, and Cajal bodies respectively. HA-UL24 staining was often juxtaposed to that of another nucleolar protein, fibrillarin. Analysis of HSV-1-induced nucleolar modifications revealed that by 18 hpi, nucleolin staining had dispersed, and fibrillarin staining went frommore » clusters of small spots to a few separate but prominent spots. Fibrillarin redistribution appeared to be independent of UL24. In contrast, cells infected with a UL24-deficient virus retained foci of nucleolin staining. Our results demonstrate involvement of UL24 in dispersal of nucleolin during infection.« less

Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1.

PubMed

Zhu, Hongwei; Li, Huixin; Han, Zongxi; Shao, Yuhao; Wang, Yu; Kong, Xiangang

2011-04-06

In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV), the causative agent of duck viral enteritis (DVE), the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. DEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (h p. i.) and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s) in the cytoplasm in the absence of any other viral protein. DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15 are very similar to those of alphaherpesviruses and most similar to the genus Mardivirus. The UL15 and/or UL15.5 accumulate(s) in the cytoplasm during early times post-infection and then are translocated to the nucleus at late times.

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Characterization of the duck enteritis virus UL55 protein

PubMed Central

2011-01-01

Background Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function. Results The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells. Conclusions In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene. PMID:21609474

TRIM56 Is an Essential Component of the TLR3 Antiviral Signaling Pathway*

PubMed Central

Shen, Yang; Li, Nan L.; Wang, Jie; Liu, Baoming; Lester, Sandra; Li, Kui

2012-01-01

Members of the tripartite motif (TRIM) proteins are being recognized as important regulators of host innate immunity. However, specific TRIMs that contribute to TLR3-mediated antiviral defense have not been identified. We show here that TRIM56 is a positive regulator of TLR3 signaling. Overexpression of TRIM56 substantially potentiated extracellular dsRNA-induced expression of interferon (IFN)-β and interferon-stimulated genes (ISGs), while knockdown of TRIM56 greatly impaired activation of IRF3, induction of IFN-β and ISGs, and establishment of an antiviral state by TLR3 ligand and severely compromised TLR3-mediated chemokine induction following infection by hepatitis C virus. The ability to promote TLR3 signaling was independent of the E3 ubiquitin ligase activity of TRIM56. Rather, it correlated with a physical interaction between TRIM56 and TRIF. Deletion of the C-terminal portion of TRIM56 abrogated the TRIM56-TRIF interaction as well as the augmentation of TLR3-mediated IFN response. Together, our data demonstrate TRIM56 is an essential component of the TLR3 antiviral signaling pathway and reveal a novel role for TRIM56 in innate antiviral immunity. PMID:22948160

The UL21 Tegument Protein of Herpes Simplex Virus 1 Is Differentially Required for the Syncytial Phenotype

PubMed Central

Starkey, Jason; Mellinger, Erica; Zhang, Dan; Chadha, Pooja; Carmichael, Jillian

2017-01-01

ABSTRACT The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20Syn and UL24Syn. To test whether UL21 is needed only for the A855V mutant, additional gBSyn derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gBSyn phenotype. This is the first example of a differential requirement for a viral protein across the four syn loci. IMPORTANCE UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations. PMID:28794039

The conserved N-terminal domain of herpes simplex virus 1 UL24 protein is sufficient to induce the spatial redistribution of nucleolin.

PubMed

Bertrand, Luc; Pearson, Angela

2008-05-01

UL24 is widely conserved among herpesviruses but its function during infection is poorly understood. Previously, we discovered a genetic link between UL24 and the herpes simplex virus 1-induced dispersal of the nucleolar protein nucleolin. Here, we report that in the absence of viral infection, transiently expressed UL24 accumulated in both the nucleus and the Golgi apparatus. In the majority of transfected cells, nuclear staining for UL24 was diffuse, but a minor staining pattern, whereby UL24 was present in nuclear foci corresponding to nucleoli, was also observed. Expression of UL24 correlated with the dispersal of nucleolin. This dispersal did not appear to be a consequence of a general disaggregation of nucleoli, as foci of fibrillarin staining persisted in cells expressing UL24. The conserved N-terminal region of UL24 was sufficient to cause this change in subcellular distribution of nucleolin. Interestingly, a bipartite nuclear localization signal predicted within the C terminus of UL24 was dispensable for nuclear localization. None of the five individual UL24 homology domains was required for nuclear or Golgi localization, but deletion of these domains resulted in the loss of nucleolin-dispersal activity. We determined that a nucleolar-targeting signal was contained within the first 60 aa of UL24. Our results show that the conserved N-terminal domain of UL24 is sufficient to specifically induce dispersal of nucleolin in the absence of other viral proteins or virus-induced cellular modifications. These results suggest that UL24 directly targets cellular factors that affect the composition of nucleoli.

Inactivation of retinoblastoma protein does not overcome the requirement for human cytomegalovirus UL97 in lamina disruption and nuclear egress.

PubMed

Reim, Natalia I; Kamil, Jeremy P; Wang, Depeng; Lin, Alison; Sharma, Mayuri; Ericsson, Maria; Pesola, Jean M; Golan, David E; Coen, Donald M

2013-05-01

Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

The Human Cytomegalovirus-Specific UL1 Gene Encodes a Late-Phase Glycoprotein Incorporated in the Virion Envelope

PubMed Central

Shikhagaie, Medya; Mercé-Maldonado, Eva; Isern, Elena; Muntasell, Aura; Albà, M. Mar; López-Botet, Miguel; Hengel, Hartmut

2012-01-01

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism. PMID:22345456

Resistance to maribavir is associated with the exclusion of pUL27 from nucleoli during human cytomegalovirus infection

PubMed Central

Hakki, Morgan; Drummond, Coyne; Houser, Benjamin; Marousek, Gail; Chou, Sunwen

2011-01-01

Select mutations in the human cytomegalovirus (HCMV) gene UL27 confer low-grade resistance to the HCMV UL97 kinase inhibitor maribavir (MBV). It has been reported that the 608-amino acid UL27 gene product (pUL27) normally localizes to cell nuclei and nucleoli, whereas its truncation at codon 415, as found in a MBV-resistant mutant, results in cytoplasmic localization. We now show that in the context of full-length pUL27, diverse single amino acid substitutions associated with MBV resistance result in loss of its nucleolar localization when visualized after transient transfection, whereas substitutions representing normal interstrain polymorphism had no such effect. The same differences in localization were observed during a complete infection cycle with recombinant HCMV strains over-expressing full-length fluorescent pUL27 variants. Nested UL27 C-terminal truncation expression plasmids showed that amino acids 596–599 were required for the nucleolar localization of pUL27. These results indicate that the loss of a nucleolar function of pUL27 may contribute to MBV resistance, and that the nucleolar localization of pUL27 during HCMV infection depends not only on a carboxy-terminal domain but also on a property of pUL27 that is affected by MBV-resistant mutations, such as an interaction with component(s) of the nucleolus. PMID:21906628

Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26).

PubMed

Apcarian, Arin; Cunningham, Anthony L; Diefenbach, Russell J

2010-11-01

In this study, fragments of the small capsid protein pUL35 (VP26) from herpes simplex virus type 1 (HSV-1) were generated to identify binding domains for a number of known ligands. Analysis of the binding of dynein light chain subunits, DYNLT1 and DYNLT3, as well the HSV-1 structural proteins pUL19 (VP5) and pUL37 was then undertaken using the LexA yeast two-hybrid assay. The N-terminal half of pUL35, in particular residues 30-43, was identified as a common region for the binding of DYNLT1 and DYNLT3. Additional distinct regions in the C terminus of pUL35 also contribute to the binding of DYNLT1 and DYNLT3. In contrast, only the C-terminal half of pUL35 was found to mediate the binding of pUL19 and pUL37 through distinct regions. The relevance of this information to the role of pUL35 in viral transport and assembly is discussed.

Rhabdomyolysis and acute kidney injury in the injured war fighter.

PubMed

Elterman, Joel; Zonies, David; Stewart, Ian; Fang, Raymond; Schreiber, Martin

2015-10-01

Rhabdomyolysis is a recognized complication of traumatic injury. The correlation of an elevated creatine kinase (CK) level and the development of acute kidney injury (AKI) has been studied in the civilian population. We sought to review the prevalence of rhabdomyolysis in injured war fighters and determine if peak CK levels correlate with AKI. This is a retrospective cohort study of patients admitted at a US military treatment facility from January to November 2010. Inclusion criteria were active duty patients transported after explosive, penetrating, or blunt injury. Patients with burns or non-trauma-related admissions were excluded. Rhabdomyolysis was defined as a CK level greater than 5,000 U/L. AKI was defined using the Kidney Disease: Improving Global Outcomes classification. Mann-Whitney U-tests were used to determine the significance for continuous data. Correlations were determined using Spearman's ρ. Significance was set at p < 0.05. Of the 318 patients included in our analysis, 310 (98%) were male, and the median age was 24 years (21-28 years). Blast was the predominant mechanism of injury (71%), with a median Injury Severity Score (ISS) of 22 (16-29). Rhabdomyolysis developed in 79 patients (24.8%). The median peak CK for all patients was 4,178 U/L and ranged from 208 U/L to 120,000 U/L. Stage 1, 2, and 3 AKI developed in 56 (17.6%), 3 (0.9%), and 7 (2.2%) patients, respectively. There was a weak but statistically significant correlation between peak CK and AKI (r = 0.26, p < 0.05). Elevated peak CK levels in the injured war fighter are weakly associated with the development of AKI but are not predictive. The development of clinical practice guidelines would help standardize treatment for rhabdomyolysis in combat casualties and would allow for standardized comparisons in future work. Epidemiologic/prognostic study, level III.

[Experience in the treatment of some complications of portal hypertension in alcoholic liver cirrhosis].

PubMed

Savić, Zeljka; Vracarić, Vladimir; Hadnadjev, Ljiljana; Petrović, Zora; Damjanov, Dragomir

2011-11-01

Portal hypertension (PH) is hemodynamical abnormality associated with the most serious complications of alcoholic liver cirrhosis (ALC): ascites, varices and variceal bleeding. The aim of this study was to determine characteristics of portal hypertension, especially of upper gastrointestinal bleedings in patients with alcoholic liver cirrhosis (ALC). A total of 237 patients with ALC were observed in a 3-year period. A total of 161 patients (68%) were hospitalized because of PH elements: 86 (36.3%) had upper gastrointestinal bleeding, 75 (31.7%) were decompensated. Only 76 (32%) of the patients had icterus. General mortality was 85 (36%). According to the source of bleeding, 61 (71%) patients bled from varices, and 25 (29%) from other sources with existing varices but non-incriminated for bleeding in 16 (64%) of those patients. Active bleeding or stigmata of recent bleeding were found in 63 (73%) cases. Endoscopic treatment of variceal bleeding along with octreotide applied in 20 (32.78%) patients, just octreotide in 32 (52.46%), and octreotid plus balloon tamponade in 9 (14.75%). According to Child-Pugh classification, 25 (29%) of the bleeding patients were in class A, score 5.4; 43 (50%) in class B, score 7.8; and 18 (21%) in class C, score 10.9. Average hemoglobin level was 93 g/L, hematocrit 0.27, AST 71.52 U/L (normal to 37 U/L), ALT 37.74 U/L (normal to 40 U/L). Until this bleeding episode, 41 (47%) of the patients already bled. In the decompensated patients 3 (4%) were in Child Pugh class A, score 6; 42 (56%) in class B, score 8.3; and 30 (40%) in class C, score 10.6. Until this decompensation episode, 7 (9.3%) patients already bled. Patients with ALC need early detection of varices, primary and secondary profilaxis of variceal bleeding and adequate therapy of ascites. When bleeding occurs, patients need urgent upper endoscopy and intensive treatment.

The C Terminus of the Large Tegument Protein pUL36 Contains Multiple Capsid Binding Sites That Function Differently during Assembly and Cell Entry of Herpes Simplex Virus

PubMed Central

Schipke, Julia; Pohlmann, Anja; Diestel, Randi; Binz, Anne; Rudolph, Kathrin; Nagel, Claus-Henning; Bauerfeind, Rudolf

2012-01-01

The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating. PMID:22258258

Localization of herpes simplex virus type 1 UL37 in the Golgi complex requires UL36 but not capsid structures.

PubMed

Desai, Prashant; Sexton, Gerry L; Huang, Eugene; Person, Stanley

2008-11-01

The herpes simplex virus type 1 (HSV-1) UL37 gene encodes a 120-kDa polypeptide which resides in the tegument structure of the virion and is important for morphogenesis. The goal of this study was to use green fluorescent protein (GFP) to follow the fate of UL37 within cells during the normal course of virus replication. GFP was inserted in frame at the C terminus of UL37 to generate a fluorescent-protein-tagged UL37 polypeptide. A virus designated K37eGFP, which replicated normally on Vero cells, was isolated and was shown to express the fusion polypeptide. When cells infected with this virus were examined by confocal microscopy, the fluorescence was observed to be predominantly cytoplasmic. As the infection progressed, fluorescence began to accumulate in a juxtanuclear structure. Mannosidase II and giantin were observed to colocalize with UL37eGFP at these structures, as judged by immunofluorescence assays. Therefore, UL37 traffics to the Golgi complex during infection. A VP26mRFP marker (red fluorescent protein fused to VP26) was recombined into K37eGFP, and when cells infected with this "dual-color" virus were examined, colocalization of the red (capsid) and green (UL37) fluorescence in the Golgi structure was observed. Null mutations in VP5 (DeltaVP5), which abolished capsid assembly, and in UL36 (Delta36) were recombined into the K37eGFP virus genome. In cells infected with K37eGFP/DeltaVP5, localization of UL37eGFP to the Golgi complex was similar to that for the parental virus (K37eGFP), indicating that trafficking of UL37eGFP to the Golgi complex did not require capsid structures. Confocal analysis of cells infected with K37eGFP/Delta36 showed that, in the absence of UL36, accumulation of UL37eGFP at the Golgi complex was not evident. This indicates an interaction between these two proteins that is important for localization of UL37 in the Golgi complex and thus possibly for cytoplasmic envelopment of the capsid. This is the first demonstration of a functional role for UL36:UL37 interaction in HSV-1-infected cells.

Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

PubMed Central

Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

2016-01-01

The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

Cyclin-dependent Kinases Phosphorylate the Cytomegalovirus RNA Export Protein pUL69 and Modulate Its Nuclear Localization and Activity*S⃞

PubMed Central

Rechter, Sabine; Scott, Gillian M.; Eickhoff, Jan; Zielke, Katrin; Auerochs, Sabrina; Müller, Regina; Stamminger, Thomas; Rawlinson, William D.; Marschall, Manfred

2009-01-01

Replication of human cytomegalovirus (HCMV) is subject to regulation by cellular protein kinases. Recently, we and others reported that inhibition of cyclin-dependent protein kinases (CDKs) or the viral CDK ortholog pUL97 can induce intranuclear speckled aggregation of the viral mRNA export factor, pUL69. Here we provide the first evidence for a direct regulatory role of CDKs on pUL69 functionality. Although replication of all HCMV strains was dependent on CDK activity, we found strain-specific differences in the amount of CDK inhibitor-induced pUL69 aggregate formation. In all cases analyzed, the inhibitor-induced pUL69 aggregates were clearly localized within viral replication centers but not subnuclear splicing, pore complex, or aggresome structures. The CDK9 and cyclin T1 proteins colocalized with these pUL69 aggregates, whereas other CDKs behaved differently. Phosphorylation analyses in vivo and in vitro demonstrated pUL69 was strongly phosphorylated in HCMV-infected fibroblasts and that CDKs represent a novel class of pUL69-phosphorylating kinases. Moreover, the analysis of CDK inhibitors in a pUL69-dependent nuclear mRNA export assay provided evidence for functional impairment of pUL69 under suppression of CDK activity. Thus, our data underline the crucial importance of CDKs for HCMV replication, and indicate a direct impact of CDK9-cyclin T1 on the nuclear localization and activity of the viral regulator pUL69. PMID:19179338

Human cytomegalovirus UL76 induces chromosome aberrations

PubMed Central

2009-01-01

Background Human cytomegalovirus (HCMV) is known to induce chromosome aberrations in infected cells, which can lead to congenital abnormalities in infected fetuses. HCMV UL76 belongs to a conserved protein family from herpesviruses. Some reported roles among UL76 family members include involvement in virulence determination, lytic replication, reactivation of latent virus, modulation of gene expression, induction of apoptosis, and perturbation of cell cycle progression, as well as potential nuclease activity. Previously, we have shown that stable expression of UL76 inhibits HCMV replication in glioblastoma cells. Methods To examine chromosomal integrity and the DNA damage signal γ-H2AX in cells constitutively expressing UL76, immunofluorescent cell staining and Western blotting were performed. The comet assay was employed to assess DNA breaks in cells transiently expressing UL76. Results We report that stably transfected cells expressing UL76 developed chromosome aberrations including micronuclei and misaligned chromosomes, lagging and bridging. In mitotic cells expressing UL76, aberrant spindles were increased compared to control cells. However, cells with supernumerary centrosomes were marginally increased in UL76-expressing cells relative to control cells. We further demonstrated that UL76-expressing cells activated the DNA damage signal γ-H2AX and caused foci formation in nuclei. In addition, the number of cells with DNA breaks increased in proportion to UL76 protein levels. Conclusion Our findings suggest that the virus-associated protein UL76 induces DNA damage and the accumulation of chromosome aberrations. PMID:19930723

Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli.

PubMed

Bender, Brian J; Coen, Donald M; Strang, Blair L

2014-10-01

Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. Importance: The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular proteins required for HCMV genome replication and replicating viral DNA. We found that UL84 localizes with viral proteins, viral DNA, and the cellular nucleolar protein nucleolin in the subnuclear replication compartments in which viral DNA replication occurs. Unexpectedly, we also found localization of UL84 with nucleolin in nucleoli and showed that the presence of nucleolin is involved in localization of UL84 to the nucleus. These results add to previous work showing the importance of nucleolin in replication compartment architecture and viral DNA synthesis and are relevant to understanding UL84 function. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

75 FR 44289 - MET Laboratories, Inc.; Application for Expansion of Recognition

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-28

... State Controls for Appliances UL 412 Refrigeration Unit Coolers UL 458* Power Converters/Inverters and Power Converter/Inverter Systems for Land Vehicles and Marine Crafts UL 466 Electric Scales UL 561 Floor...

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

Herpes Simplex Virus Processivity Factor UL42 Imparts Increased DNA-Binding Specificity to the Viral DNA Polymerase and Decreased Dissociation from Primer-Template without Reducing the Elongation Rate

PubMed Central

Weisshart, Klaus; Chow, Connie S.; Coen, Donald M.

1999-01-01

Herpes simplex virus DNA polymerase consists of a catalytic subunit, Pol, and a processivity subunit, UL42, that, unlike other established processivity factors, binds DNA directly. We used gel retardation and filter-binding assays to investigate how UL42 affects the polymerase-DNA interaction. The Pol/UL42 heterodimer bound more tightly to DNA in a primer-template configuration than to single-stranded DNA (ssDNA), while Pol alone bound more tightly to ssDNA than to DNA in a primer-template configuration. The affinity of Pol/UL42 for ssDNA was reduced severalfold relative to that of Pol, while the affinity of Pol/UL42 for primer-template DNA was increased ∼15-fold relative to that of Pol. The affinity of Pol/UL42 for circular double-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, but the affinity of Pol/UL42 for short primer-templates was increased modestly relative to that of UL42. Pol/UL42 associated with primer-template DNA ∼2-fold faster than did Pol and dissociated ∼10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar Kd. Despite such stable binding, rapid-quench analysis revealed that the rates of elongation of Pol/UL42 and Pol were essentially the same, ∼30 nucleotides/s. Taken together, these studies indicate that (i) Pol/UL42 is more likely than its subunits to associate with DNA in a primer-template configuration rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduces the rate of dissociation from primer-template DNA but not the rate of elongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented. PMID:9847307

Elimination of mitochondrial DNA is not required for herpes simplex virus 1 replication.

PubMed

Duguay, Brett A; Saffran, Holly A; Ponomarev, Alina; Duley, Shayla A; Eaton, Heather E; Smiley, James R

2014-03-01

Infection with herpes simplex virus type 1 (HSV-1) results in the rapid elimination of mitochondrial DNA (mtDNA) from host cells. It is known that a mitochondrial isoform of the viral alkaline nuclease (UL12) called UL12.5 triggers this process. However, very little is known about the impact of mtDNA depletion on viral replication or the biology of HSV-1 infections. These questions have been difficult to address because UL12.5 and UL12 are encoded by overlapping transcripts that share the same open reading frame. As a result, mutations that alter UL12.5 also affect UL12, and UL12 null mutations severely impair viral growth by interfering with the intranuclear processing of progeny viral genomes. Therefore, to specifically assess the impact of mtDNA depletion on viral replication, it is necessary to eliminate the activity of UL12.5 while preserving the nuclear functions of UL12. Previous work has shown that the human cytomegalovirus alkaline nuclease UL98 can functionally substitute for UL12 during HSV-1 replication. We found that UL98 is unable to deplete mtDNA in transfected cells and therefore generated an HSV-1 variant in which UL98 coding sequences replace the UL12/UL12.5 open reading frame. The resulting virus was severely impaired in its ability to trigger mtDNA loss but reached titers comparable to those of wild-type HSV-1 in one-step and multistep growth experiments. Together, these observations demonstrate that the elimination of mtDNA is not required for HSV-1 replication in cell culture. Herpes simplex virus types 1 and 2 destroy the DNA of host cell mitochondria, the powerhouses of cells. Epstein-Barr virus, a distantly related herpesvirus, has a similar effect, indicating that mitochondrial DNA destruction is under positive selection and thus confers a benefit to the virus. The present work shows that mitochondrial DNA destruction is not required for efficient replication of herpes simplex virus type 1 in cultured Vero kidney epithelial cells, suggesting that this activity likely benefits the virus in other cell types or in the intact human host.

Herpes simplex virus 2 UL13 protein kinase disrupts nuclear lamins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cano-Monreal, Gina L.; Wylie, Kristine M.; Cao, Feng

2009-09-15

Herpesviruses must cross the inner nuclear membrane and underlying lamina to exit the nucleus. HSV-1 US3 and PKC can phosphorylate lamins and induce their dispersion but do not elicit all of the phosphorylated lamin species produced during infection. UL13 is a serine threonine protein kinase conserved among many herpesviruses. HSV-1 UL13 phosphorylates US3 and thereby controls UL31 and UL34 nuclear rim localization, indicating a role in nuclear egress. Here, we report that HSV-2 UL13 alone induced conformational changes in lamins A and C and redistributed lamin B1 from the nuclear rim to intranuclear granular structures. HSV-2 UL13 directly phosphorylated laminsmore » A, C, and B1 in vitro, and the lamin A1 tail domain. HSV-2 infection recapitulated the lamin alterations seen upon expression of UL13 alone, and other alterations were also observed, indicating that additional viral and/or cellular proteins cooperate with UL13 to alter lamins during HSV-2 infection to allow nuclear egress.« less

Application of Subspace Detection to the 6 November 2011 M5.6 Prague, Oklahoma Aftershock Sequence

NASA Astrophysics Data System (ADS)

McMahon, N. D.; Benz, H.; Johnson, C. E.; Aster, R. C.; McNamara, D. E.

2015-12-01

Subspace detection is a powerful tool for the identification of small seismic events. Subspace detectors improve upon single-event matched filtering techniques by using multiple orthogonal waveform templates whose linear combinations characterize a range of observed signals from previously identified earthquakes. Subspace detectors running on multiple stations can significantly increasing the number of locatable events, lowering the catalog's magnitude of completeness and thus providing extraordinary detail on the kinematics of the aftershock process. The 6 November 2011 M5.6 earthquake near Prague, Oklahoma is the largest earthquake instrumentally recorded in Oklahoma history and the largest earthquake resultant from deep wastewater injection. A M4.8 foreshock on 5 November 2011 and the M5.6 mainshock triggered tens of thousands of detectable aftershocks along a 20 km splay of the Wilzetta Fault Zone known as the Meeker-Prague fault. In response to this unprecedented earthquake, 21 temporary seismic stations were deployed surrounding the seismic activity. We utilized a catalog of 767 previously located aftershocks to construct subspace detectors for the 21 temporary and 10 closest permanent seismic stations. Subspace detection identified more than 500,000 new arrival-time observations, which associated into more than 20,000 locatable earthquakes. The associated earthquakes were relocated using the Bayesloc multiple-event locator, resulting in ~7,000 earthquakes with hypocentral uncertainties of less than 500 m. The relocated seismicity provides unique insight into the spatio-temporal evolution of the aftershock sequence along the Wilzetta Fault Zone and its associated structures. We find that the crystalline basement and overlying sedimentary Arbuckle formation accommodate the majority of aftershocks. While we observe aftershocks along the entire 20 km length of the Meeker-Prague fault, the vast majority of earthquakes were confined to a 9 km wide by 9 km deep surface striking N54°E and dipping 83° to the northwest near the junction of the splay with the main Wilzetta fault structure. Relocated seismicity shows off-fault stress-related interaction to distances of 10 km or more from the mainshock, including clustered seismicity to the northwest and southeast of the mainshock.

75 FR 47674 - In the Matter of the Designation of Harakat-ul Jihad Islami, Also Known as HUJI, Also Known as...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-06

... DEPARTMENT OF STATE [Public Notice 7102] In the Matter of the Designation of Harakat-ul Jihad Islami, Also Known as HUJI, Also Known as Movement of Islamic Holy War, Also Known as Harkat-ul-Jihad-al... Islamic Holy War, also known as Harkat-ul-Jihad-al Islami, also known as Harkat-al-Jihad-ul Islami, also...

75 FR 47674 - In the Matter of the Designation of Harakat-ul Jihad Islami, Also Known as HUJI, Also Known as...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-06

... DEPARTMENT OF STATE [Public Notice 7101] In the Matter of the Designation of Harakat-ul Jihad Islami, Also Known as HUJI, Also Known as Movement of Islamic Holy War, Also Known as Harkat-ul-Jihad-al... Movement of Islamic Holy War, also known as Harkat-ul-Jihad-al Islami, also known as Harkat-al-Jihad-ul...

The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela, E-mail: angela.pearson@iaf.inrs.ca

2013-09-15

Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs.more » Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.« less

Vitamin E has a beneficial effect on nonalcoholic fatty liver disease: a meta-analysis of randomized controlled trials.

PubMed

Sato, Ken; Gosho, Masahiko; Yamamoto, Takaya; Kobayashi, Yuji; Ishii, Norimitsu; Ohashi, Tomohiko; Nakade, Yukiomi; Ito, Kiyoaki; Fukuzawa, Yoshitaka; Yoneda, Masashi

2015-01-01

Vitamin E is often used in the treatment of nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH); however, the magnitude of treatment response associated with vitamin E in improving liver function and histology in NAFLD/NASH has not, to our knowledge, been quantified systematically. Thus, we conducted a meta-analysis of randomized controlled trials (RCTs) using vitamin E in the treatment of NAFLD/NASH. PubMed, Medline, and Cochrane Library Full Text Database, and Japan Medical-Literature Database (Igaku Chuo Zasshi) were searched until March 2014, and five RCTs were identified for meta-analysis. According to a random effect model analysis of the five studies, vitamin E significantly reduced aspartate transaminase (AST) by -19.43 U/L, alanine aminotransferase (ALT) by -28.91 U/L, alkaline phosphatase (ALP) by -10.39 U/L, steatosis by -0.54 U/L, inflammation by -0.20 U/L, and hepatocellular ballooning by -0.34 U/L compared with the control group. Vitamin E treatment with NASH adult patients showed obvious reductions in not only AST of -13.91 U/L, ALT by -22.44 U/L, steatosis of -0.67 U/L, inflammation of -0.20 U/L, but also fibrosis of -0.30 U/L compared to the control treatment. Vitamin E significantly improved liver function and histologic changes in patients with NAFLD/NASH. Copyright © 2015 Elsevier Inc. All rights reserved.

UL74 of Human Cytomegalovirus Contributes to Virus Release by Promoting Secondary Envelopment of Virions▿ †

PubMed Central

Jiang, Xiao Jing; Adler, Barbara; Sampaio, Kerstin Laib; Digel, Margarete; Jahn, Gerhard; Ettischer, Nicole; Stierhof, York-Dieter; Scrivano, Laura; Koszinowski, Ulrich; Mach, Michael; Sinzger, Christian

2008-01-01

The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC. PMID:18184717

Uterine leiomyomata: a retrospective study of correlations with hypertension and diabetes mellitus from the Japan Nurses' Health Study.

PubMed

Yasui, Toshiyuki; Hayashi, Kunihiko; Okano, Hiroya; Kamio, Masayo; Mizunuma, Hideki; Kubota, Toshiro; Lee, Jung-Su; Suzuki, Shosuke

2018-06-08

We performed a scrutiny survey of self-reported uterine leiomyomata (UL) to investigate the associations of parental history with hypertension and personal history of hypertension in the UL cases in Japanese women. Questionnaires that included items on the sites of UL determined by imaging techniques and surgical procedure were mailed to 2015 women with a self-reported UL at a baseline survey of the Japan Nurses' Health Study (n = 15,019). We found that women with a past history and a maternal history of hypertension had an increase in their risk of UL. A maternal history of hypertension was significantly associated with an increase in the risk of UL in women without a past history of hypertension but not in the women with a past history of hypertension. A past history and a parental history of diabetes mellitus were not associated with an increase in the risk of UL. Women of reproductive age with a maternal history of hypertension may be at a higher risk for hypertension and UL. Impact Statement What is already known on this subject? A positive association of uterine leiomyomata (UL) with a past history of hypertension has been found but the association of a parental history of hypertension with UL has not yet been clarified. What do the results of this study add? Maternal hypertension, as well as a personal history of hypertension, was associated with an increased risk of UL and a past history and a parental history of diabetes mellitus were not associated with an increase in the risk of UL. What are the implications of these findings for clinical practice and/or further research? Women of a reproductive age with a maternal history of hypertension may be at a higher risk for hypertension and UL.

DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms.

PubMed

Kim, Woohyun; Lee, Sooyoung; Son, Yeongnam; Ko, Chunkyu; Ryu, Wang-Shick

2016-11-01

HBx, a small regulatory protein of hepatitis B virus (HBV), augments viral DNA replication by stimulating viral transcription. Among numerous reported HBx-binding proteins, DDB1 has drawn attention, because DDB1 acts as a substrate receptor of the Cul4-DDB1 ubiquitin E3 ligase. Previous work reported that the DDB1-HBx interaction is indispensable for HBx-stimulated viral DNA replication, suggesting that the Cul4-DDB1 ubiquitin E3 ligase might target cellular restriction factors for ubiquitination and proteasomal degradation. To gain further insight into the DDB1-HBx interaction, we generated HBx mutants deficient for DDB1 binding (i.e., R96A, L98A, and G99A) and examined whether they support HBx-stimulated viral DNA replication. In contrast to data from previous reports, our results showed that the HBx mutants deficient for DDB1 binding supported viral DNA replication to nearly wild-type levels, revealing that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, we found that DDB1 directly stimulates viral transcription regardless of HBx expression. Through an HBV infection study, importantly, we demonstrated that DDB1 stimulates viral transcription from covalently closed circular DNA, a physiological template for viral transcription. Overall, we concluded that DDB1 stimulates viral transcription via a mechanism that does not involve an interaction with HBx. DDB1 constitutes a cullin-based ubiquitin E3 ligase, where DDB1 serves as an adaptor linking the cullin scaffold to the substrate receptor. Previous findings that the DDB1-binding ability of HBx is essential for HBx-stimulated viral DNA replication led to the hypothesis that HBx could downregulate host restriction factors that limit HBV replication through the cullin ubiquitin E3 ligase that requires the DDB1-HBx interaction. Consistent with this hypothesis, recent work identified Smc5/6 as a host restriction factor that is regulated by the viral cullin ubiquitin E3 ligase. In contrast, here we found that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, our results clearly showed that DDB1, regardless of HBx expression, enhances viral transcription. Overall, besides its role in the viral cullin ubiquitin E3 ligase, DDB1 itself stimulates viral transcription via HBx-independent mechanisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

46 CFR 129.410 - Lighting fixtures.

Code of Federal Regulations, 2010 CFR

2010-10-01

... COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS ELECTRICAL... electrical system operating at more than 50 volts must comply with UL 595, “Marine Type Electric Lighting... comply with UL 57, “Electric Lighting Fixtures,” UL 1570, “Fluorescent Lighting Fixtures,” UL 1571...

Increased urinary levels of tissue polypeptide specific antigen (TPS) in alcoholics.

PubMed

Barros, Paula; Gonzalez-Quintela, Arturo; Mella, Carmen; Perez, Luis-Fernando

2006-01-01

Urinary levels of tissue polypeptide specific antigen (TPS, cytokeratin-18) have been proposed as a marker of urothelial malignancies. Previous studies have shown that serum TPS levels are elevated in alcoholics. This study was designed to determine whether alcoholics had elevated urinary TPS levels as well. Serum and urinary TPS levels were determined in 24 alcoholics and 15 healthy controls by means of a commercial chemiluminiscent immunoassay. Serum TPS levels were higher in alcoholics than in controls (median 332 U/L, range 51-21241 U/L versus median 17 U/L, range 15-65 U/L, respectively, p<0.001). Urinary TPS levels were also higher in alcoholics than in controls (median 244 U/L, range 22-1267 U/L versus median 66.5 U/L, range 15-600 U/L, respectively, p=0.001). Urinary TPS levels were correlated with serum TPS levels in alcoholics. Urinary TPS levels are elevated in alcoholics. Consequently, the specificity of urinary TPS as a tumor marker may be limited in alcoholics.

Characterization of molecular determinants for nucleocytoplasmic shuttling of PRV UL54

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Meili; Wang Shuai; Cai Mingsheng

2011-09-01

The pseudorabies virus (PRV) early protein UL54 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein and essential for HSV-1 infection. To determine if UL54 might shuttle between the nucleus and cytoplasm, as has been shown for its homologues in human herpesviruses, the molecular determinants for its nucleocytoplasmic shuttling were investigated. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of chromosome region maintenance 1 (CRM1). However, TAP/NXF1 promoted the nuclear export of UL54 and interacted with UL54,more » suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP/NXF1, but not CRM1, dependent nuclear export pathway. Furthermore, UL54 was demonstrated to target to the nucleus through a classic Ran-, importin {beta}1- and {alpha}5-dependent nuclear import mechanism.« less

Novel Structure and Unexpected RNA-Binding Ability of the C-Terminal Domain of Herpes Simplex Virus 1 Tegument Protein UL21

DOE Office of Scientific and Technical Information (OSTI.GOV)

Metrick, Claire M.; Heldwein, Ekaterina E.; Sandri-Goldin, R. M.

Proteins forming the tegument layers of herpesviral virions mediate many essential processes in the viral replication cycle, yet few have been characterized in detail. UL21 is one such multifunctional tegument protein and is conserved among alphaherpesviruses. While UL21 has been implicated in many processes in viral replication, ranging from nuclear egress to virion morphogenesis to cell-cell spread, its precise roles remain unclear. Here we report the 2.7-Å crystal structure of the C-terminal domain of herpes simplex virus 1 (HSV-1) UL21 (UL21C), which has a unique α-helical fold resembling a dragonfly. Analysis of evolutionary conservation patterns and surface electrostatics pinpointed fourmore » regions of potential functional importance on the surface of UL21C to be pursued by mutagenesis. In combination with the previously determined structure of the N-terminal domain of UL21, the structure of UL21C provides a 3-dimensional framework for targeted exploration of the multiple roles of UL21 in the replication and pathogenesis of alphaherpesviruses. Additionally, we describe an unanticipated ability of UL21 to bind RNA, which may hint at a yet unexplored function. IMPORTANCEDue to the limited genomic coding capacity of viruses, viral proteins are often multifunctional, which makes them attractive antiviral targets. Such multifunctionality, however, complicates their study, which often involves constructing and characterizing null mutant viruses. Systematic exploration of these multifunctional proteins requires detailed road maps in the form of 3-dimensional structures. In this work, we determined the crystal structure of the C-terminal domain of UL21, a multifunctional tegument protein that is conserved among alphaherpesviruses. Structural analysis pinpointed surface areas of potential functional importance that provide a starting point for mutagenesis. In addition, the unexpected RNA-binding ability of UL21 may expand its functional repertoire. The structure of UL21C and the observation of its RNA-binding ability are the latest additions to the navigational chart that can guide the exploration of the multiple functions of UL21.« less

Usefulness of model-based iterative reconstruction in semi-automatic volumetry for ground-glass nodules at ultra-low-dose CT: a phantom study.

PubMed

Maruyama, Shuki; Fukushima, Yasuhiro; Miyamae, Yuta; Koizumi, Koji

2018-06-01

This study aimed to investigate the effects of parameter presets of the forward projected model-based iterative reconstruction solution (FIRST) on the accuracy of pulmonary nodule volume measurement. A torso phantom with simulated nodules [diameter: 5, 8, 10, and 12 mm; computed tomography (CT) density: - 630 HU] was scanned with a multi-detector CT at tube currents of 10 mA (ultra-low-dose: UL-dose) and 270 mA (standard-dose: Std-dose). Images were reconstructed with filtered back projection [FBP; standard (Std-FBP), ultra-low-dose (UL-FBP)], FIRST Lung (UL-Lung), and FIRST Body (UL-Body), and analyzed with a semi-automatic software. The error in the volume measurement was determined. The errors with UL-Lung and UL-Body were smaller than that with UL-FBP. The smallest error was 5.8% ± 0.3 for the 12-mm nodule with UL-Body (middle lung). Our results indicated that FIRST Body would be superior to FIRST Lung in terms of accuracy of nodule measurement with UL-dose CT.

Crystal Structure of the N-Terminal Half of the Traffic Controller UL37 from Herpes Simplex Virus 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koenigsberg, Andrea L.; Heldwein, Ekaterina E.; Sandri-Goldin, Rozanne M.

Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here,more » we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distributions of surface charges, and locations of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region, whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37. IMPORTANCEThe ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as human herpes simplex virus 1 (HSV-1), HSV-2, and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires detailed navigational charts in the form of their three-dimensional structures. Here, we determined the crystal structure of the N-terminal half of a conserved tegument protein, UL37, from HSV-1. This structure, along with our previously reported structure of the UL37 homolog from PRV, provides a much needed 3-dimensional template for the dissection of both conserved and virus-specific functions of UL37 in intracellular capsid trafficking.« less

Herpes Simplex Virus 1 UL37 Protein Tyrosine Residues Conserved among All Alphaherpesviruses Are Required for Interactions with Glycoprotein K, Cytoplasmic Virion Envelopment, and Infectious Virus Production

PubMed Central

Chouljenko, Dmitry V.; Jambunathan, Nithya; Chouljenko, Vladimir N.; Naderi, Misagh; Brylinski, Michal; Caskey, John R.

2016-01-01

ABSTRACT The herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927–5935, 2014, http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production. IMPORTANCE The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with gK. We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK. PMID:27630233

Herpes simplex virus type 1 gene UL14: phenotype of a null mutant and identification of the encoded protein.

PubMed

Cunningham, C; Davison, A J; MacLean, A R; Taus, N S; Baines, J D

2000-01-01

Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.

A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens

PubMed Central

Mahairas, Gregory G.; Shaw, Carolyn E.; Huang, Meei-Li; Koelle, David M.; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M.

2015-01-01

ABSTRACT We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4+ and CD8+ T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. IMPORTANCE HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and acquisition of HIV-1 infection 3- to 4-fold. A herpes vaccine that prevents genital lesions and asymptomatic genital shedding will have a substantial impact on two epidemics, i.e., both the HSV-2 and HIV-1 epidemics. We previously reported that a vaccine containing HSV-2 glycoprotein C (gC2) and glycoprotein D (gD2) reduced genital lesions and asymptomatic HSV-2 genital shedding in guinea pigs, yet the protection was not complete. We evaluated whether adding the T cell immunogens UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) would enhance the protection provided by the gC2/gD2 vaccine, which produces potent antibody responses. Here we report the efficacy of a combination vaccine containing gC2/gD2 and UL19/UL47 for prevention of genital disease, vaginal shedding of HSV-2 DNA, and latent infection of dorsal root ganglia in guinea pigs. PMID:26041292

Regulation of 2-5A Dependent RNase at the Level of its Phosphorylation

DTIC Science & Technology

1991-06-26

extract as follows: 25 ul wheat germ extract 10 ul H2O 1 ul RNasin ribonuclease inhibitor (40 u/ml) 7 ul ImM amino acid mixture 1 ul IM...diacylglycerol (DAG) 2. TPA 3. Indolactam Figure 6. Chemical structure of: 1. H-7 (A kinase inhibitor) 2. okadaic acid (A phosphatase inhibitor) Figure 7...elevating agents: Forskolin and Cholera toxin Figure 17. Down-regulation of 2-5A-depRNase by Okadaic 77 acid : A phosphatase inhibitor Figure 18

78 FR 28812 - Energy Efficiency Program for Industrial Equipment: Petition of UL Verification Services Inc. for...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-16

... are engineers. UL today is comprised of five businesses, Product Safety, Verification Services, Life..., Director--Global Technical Research, UL Verification Services. Subscribed and sworn to before me this 20... (431.447(c)(4)) General Personnel Overview UL is a global independent safety science company with more...

Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.

PubMed

Conwell, Sara E; White, Anne E; Harper, J Wade; Knipe, David M

2015-01-01

The herpes simplex virus 1 (HSV-1) immediate early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification and eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBN and MRE11A, regulators of apoptosis, including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal coimmunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions. During productive infection, a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To orchestrate such complex regulation, viruses, including herpes simplex virus 1 (HSV-1), rely on multifunctional proteins such as the E3 ubiquitin ligase ICP0. This protein regulates various cellular pathways concurrently by targeting a diverse set of cellular factors for degradation. While some of these targets have been previously identified and characterized, we undertook a proteomic screen to identify additional targets of this activity to further characterize ICP0's role during infection. We describe a set of candidate interacting proteins of ICP0 identified through this approach and our characterization of the most statistically significant result, the cellular transcriptional repressor TRIM27. We present TRIM27 as a novel degradation target of ICP0 and describe the relationship of these two proteins during infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The zinc fingers of the Small Optic Lobes (SOL) calpain bind polyubiquitin.

PubMed

Hastings, Margaret H; Qiu, Alvin; Zha, Congyao; Farah, Carole A; Mahdid, Yacine; Ferguson, Larissa; Sossin, Wayne S

2018-05-28

The Small Optic Lobes (SOL) calpain is a highly conserved member of the calpain family expressed in the nervous system. A dominant negative form of the SOL calpain inhibited consolidation of one form of synaptic plasticity, non-associative facilitation, in sensory-motor neuronal cultures in Aplysia, presumably by inhibiting cleavage of protein kinase Cs (PKCs) into constitutively active protein kinase Ms (PKMs) (Hu et al, 2017a). SOL calpains have a conserved set of 5-6 N-terminal zinc fingers. Bioinformatic analysis suggests that these zinc fingers could bind to ubiquitin. In this study, we show that both the Aplysia and mouse SOL calpain (also known as Calpain 15) zinc fingers bind ubiquitinated proteins, and we confirm that Aplysia SOL binds poly- but not mono or di-ubiquitin. No specific zinc finger is required for polyubiquitin binding. Neither polyubiquitin nor calcium was sufficient to induce purified Aplysia SOL calpain to autolyse or to cleave the atypical PKC to PKM in vitro. In Aplysia, overexpression of the atypical PKC in sensory neurons leads to an activity-dependent cleavage event and an increase in nuclear ubiquitin staining. Activity-dependent cleavage is partially blocked by a dominant negative SOL calpain, but not by a dominant negative classical calpain. The cleaved PKM was stabilized by the dominant negative classical calpain and destabilized by a dominant negative form of the PKM stabilizing proteinKIdney/BRAin protein(KIBRA). These studies provide new insight into SOL calpain's function and regulation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

PubMed Central

Graf, Laura; Webel, Rike; Wagner, Sabrina; Hamilton, Stuart T.; Rawlinson, William D.; Sticht, Heinrich; Marschall, Manfred

2013-01-01

The human cytomegalovirus (HCMV)-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK) ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins. PMID:24351800

In Vivo Imaging of Glial Activation after Unilateral Labyrinthectomy in the Rat: A [18F]GE180-PET Study.

PubMed

Zwergal, Andreas; Günther, Lisa; Brendel, Matthias; Beck, Roswitha; Lindner, Simon; Xiong, Guoming; Eilles, Eva; Unterrainer, Marcus; Albert, Nathalie Lisa; Becker-Bense, Sandra; Brandt, Thomas; Ziegler, Sibylle; la Fougère, Christian; Dieterich, Marianne; Bartenstein, Peter

2017-01-01

The functional relevance of reactive gliosis for recovery from acute unilateral vestibulopathy is unknown. In the present study, glial activation was visualized in vivo by [ 18 F]GE180-PET in a rat model of unilateral labyrinthectomy (UL) and compared to behavioral vestibular compensation (VC) overtime. 14 Sprague-Dawley rats underwent a UL by transtympanic injection of bupivacaine/arsenilate, 14 rats a SHAM UL (injection of normal saline). Glial activation was depicted with [ 18 F]GE180-PET and ex vivo autoradiography at baseline and 7, 15, 30 days after UL/SHAM UL. Postural asymmetry and nystagmus were registered at 1, 2, 3, 7, 15, 30 days after UL/SHAM UL. Signs of vestibular imbalance were found only after UL, which significantly decreased until days 15 and 30. In parallel, [ 18 F]GE180-PET and ex vivo autoradiography depicted glial activation in the ipsilesional vestibular nerve and nucleus on days 7 and 15 after UL. Correlation analysis revealed a strong negative association of [ 18 F]GE180 uptake in the ipsilesional vestibular nucleus on day 7 with the rate of postural recovery ( R  = -0.90, p  < 0.001), suggesting that glial activation accelerates VC. In conclusion, glial activation takes place in the ipsilesional vestibular nerve and nucleus within the first 30 days after UL in the rat and can be visualized in vivo by [ 18 F]GE180-PET.

In Vivo Imaging of Glial Activation after Unilateral Labyrinthectomy in the Rat: A [18F]GE180-PET Study

PubMed Central

Zwergal, Andreas; Günther, Lisa; Brendel, Matthias; Beck, Roswitha; Lindner, Simon; Xiong, Guoming; Eilles, Eva; Unterrainer, Marcus; Albert, Nathalie Lisa; Becker-Bense, Sandra; Brandt, Thomas; Ziegler, Sibylle; la Fougère, Christian; Dieterich, Marianne; Bartenstein, Peter

2017-01-01

The functional relevance of reactive gliosis for recovery from acute unilateral vestibulopathy is unknown. In the present study, glial activation was visualized in vivo by [18F]GE180-PET in a rat model of unilateral labyrinthectomy (UL) and compared to behavioral vestibular compensation (VC) overtime. 14 Sprague-Dawley rats underwent a UL by transtympanic injection of bupivacaine/arsenilate, 14 rats a SHAM UL (injection of normal saline). Glial activation was depicted with [18F]GE180-PET and ex vivo autoradiography at baseline and 7, 15, 30 days after UL/SHAM UL. Postural asymmetry and nystagmus were registered at 1, 2, 3, 7, 15, 30 days after UL/SHAM UL. Signs of vestibular imbalance were found only after UL, which significantly decreased until days 15 and 30. In parallel, [18F]GE180-PET and ex vivo autoradiography depicted glial activation in the ipsilesional vestibular nerve and nucleus on days 7 and 15 after UL. Correlation analysis revealed a strong negative association of [18F]GE180 uptake in the ipsilesional vestibular nucleus on day 7 with the rate of postural recovery (R = −0.90, p < 0.001), suggesting that glial activation accelerates VC. In conclusion, glial activation takes place in the ipsilesional vestibular nerve and nucleus within the first 30 days after UL in the rat and can be visualized in vivo by [18F]GE180-PET. PMID:29312111

Elucidation of the Block to Herpes Simplex Virus Egress in the Absence of Tegument Protein UL16 Reveals a Novel Interaction with VP22

PubMed Central

Starkey, Jason L.; Han, Jun; Chadha, Pooja; Marsh, Jacob A.

2014-01-01

UL16 is a tegument protein of herpes simplex virus (HSV) that is conserved among all members of the Herpesviridae, but its function is poorly understood. Previous studies revealed that UL16 is associated with capsids in the cytoplasm and interacts with the membrane protein UL11, which suggested a “bridging” function during cytoplasmic envelopment, but this conjecture has not been tested. To gain further insight, cells infected with UL16-null mutants were examined by electron microscopy. No defects in the transport of capsids to cytoplasmic membranes were observed, but the wrapping of capsids with membranes was delayed. Moreover, clusters of cytoplasmic capsids were often observed, but only near membranes, where they were wrapped to produce multiple capsids within a single envelope. Normal virion production was restored when UL16 was expressed either by complementing cells or from a novel position in the HSV genome. When the composition of the UL16-null viruses was analyzed, a reduction in the packaging of glycoprotein E (gE) was observed, which was not surprising, since it has been reported that UL16 interacts with this glycoprotein. However, levels of the tegument protein VP22 were also dramatically reduced in virions, even though this gE-binding protein has been shown not to depend on its membrane partner for packaging. Cotransfection experiments revealed that UL16 and VP22 can interact in the absence of other viral proteins. These results extend the UL16 interaction network beyond its previously identified binding partners to include VP22 and provide evidence that UL16 plays an important function at the membrane during virion production. PMID:24131716

Comparison of Self-Report Versus Sensor-Based Methods for Measuring the Amount of Upper Limb Activity Outside the Clinic.

PubMed

Waddell, Kimberly J; Lang, Catherine E

2018-03-10

To compare self-reported with sensor-measured upper limb (UL) performance in daily life for individuals with chronic (≥6mo) UL paresis poststroke. Secondary analysis of participants enrolled in a phase II randomized, parallel, dose-response UL movement trial. This analysis compared the accuracy and consistency between self-reported UL performance and sensor-measured UL performance at baseline and immediately post an 8-week intensive UL task-specific intervention. Outpatient rehabilitation. Community-dwelling individuals with chronic (≥6mo) UL paresis poststroke (N=64). Not applicable. Motor Activity Log amount of use scale and the sensor-derived use ratio from wrist-worn accelerometers. There was a high degree of variability between self-reported UL performance and the sensor-derived use ratio. Using sensor-based values as a reference, 3 distinct categories were identified: accurate reporters (reporting difference ±0.1), overreporters (difference >0.1), and underreporters (difference <-0.1). Five of 64 participants accurately self-reported UL performance at baseline and postintervention. Over half of participants (52%) switched categories from pre-to postintervention (eg, moved from underreporting preintervention to overreporting postintervention). For the consistent reporters, no participant characteristics were found to influence whether someone over- or underreported performance compared with sensor-based assessment. Participants did not consistently or accurately self-report UL performance when compared with the sensor-derived use ratio. Although self-report and sensor-based assessments are moderately associated and appear similar conceptually, these results suggest self-reported UL performance is often not consistent with sensor-measured performance and the measures cannot be used interchangeably. Copyright © 2018 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

Human Cytomegalovirus pUL97 Regulates the Viral Major Immediate Early Promoter by Phosphorylation-Mediated Disruption of Histone Deacetylase 1 Binding

PubMed Central

Bigley, Tarin M.; Reitsma, Justin M.; Mirza, Shama P.

2013-01-01

Human cytomegalovirus (HCMV) is a common agent of congenital infection and causes severe disease in immunocompromised patients. Current approved therapies focus on inhibiting viral DNA replication. The HCMV kinase pUL97 contributes to multiple stages of viral infection including DNA replication, controlling the cell cycle, and virion maturation. Our studies demonstrate that pUL97 also functions by influencing immediate early (IE) gene expression during the initial stages of infection. Inhibition of kinase activity using the antiviral compound maribavir or deletion of the UL97 gene resulted in decreased expression of viral immediate early genes during infection. Expression of pUL97 was sufficient to transactivate IE1 gene expression from the viral genome, which was dependent on viral kinase activity. We observed that pUL97 associates with histone deacetylase 1 (HDAC1). HDAC1 is a transcriptional corepressor that acts to silence expression of viral genes. We observed that inhibition or deletion of pUL97 kinase resulted in increased HDAC1 and decreased histone H3 lysine 9 acetylation associating with the viral major immediate early (MIE) promoter. IE expression during pUL97 inhibition or deletion was rescued following inhibition of deacetylase activity. HDAC1 associates with chromatin by protein-protein interactions. Expression of active but not inactive pUL97 kinase decreased HDAC1 interaction with the transcriptional repressor protein DAXX. Finally, using mass spectrometry, we found that HDAC1 is uniquely phosphorylated upon expression of pUL97. Our results support the conclusion that HCMV pUL97 kinase regulates viral immediate early gene expression by phosphorylation-mediated disruption of HDAC1 binding to the MIE promoter. PMID:23616659

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery.

PubMed

Adelman, K; Salmon, B; Baines, J D

2001-03-13

The product of the herpes simplex virus type 1 U(L)28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of U(L)28 in this process, purified U(L)28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for U(L)28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by U(L)28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by U(L)28 protein. To determine the relevance of the observed U(L)28 protein-pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of U(L)28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U(L)28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the U(L)28 protein-pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the U(L)28-encoded component of the herpesvirus cleavage and packaging machinery.

The herpes simplex virus 1 UL51 protein interacts with the UL7 protein and plays a role in its recruitment into the virion.

PubMed

Roller, Richard J; Fetters, Rachel

2015-03-01

The alphaherpesvirus UL51 protein is a tegument component that interacts with the viral glycoprotein E and functions at multiple steps in virus assembly and spread in epithelial cells. We show here that pUL51 forms a complex in infected cells with another conserved tegument protein, pUL7. This complex can form in the absence of other viral proteins and is largely responsible for recruitment of pUL7 to cytoplasmic membranes and into the virion tegument. Incomplete colocalization of pUL51 and pUL7 in infected cells, however, suggests that a significant fraction of the population of each protein is not complexed with the other and that they may accomplish independent functions. The ability of herpesviruses to spread from cell to cell in the face of an immune response is critical for disease and shedding following reactivation from latency. Cell-to-cell spread is a conserved ability of herpesviruses, and the identification of conserved viral genes that mediate this process will aid in the design of attenuated vaccines and of novel therapeutics. The conserved UL51 gene of herpes simplex virus 1 plays important roles in cell-to-cell spread and in virus assembly in the cytoplasm, both of which likely depend on specific interactions with other viral and cellular proteins. Here we identify one of those interactions with the product of another conserved herpesvirus gene, UL7, and show that formation of this complex mediates recruitment of UL7 to membranes and to the virion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Herpesvirus capsid assembly and DNA packaging

PubMed Central

Heming, Jason D.; Conway, James F.; Homa, Fred L.

2017-01-01

Herpes simplex virus type I (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. During productive lytic infection, over 80 viral proteins are expressed in a highly regulated manner, resulting in the replication of viral genomes and assembly of progeny virions. The virion of all herpesviruses consists of an external membrane envelope, a proteinaceous layer called the tegument, and an icosahedral capsid containing the double-stranded linear DNA genome. The capsid shell of HSV-1 is built from four structural proteins: a major capsid protein, VP5, which forms the capsomers (hexons and pentons), the triplex consisting of VP19C and VP23 found between the capsomers, and VP26 which binds to VP5 on hexons but not pentons. In addition, the dodecameric pUL6 portal complex occupies one of the 12 capsid vertices, and the capsid vertex specific component (CVSC), a heterotrimer complex of pUL17, pUL25 and pUL36 binds specifically to the triplexes adjacent to each penton. The capsid is assembled in the nucleus where the viral genome is packaged into newly assembled closed capsid shells. Cleavage and packaging of replicated, concatemeric viral DNA requires the seven viral proteins encoded by the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes. Considerable advances have been made in understanding the structure of the herpesvirus capsid and the function of several of the DNA packaging proteins by applying biochemical, genetic, and structural techniques. This review is a summary of recent advances with respect to the structure of the HSV-1 virion capsid and what is known about the function of the seven packaging proteins and their interactions with each other and with the capsid shell. PMID:28528442

Expression and distribution of the duck enteritis virus UL51 protein in experimentally infected ducks.

PubMed

Shen, Chanjuan; Cheng, Anchun; Wang, Mingshu; Xu, Chao; Jia, Renyong; Chen, Xiaoyue; Zhu, Dekang; Luo, Qihui; Cui, Hengmin; Zhou, Yi; Wang, Yin; Xu, Zhiwen; Chen, Zhengli; Wang, Xiaoyu

2010-06-01

To determine the expression and distribution of tegument proteins encoded by duck enteritis virus (DEV) UL51 gene in tissues of experimentally infected ducks, for the first time, an immunoperoxidase staining method to detect UL51 protein (UL51p) in paraffin-embedded tissues is reported. A rabbit anti-UL51 polyclonal serum, raised against a recombinant 6-His-UL51 fusion protein expressed in Escherichia coli, was prepared, purified, and used as primary antibodies. Fifty-eight 30-day-old DEV-free ducks were intramuscularly inoculated with the pathogenic DEV CHv strain as infection group, and two ducks were selected as preinfection group. The tissues were collected at sequential time points between 2 and 480 hr postinoculation (PI) and prepared for immunoperoxidase staining. DEV UL51p was first found in the spleen and liver at 8 hr PI; in the bursa of Fabricius and thymus at 12 hr PI; in the Harders glands, esophagus, small intestine (including the duodenum, jejunum, and ileum), and large intestine (including the caecum and rectum) at 24 hr PI; in the glandularis ventriculus at 48 hr PI; and in the pancreas, cerebrum, kidney, lung, and myocardium at 72 hr PI. Throughout the infection process, the UL51p was not seen in the muscle. Furthermore, the intensity of positive staining of DEV UL51p antigen in various tissues increased sharply from 8 to 96 hr PI, peaked during 120-144 hr PI, and then decreased steadily from 216 to 480 hr PI, suggesting that the expressional levels of DEV UL51p in systemic organs have a close correlation with the progression of duck virus enteritis (DVE) disease. A number of DEV UL51p was distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, small intestine, and large intestine of DEV-infected ducks, whereas less DEV UL51p was distributed in the Harders glands, glandularis ventriculus, cerebrum, kidney, lung, pancreas, and myocardium of DEV-infected ducks. Moreover, DEV UL51p can be expressed in the cytoplasm of various types of cells, especially most abundantly in the cytoplasm of lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes. The present study may be useful not only for describing the characteristics of UL51p expression and distribution in vivo but also for a greater understanding of the pathogenesis of this DVE.

SOVRaD - A Digest of Recent Soviet R and D Articles. Volume 1, Numbers 5-6, May-June 1975

DTIC Science & Technology

1975-06-01

oreoared bv nnW i ^ • y^ ! . S^ aryl.b.s.(cYanamid^ P ulN ^Tu^^Y^Tml^T "’ lleterojunction Solar Cell (verbatim^ forward segment of...Wolf numbers for 1920 -- 1965; Wolf number R = ’’(lOg + f), where f = the number of spots in the solar disc, g = tie numbo’" of soot groups...an increase in the intensity of solar activity, as detcrm’^wd by the mean annual Wolf number. The maximum rate of ball lightning repeats

Evaluation of Interacavitary Chemotherapy Delivery for Treatment of Mammary Carcinoma

DTIC Science & Technology

2005-04-01

Celltiter 96 Aqueous one solution cell proliferation assay - Promega) in 96 well plates were used, each well received 100 ul of cell culture medium and...treatments: a) polotax (200 ul of 22% poloxamer/5.4mg/ml taxol suspension) in wound, b) 200 ul polotax remote (between 2 scapulae ), c) 200 ul 22% poloxamer in

Identification of Arabidopsis MYB56 as a novel substrate for CRL3(BPM) E3 ligases.

PubMed

Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

2015-02-01

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Relocation of the 2010-2013 near the north coast of Papua earthquake sequence using Modified Joint Hypocenter Determination (MJHD) method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salomo, Dimas, E-mail: dimas.salomo@gmail.com; Daryono,; Subakti, Hendri

The accuracy of earthquake hypocenter position is necessary to analyze the tectonic conditions. This study aims to: (1) relocate the mainshock and aftershocks of the large earthquakes in Papua region i.e. June 16, 2010, April 21, 2012 and April 06, 2013 earthquake (2) determine the true fault plane, (3) estimate the area of the fracture, and (4) analyze the advantages and disadvantages of relocation with MJHD method in benefits for tectonic studies. This study used Modified Joint Hypocenter Determination (MJHD) method. Using P arrival phase data reported by the BMKG and openly available from website repogempa.bmkg.go.id, we relocated the mainshockmore » of this large significant earthquake and its aftershocks. Then we identified the prefered fault planes from the candidate fault planes provided by the global CMT catalogue. The position of earthquakes was successfully relocated. The earthquakes mostly were clustered around the mainshock. Earthquakes that not clustered around mainshock are considered to be different mechanism from the mainshock. Relocation results indicate that the mainshock fault plane of June 16, 2010 earthquake is a field with strike 332o, dip 80o and −172o slip, the mainshock fault plane of April 21, 2012 earthquake is a field with strike 82o, dip 84o and 2o slip, the mainshock fault plane of April 06, 2013 earthquake is a field with strike 339o, dip 56o and −137o slip. Fault plane area estimated by cross section graphical method is an area of 2816.0 km2 (June 16, 2010), 906.2 km2 (April 21, 2012) and 1984.3 km2 (April 06, 2013). MJHD method has the advantage that it can calculate a lot of earthquakes simultaneously and has a station correction to account for lateral heterogeneity of the earth. This method successfully provides significant changes to improve the position of the depth of earthquakes that most of the hypocenter depth manually specified as a fixed depth (± 10 km). But this method cannot be sure that the hypocenters derived from the same earthquake mechanism.« less

Clinical features and risk factors of acute hepatitis E with severe jaundice.

PubMed

Xu, Bin; Yu, Hai-Bin; Hui, Wei; He, Jia-Li; Wei, Lin-Lin; Wang, Zheng; Guo, Xin-Hui

2012-12-28

To compares the clinical features of patients infected with hepatitis E virus (HEV) with or without severe jaundice. In addition, the risk factors for HEV infection with severe jaundice were investigated. We enrolled 235 patients with HEV into a cross-sectional study using multi-stage sampling to select the study group. Patients with possible acute hepatitis E showing elevated liver enzyme levels were screened for HEV infection using serologic and molecular tools.HEV infection was documented by HEV antibodies and by the detection of HEV-RNA in serum. We used χ(2) analysis, Fisher's exact test, and Student's t test where appropriate in this study. Significant predictors in the univariate analysis were then included in a forward, stepwise multiple logistic regression model. No significant differences in symptoms, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, or hepatitis B virus surface antigen between the two groups were observed. HEV infected patients with severe jaundice had significantly lower peak serum levels of γ-glutamyl-transpeptidase (GGT) (median: 170.31 U/L vs 237.96 U/L, P = 0.007), significantly lower ALB levels (33.84 g/L vs 36.89 g/L, P = 0.000), significantly lower acetylcholine esterase (CHE) levels (4500.93 U/L vs 5815.28 U/L, P = 0.000) and significantly higher total bile acid (TBA) levels (275.56 μmol/L vs 147.03 μmol/L, P = 0.000) than those without severe jaundice. The median of the lowest point time tended to be lower in patients with severe jaundice (81.64% vs 96.12%, P = 0.000). HEV infected patients with severe jaundice had a significantly higher viral load (median: 134 vs 112, P = 0.025) than those without severe jaundice. HEV infected patients with severe jaundice showed a trend toward longer median hospital stay (38.17 d vs 18.36 d, P = 0.073). Multivariate logistic regression indicated that there were significant differences in age, sex, viral load, GGT, albumin, TBA, CHE, prothrombin index, alcohol overconsumption, and duration of admission between patients infected with acute hepatitis E with and without severe jaundice. Acute hepatitis E patients may naturally present with severe jaundice.

Deformational behaviour of knee cartilage and changes in serum cartilage oligomeric matrix protein (COMP) after running and drop landing.

PubMed

Niehoff, A; Müller, M; Brüggemann, L; Savage, T; Zaucke, F; Eckstein, F; Müller-Lung, U; Brüggemann, G-P

2011-08-01

To investigate (1) the effect of running and drop landing interventions on knee cartilage deformation and serum cartilage oligomeric matrix protein (COMP) concentration and (2) if the changes in cartilage volume correlate with the changes in serum COMP level. Knee joint cartilage volume and thickness were determined using magnetic resonance imaging (MRI) as well as COMP concentration from serum samples before and after in vivo loading of 14 healthy adults (seven male and seven female). Participants performed different loading interventions of 30 min duration on three different days: (1) 100 vertical drop landings from a 73 cm high platform, (2) running at a velocity of 2.2m/s (3.96 km), and (3) resting on a chair. Blood samples were taken immediately before, immediately after and 0.5h, 1h, 2h and 3h post intervention. Pre- and post-loading coronal and axial gradient echo MR images with fat suppression were used to determine the patellar, tibial and femoral cartilage deformation. Serum COMP levels increased immediately after the running (+30.7%, pre: 7.3U/l, 95% confidence interval (CI): 5.6, 8.9, post: 9.1U/l, 95% CI: 7.2, 11.0, P=0.001) and after drop landing intervention (+32.3%, pre: 6.8U/l, 95% CI: 5.3, 8.4; post: 8.9U/l, 95% CI: 6.8, 10.9, P=0.001). Cartilage deformation was more pronounced after running compared to drop landing intervention, with being significant (volume: P=0.002 and thickness: P=0.001) only in the lateral tibia. We found a significant correlation (r(2)=0.599, P=0.001) between changes in serum COMP (%) and in cartilage volume (%) after the drop landing intervention, but not after running. In vivo exercise interventions differentially regulate serum COMP concentrations and knee cartilage deformations. The relation between changes in COMP and in cartilage volume seems to depend on both mechanical and biochemical factors. Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

75 FR 67095 - Charles M. Russell National Wildlife Refuge and UL Bend National Wildlife Refuge, Montana

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-01

...] Charles M. Russell National Wildlife Refuge and UL Bend National Wildlife Refuge, Montana AGENCY: Fish and... conservation plan (CCP) and environmental impact statement (EIS) for Charles M. Russell and UL Bend National... are extending the comment period for review of the draft CCP and EIS for Charles M. Russell NWR and UL...

Reachability in K 3,3-Free Graphs and K 5-Free Graphs Is in Unambiguous Log-Space

NASA Astrophysics Data System (ADS)

Thierauf, Thomas; Wagner, Fabian

We show that the reachability problem for directed graphs that are either K 3,3-free or K 5-free is in unambiguous log-space, UL ∩ coUL. This significantly extends the result of Bourke, Tewari, and Vinodchandran that the reachability problem for directed planar graphs is in UL ∩ coUL.

Human Cytomegalovirus UL18 Utilizes US6 for Evading the NK and T-Cell Responses

PubMed Central

Kim, Youngkyun; Park, Boyoun; Cho, Sunglim; Shin, Jinwook; Cho, Kwangmin; Jun, Youngsoo; Ahn, Kwangseog

2008-01-01

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses. PMID:18688275

Identification of multiple sites suitable for insertion of foreign genes in herpes simplex virus genomes.

PubMed

Morimoto, Tomomi; Arii, Jun; Akashi, Hiroomi; Kawaguchi, Yasushi

2009-03-01

Information on sites in HSV genomes at which foreign gene(s) can be inserted without disrupting viral genes or affecting properties of the parental virus are important for basic research on HSV and development of HSV-based vectors for human therapy. The intergenic region between HSV-1 UL3 and UL4 genes has been reported to satisfy the requirements for such an insertion site. The UL3 and UL4 genes are oriented toward the intergenic region and, therefore, insertion of a foreign gene(s) into the region between the UL3 and UL4 polyadenylation signals should not disrupt any viral genes or transcriptional units. HSV-1 and HSV-2 each have more than 10 additional regions structurally similar to the intergenic region between UL3 and UL4. In the studies reported here, it has been demonstrated that insertion of a reporter gene expression cassette into several of the HSV-1 and HSV-2 intergenic regions has no effect on viral growth in cell culture or virulence in mice, suggesting that these multiple intergenic regions may be suitable HSV sites for insertion of foreign genes.

Measurement of free radicals using electron paramagnetic resonance spectroscopy during open aorto-iliac arterial reconstruction.

PubMed

Majewski, Wacław; Krzyminiewski, Ryszard; Stanisić, Michał; Iskra, Maria; Krasiński, Zbigniew; Nowak, Marek; Dobosz, Bernadeta

2014-11-27

Aortic cross-clamping during abdominal aortic aneurysm (AAA) open repair leads to development of ischemia-reperfusion injury. Electron paramagnetic resonance spectroscopy (EPR) spin-trapping is a valuable method of direct measurement of free radicals. The objective of the study was to evaluate the results of EPR as a direct method of free radical measurement and degree of inflammatory response in open operative treatment of patients with AAA and aorto-iliac occlusive disease (AIOD). The study was performed on a group of 32 patients with AAA and 25 patients with AIOD scheduled for open repair. Peripheral venous blood for EPR spectroscopy and for SOD, GPx, ox-LDL, Il-6, TNF-alfa, CRP, and HO-1 were harvested. Selected parameters were established accordingly to specified EPR and immunohistochemical methods and analyzed between groups by Mann-Whitney U test and Wilcoxon matched-pairs signed-ranks test with Bonferroni correction. Free radicals level was correlated with the time of the aortic cross-clamping after the reperfusion of he first and second leg in AAA (r=0.7; r=0.47). ox-LDL in AAA decreased 5 min after reperfusion of the first leg (32.99 U/L, range: 14.09-77.12) and 5 min after reperfusion of the second leg (26.75 U/L, range: 11.56-82.12) and 24 h after the operation (25.85 U/L, range: 14.29-49.70). HO-1 concentration increased to above the level before intervention 24 h after surgery. The activities of GPx and SOD decreased 5 min after the first-leg reperfusion in AAA. Twenty-four hours after surgery, inflammatory markers increased in AAA to CRP was 14.76 ml/l (0.23-38.55), IL-6 was 141.22 pg/ml (84.3-591.03), TNF-alfa was 6.82 pg/ml (1.76-80.01) and AIOD: CRP was 18.44 mg/l (2.56-33.14), IL-6: 184.1 pg/ml (128.46-448.03), TNF-alfa was 7.74 pg/ml (1.74-74.74). EPR spin-trapping demonstrates temporarily elevated level of free radicals in early phase of reperfusion, leading to decrease antioxidants in AAA. Elevated free radical levels decreased 24 h after surgery due to various endogenous antioxidants and therapies.

Liver enzyme elevation induced by hyperemesis gravidarum: aetiology, diagnosis and treatment.

PubMed

Conchillo, J M; Pijnenborg, J M A; Peeters, P; Stockbrügger, R W; Fevery, J; Koek, G H

2002-10-01

Three primigravidae were admitted during the first trimester of pregnancy with nausea, vomiting, ketonuria and liver enzyme elevation of varying severity. A 29-year-old woman had elevated aminotransferase values, at levels described in the literature (ASAT 112 U/l, ALAT 214 U/l). The second patient, a woman aged 26 years, had undergone in vitro fertilisation and showed higher liver enzyme elevation, including the total bilirubin level (ASAT 250 U/l, ALAT 474 U/l, total bilirubin 59.8 micromol/l). A 30-year-old woman had extremely high aminotransferase values (ASAT 705 U/l, ALAT 1674 U/l) and she is the first reported patient with ALAT values exceeding 1,000 U/l in connection with hyperemesis gravidarum. Gallstone disease, viral and drug-induced hepatitis were excluded in all of these patients. Treatment was symptomatic and the abnormal liver tests returned to normal promptly when the vomiting resolved, independent of the severity of liver enzyme elevation. The pregnancies proceeded normally and all three patients delivered healthy babies.

A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens.

PubMed

Awasthi, Sita; Mahairas, Gregory G; Shaw, Carolyn E; Huang, Meei-Li; Koelle, David M; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M

2015-08-01

We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4(+) and CD8(+) T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and acquisition of HIV-1 infection 3- to 4-fold. A herpes vaccine that prevents genital lesions and asymptomatic genital shedding will have a substantial impact on two epidemics, i.e., both the HSV-2 and HIV-1 epidemics. We previously reported that a vaccine containing HSV-2 glycoprotein C (gC2) and glycoprotein D (gD2) reduced genital lesions and asymptomatic HSV-2 genital shedding in guinea pigs, yet the protection was not complete. We evaluated whether adding the T cell immunogens UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) would enhance the protection provided by the gC2/gD2 vaccine, which produces potent antibody responses. Here we report the efficacy of a combination vaccine containing gC2/gD2 and UL19/UL47 for prevention of genital disease, vaginal shedding of HSV-2 DNA, and latent infection of dorsal root ganglia in guinea pigs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Implications of the ’Nuclear Winter’ Thesis.

DTIC Science & Technology

1985-06-24

not provide significant relief by warming the planet o ozone depletion would increase exposure to ultraviolet light ( UV -B) Technical Uncertainties in...pgs. 37. - ------------ "When Light is Put Away, Ecological Effects of Nuclear War," The Counterfeit Ark: Crisis Relocation for Nuclear War, 1984, 13...Mary, "Biologists Paint an Icy Picture . . ., Washington Post, November 1, 1983, 1 pg. 56. McWilliams, Rita, "Hill Told Money Won’t Buy Nuclear

Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

PubMed

Tudor, E L; Galtrey, C M; Perkinton, M S; Lau, K-F; De Vos, K J; Mitchell, J C; Ackerley, S; Hortobágyi, T; Vámos, E; Leigh, P N; Klasen, C; McLoughlin, D M; Shaw, C E; Miller, C C J

2010-05-19

Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores

PubMed Central

Huffman, Jamie B.; Daniel, Gina R.; Falck-Pedersen, Erik; Huet, Alexis

2017-01-01

ABSTRACT The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids. IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release. PMID:28490590

Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization.

PubMed

Maschkowitz, Gregor; Gärtner, Sabine; Hofmann-Winkler, Heike; Fickenscher, Helmut; Winkler, Michael

2018-05-01

Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans -Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization. IMPORTANCE Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects the localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication. Copyright © 2018 American Society for Microbiology.

The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemčovičová, Ivana; Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava; Zajonc, Dirk M., E-mail: dzajonc@liai.org

2014-03-01

The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155more » as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor interactions are evolutionary conserved.« less

Serum Alkaline Phosphatase Levels Predict Infection-Related Mortality and Hospitalization in Peritoneal Dialysis Patients.

PubMed

Hwang, Seun Deuk; Kim, Su-Hyun; Kim, Young Ok; Jin, Dong Chan; Song, Ho Chul; Choi, Euy Jin; Kim, Yong-Lim; Kim, Yon-Su; Kang, Shin-Wook; Kim, Nam-Ho; Yang, Chul Woo; Kim, Yong Kyun

2016-01-01

Serum alkaline phosphatase (ALP) levels have been reported to be associated with all-cause and cardiovascular mortality in peritoneal dialysis (PD) patients. However, it is unclear whether serum ALP levels predict infection-related clinical outcomes in PD patients. The aim of this study was to determine the relationships between serum ALP levels, infection-related mortality and hospitalization in PD patients. PD patients from the Clinical Research Center registry for end-stage renal disease, a multicenter prospective observational cohort study in Korea, were included in the present study. Patients were categorized into three groups by serum ALP tertiles as follows: Tertile 1, ALP <78 U/L; Tertile 2, ALP = 78-155 U/L; Tertile 3, ALP >155 U/L. Tertile 1 was used as the reference category. The primary outcomes were infection-related mortality and hospitalization. A total of 1,455 PD patients were included. The median follow-up period was 32 months. The most common cause of infection-related mortality and hospitalization was PD-related peritonitis. Multivariate Cox regression analyses showed that patients in the highest tertiles of serum ALP levels were at higher risk of infection-related mortality (HR 2.29, 95% CI, 1.42-5.21, P = 0.008) after adjustment for clinical variables. Higher tertiles of serum ALP levels were associated with higher risk of infection-related hospitalization (Tertile 2: HR 1.56, 95% CI, 1.18-2.19, P = 0.009, tertile 3: HR 1.34, 95% CI, 1.03-2.62, P = 0.031). Our data showed that elevated serum ALP levels were independently associated with a higher risk of infection-related mortality and hospitalization in PD patients.

Hepatoprotective effect of ethanol extract from Berchemia lineate against CCl4-induced acute hepatotoxicity in mice.

PubMed

Li, Cong; Yi, Li-Tao; Geng, Di; Han, Yuan-Yuan; Weng, Lian-Jin

2015-05-01

The roots of Berchemia lineate (L.) DC. (Rhamnaceae) have been long used as a remedy for the treatment of some diseases in Guangxi Province, China. The present study investigates the hepatoprotective effect of Berchemia lineate ethanol extract (BELE) on CCl4-induced acute liver damage in mice. Effect of BELE administrated for 7 consecutive days was evaluated in mice by the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), albulin (ALB), globulin (GLB), and total protein (TP) levels, as well as liver superoxide dismutase (SOD) activity and malondialdehyde (MDA) level. Moreover, histopathological examinations were also taken. Compared with the model group, administration of 400 mg/kg BELE for 7 d in mice significantly decreased the serum ALT (56.25 U/L), AST (297.67 U/L), ALP (188.20 U/L), and TBIL (17.90 mol/L), along with the elevation of TP (64.67 g/L). In addition, BELE (100, 200, and 400 mg/kg, i.g.) treated mice recorded a dose-dependent increment of SOD (291.17, 310.32, and 325.67 U/mg prot) and reduction of MDA (7.27, 6.77, and 5.33 nmol/mg prot) levels. Histopathological examinations also confirmed that BELE can ameliorate CCl4-induced liver injuries, characterized by extensive hepatocellular degeneration/necrosis, inflammatory cell infiltration, congestion, and sinusoidal dilatation. The results indicated that BELE possessed remarkable protective effect against acute hepatotoxicity and oxidative injuries induced by CCl4, and that the hepatoprotective effects of BELE may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity.

Manganese content of soy or rice beverages is high in comparison to infant formulas.

PubMed

Cockell, Kevin A; Bonacci, Giuseppe; Belonje, Bartholomeus

2004-04-01

Well-meaning but inadequately informed parents may perceive plant-based beverages such as soy beverages (SB) or rice beverages (RB) as an alternative to infant formula. Manganese (Mn) is an essential mineral nutrient found at high levels in plants such as soy and rice. Excessive Mn exposure increases the risk of adverse neurological effects. We analysed, by atomic absorption spectrometry, the Mn content of 36 SB, 5 RB, 6 evaporated milks (EM), 14 soy-based infant formulas (SF) and 16 milk-based infant formulas (MF), obtained from commercial outlets in Ottawa, Canada. SB had the highest levels of Mn (16.5 +/- 8.6 micro g/g dry wt, mean +/- s.d.), followed by RB (9.9 +/- 1.7 micro g/g dry wt). Mn levels of individual SB/RB ranged from 2 to 17 times the mean Mn content of SF (2.4 +/- 0.7 micro g/g dry wt) and 7 to 56 times that of MF (0.70 +/- 0.35 micro g/g dry wt). EM contained very little Mn (0.02 +/- 0.03 micro g/g dry wt). Calculated mean Mn intakes from SB/RB by infants up to 6 months of age, assuming complete substitution of these products (0.78 L/day), approached the Tolerable Upper Intake Level (UL) for 1-3 year olds (no UL for Mn is available for infants under 1 year of age). Expressed as micro g Mn/100 kcal, SB/RB exceeded the range derived from ULs and typical energy intakes of 1-3 year olds. SB/RB should not be fed to infants because they are nutritionally inadequate and contain Mn at levels which may present an increased risk of adverse neurological effects if used as a sole source of nutrition.

Immunization with herpes simplex virus 2 (HSV-2) genes plus inactivated HSV-2 is highly protective against acute and recurrent HSV-2 disease.

PubMed

Morello, Christopher S; Levinson, Michael S; Kraynyak, Kimberly A; Spector, Deborah H

2011-04-01

To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.

Expanding the 2011 Prague, OK Event Catalog: Detections, Relocations, and Stress Drop Estimates

NASA Astrophysics Data System (ADS)

Clerc, F.; Cochran, E. S.; Dougherty, S. L.; Keranen, K. M.; Harrington, R. M.

2016-12-01

The Mw 5.6 earthquake occurring on 6 Nov. 2011, near Prague, OK, is thought to have been triggered by a Mw 4.8 foreshock, which was likely induced by fluid injection into local wastewater disposal wells [Keranen et al., 2013; Sumy et al., 2014]. Previous stress drop estimates for the sequence have suggested values lower than those for most Central and Eastern U.S. tectonic events of similar magnitudes [Hough, 2014; Sun & Hartzell, 2014; Sumy & Neighbors et al., 2016]. Better stress drop estimates allow more realistic assessment of seismic hazard and more effective regulation of wastewater injection. More reliable estimates of source properties may help to differentiate induced events from natural ones. Using data from local and regional networks, we perform event detections, relocations, and stress drop calculations of the Prague aftershock sequence. We use the Match & Locate method, a variation on the matched-filter method which detects events of lower magnitudes by stacking cross-correlograms from different stations [Zhang & Wen, 2013; 2015], in order to create a more complete catalog from 6 Nov to 31 Dec 2011. We then relocate the detected events using the HypoDD double-difference algorithm. Using our enhanced catalog and relocations, we examine the seismicity distribution for evidence of migration and investigate implications for triggering mechanisms. To account for path and site effects, we calculate stress drops using the Empirical Green's Function (EGF) spectral ratio method, beginning with 2730 previously relocated events. We determine whether there is a correlation between the stress drop magnitudes and the spatial and temporal distribution of events, including depth, position relative to existing faults, and proximity to injection wells. Finally, we consider the range of stress drop values and scaling with respect to event magnitudes within the context of previously published work for the Prague sequence as well as other induced and natural sequences.

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

PubMed

Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

2016-03-10

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

Modeling fortification of corn masa flour with folic acid: the potential impact on exceeding the tolerable upper intake level for folic acid, NHANES 2001–2008

PubMed Central

Hamner, Heather C.; Tinker, Sarah C.; Berry, R.J.; Mulinare, Joe

2013-01-01

Background The Institute of Medicine set a tolerable upper intake level (UL) for usual daily total folic acid intake (1,000 µg). Less than 3% of US adults currently exceed the UL. Objective The objective of this study was to determine if folic acid fortification of corn masa flour would increase the percentage of the US population who exceed the UL. Design We used dietary intake data from NHANES 2001–2008 to estimate the percentage of adults and children who would exceed the UL if corn masa flour were fortified at 140 µg of folic acid/100 g. Results In 2001–2008, 2.5% of the US adult population (aged≥19 years) exceeded the UL, which could increase to 2.6% if fortification of corn masa flour occurred. With corn masa flour fortification, percentage point increases were small and not statistically significant for US adults exceeding the UL regardless of supplement use, sex, race/ethnicity, or age. Children aged 1–8 years, specifically supplement users, were the most likely to exceed their age-specific UL. With fortification of corn masa flour, there were no statistically significant increases in the percentage of US children who were exceeding their age-specific UL, and the percentage point increases were small. Conclusions Our results suggest that fortification of corn masa flour would not significantly increase the percentage of individuals who would exceed the UL. Supplement use was the main factor related to exceeding the UL with or without fortification of corn masa flour and within all strata of sex, race/ethnicity, and age group. PMID:23316130

Model-based iterative reconstruction and adaptive statistical iterative reconstruction: dose-reduced CT for detecting pancreatic calcification.

PubMed

Yasaka, Koichiro; Katsura, Masaki; Akahane, Masaaki; Sato, Jiro; Matsuda, Izuru; Ohtomo, Kuni

2016-01-01

Iterative reconstruction methods have attracted attention for reducing radiation doses in computed tomography (CT). To investigate the detectability of pancreatic calcification using dose-reduced CT reconstructed with model-based iterative construction (MBIR) and adaptive statistical iterative reconstruction (ASIR). This prospective study approved by Institutional Review Board included 85 patients (57 men, 28 women; mean age, 69.9 years; mean body weight, 61.2 kg). Unenhanced CT was performed three times with different radiation doses (reference-dose CT [RDCT], low-dose CT [LDCT], ultralow-dose CT [ULDCT]). From RDCT, LDCT, and ULDCT, images were reconstructed with filtered-back projection (R-FBP, used for establishing reference standard), ASIR (L-ASIR), and MBIR and ASIR (UL-MBIR and UL-ASIR), respectively. A lesion (pancreatic calcification) detection test was performed by two blinded radiologists with a five-point certainty level scale. Dose-length products of RDCT, LDCT, and ULDCT were 410, 97, and 36 mGy-cm, respectively. Nine patients had pancreatic calcification. The sensitivity for detecting pancreatic calcification with UL-MBIR was high (0.67-0.89) compared to L-ASIR or UL-ASIR (0.11-0.44), and a significant difference was seen between UL-MBIR and UL-ASIR for one reader (P = 0.014). The area under the receiver-operating characteristic curve for UL-MBIR (0.818-0.860) was comparable to that for L-ASIR (0.696-0.844). The specificity was lower with UL-MBIR (0.79-0.92) than with L-ASIR or UL-ASIR (0.96-0.99), and a significant difference was seen for one reader (P < 0.01). In UL-MBIR, pancreatic calcification can be detected with high sensitivity, however, we should pay attention to the slightly lower specificity.

Identification of host cell proteins which interact with herpes simplex virus type 1 tegument protein pUL37.

PubMed

Kelly, Barbara J; Diefenbach, Eve; Fraefel, Cornel; Diefenbach, Russell J

2012-01-20

The herpes simplex virus type 1 (HSV-1) structural tegument protein pUL37, which is conserved across the Herpesviridae family, is known to be essential for secondary envelopment during the egress of viral particles. To shed light on additional roles of pUL37 during viral replication a yeast two-hybrid screen of a human brain cDNA library was undertaken. This screen identified ten host cell proteins as potential pUL37 interactors. One of the interactors, serine threonine kinase TAOK3, was subsequently confirmed to interact with pUL37 using an in vitro pulldown assay. Such host cell/pUL37 interactions provide further insights into the multifunctional role of this herpesviral tegument protein. Copyright © 2011 Elsevier Inc. All rights reserved.

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth*

PubMed Central

Mertz, Joseph; Tan, Haiyan; Pagala, Vishwajeeth; Bai, Bing; Chen, Ping-Chung; Li, Yuxin; Cho, Ji-Hoon; Shaw, Timothy; Wang, Xusheng; Peng, Junmin

2015-01-01

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development. PMID:25931508

Association of FAS A-670G Polymorphism and Risk of Uterine Leiomyoma in a Southeast Iranian Population

PubMed Central

Mohammadpour-Gharehbagh, Abbas; Salimi, Saeedeh; Keshavarzi, Farshid; Zakerian, Sepideh; Sajadian, Mojtaba; Mokhtari, Mojgan

2016-01-01

Background: Uterine leiomyoma (UL) is a benign tumor of uterine smooth muscle that affects women in reproductive ages. FAS has an important role in initial stages of apoptosis. Previous studies have shown an association between the FAS gene and tumorigenesis. In the present study, we evaluated the relationship between FAS A-670G (rs 1800682) and UL risk Methods: The FAS gene polymorphism of 155 women with UL and 157 healthy controls was analyzed by the polymerase chain reaction restriction fragment length polymorphism method Results: The AA, AG, and GG genotype frequencies of the FAS A-670G polymorphism were respectively 37.4, 42.6, and 20% in women with UL, and 46, 42.6, and 11.5% in healthy controls. The risk of UL in women was 1.5-fold greater in GG-genotype women than in AA-genotype women. The G allele frequencies were 41% in women with UL and 33% in healthy controls and statistically different (P = 0.03) Conclusion: The FAS polymorphism was associated with the risk of UL in a sample of Iranian women. PMID:28070535

The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Michael A.; Bucks, Michelle A.; O'Regan, Kevin J.

2008-07-05

The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 formore » capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.« less

Screening and identification of host factors interacting with UL14 of herpes simplex virus 1.

PubMed

Wu, Fuqing; Xing, Junji; Wang, Shuai; Li, Meili; Zheng, Chunfu

2011-08-01

The UL14 protein of herpes simplex virus type 1 (HSV-1) is highly conserved in herpesvirus family. However, its exact function during the HSV-1 replication cycle is little known. In the present study, a high throughput yeast two-hybrid system was employed to screen the cellular factors interacting with UL14, and five target candidates were yielded: (1) TSC22 domain family protein 3 (TSC22D3); (2) Mediator of RNA polymerase II transcription subunit 8 isoform 1(MED8); (3) Runt-related transcription factor 3 (RUNX3); (4) Arrestin beta-2 (ARRB2); (5) Cereblon (CRBN). Indirect immunofluorescent assay showed that both TSC22D3 and MED8 co-localized with UL14. Co-immunoprecipitation assay demonstrated that UL14 could be immunoprecipitated by TSC22D3, suggesting that UL14 interacted with TSC22D3 under physiological condition. In summary, this study opened up new avenues toward delineating the function and physiological significance of UL14 during the HSV-1 replication cycle.

The Cytomegalovirus protein pUL37×1 targets mitochondria to mediate neuroprotection

PubMed Central

Hong, Chien Tai; Chau, Kai-Yin; Schapira, Anthony H. V.

2016-01-01

There is substantial evidence that mitochondrial dysfunction plays a significant role in the pathogenesis of Parkinson disease (PD). This contribution probably encompasses defects of oxidative phosphorylation, mitochondrial turnover (mitophagy), mitochondrial derived oxidative stress, and apoptotic signalling. Human cytomegalovirus immediate-early protein pUL37 × 1 induces Bax mitochondrial translocation and inactivation to prevent apoptosis. Over-expressing pUL37 × 1 in neuronal cells protects against staurosporin and 6-hydroxydopamine induced apoptosis and cell death. Protection is not enhanced by bax silencing in pUL37 × 1 over-expressing cells, suggesting a bax-dependent mechanism of action. pUL37 × 1 increases glycolysis and induces mitochondrial hyperpolarization, a bax independent anti-apoptotic action. pUL37 × 1 increases glycolysis through activation of phosphofructokinase by a calcium-dependent pathway. The dual anti-apoptotic mechanism of pUL37 × 1 may be considered a novel neuroprotective strategy in diseases where mitochondrial dysfunction and apoptotic pathways are involved. PMID:27562039

78 FR 52568 - TUV SUD America, Inc.: Modification of Scope of Recognition

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-23

... three test standards from the scope of recognition of the Nationally Recognized Testing Laboratory (NRTL... standards (1) UL 551 Transformer-type Arc-welding Machine, (2) UL 1484 Residential Gas Detectors, and (3) UL...

Polyamines in the lateral vestibular nuclei of the squirrel monkey and their potential role in vestibular compensation

NASA Technical Reports Server (NTRS)

Henley, C.; Igarashi, M.

1993-01-01

Polyamine synthesis increases in response to injurious stimuli including axotomy and denervation. Reduced eye nystagmus and head-deviation have been observed in unilateral labyrinthectomized (UL) guinea pigs treated with an inhibitor of polyamine synthesis, alpha-difluoromethylornithine (DFMO). We quantified polyamines in the lateral vestibular nuclei (LVN) of control and UL squirrel monkeys during the phase of vestibular compensation (VC) and performed an experiment to determine if DFMO reduces nystagmus previously observed in the guinea pig. Polyamines were detected in the LVN of control and UL squirrel monkeys. Putrescine and spermidine increased in the ipsilateral LVN 3 days after UL with no change in the contralateral LVN. No left-right differences were noted in the 5-day post-UL monkey. DFMO reduced nystagmus in a UL squirrel monkey. These findings suggest that polyamines are important in vestibular function and may contribute to nystagmus observed in VC.

Procedures involving lipid media for detection of bacterial contamination in breweries.

PubMed

Van Vuuren, H J; Louw, H A; Loos, M A; Meisel, R

1977-02-01

The liquid equivalent of universal beer agar, designated universal beer liquid medium, and its beer-free equivalent, universal liquid medium (UL), were equally effective in demonstrating bacterial contamination in 120 of 200 samples from different stages of commercial brewing process. Growth of the contaminants after 3 days was consistently more luxuriant in the UL medium. A yeast-water substrate medium failed to reveal many contaminants detected with UL in 392 samples from three breweries and revealed only a few not detected with UL. The use of UL and a lactose-peptone medium, with microscope examination of the media for bacterial growth, permitted detection of 93% of the known contaminants compared to 87%, detected with UL alone; this combination or universal beer liquid medium plus lactose-peptone medium can therefore be recommended for the detection of bacterial contaminants in brewery samples. Bacterial contamination of pitching yeasts appeared to be a particular problem in the breweries investigated.

Histopathology and biochemistry analysis of the interaction between sunitinib and paracetamol in mice.

PubMed

Lim, Adeline Yl; Segarra, Ignacio; Chakravarthi, Srikumar; Akram, Sufyan; Judson, John P

2010-10-15

Sunitinib, a tyrosine kinase inhibitor to treat GIST and mRCC may interact with paracetamol as both undergo P450 mediated biotransformation and P-glycoprotein transport. This study evaluates the effects of sunitinib-paracetamol coadministration on liver and renal function biomarkers and liver, kidney, brain, heart and spleen histopathology. ICR male mice (n = 6 per group/dose) were administered saline (group-A) or paracetamol 500 mg/kg IP (group-B), or sunitinib at 25, 50, 80, 100, 140 mg/kg PO (group-C) or coadministered sunitinib at 25, 50, 80, 100, 140 mg/kg PO and paracetamol IP at fixed dose 500 mg/kg (group-D). Paracetamol was administered 15 min before sunitinib. Mice were sacrificed 4 h post sunitinib administration. Group-A serum ALT and AST levels were 14.29 ± 2.31 U/L and 160.37 ± 24.74 U/L respectively and increased to 249.6 ± 222.7 U/L and 377.1 ± 173.6 U/L respectively in group-B; group-C ALT and AST ranged 36.75-75.02 U/L and 204.4-290.3 U/L respectively. After paracetamol coadministration with low sunitinib doses (group-D), ALT and AST concentrations ranged 182.79-221.03 U/L and 259.7-264.4 U/L respectively, lower than group-B. Paracetamol coadministration with high sunitinib doses showed higher ALT and AST values (range 269.6-349.2 U/L and 430.2-540.3 U/L respectively), p < 0.05. Hepatic histopathology showed vascular congestion in group-B; mild congestion in group-C (but lesser than in group-B and D). In group-D, at low doses of sunitinib, lesser damage than in group-B occurred but larger changes including congestion were observed at high sunitinib doses. BUN levels were higher (p < 0.05) for group-B (33.81 ± 5.68 mg/dL) and group-D (range 35.01 ± 6.95 U/L to 52.85 ± 12.53 U/L) compared to group-A (15.60 ± 2.17 mg/dL) and group-C (range 17.50 ± 1.25 U/L to 26.68 ± 6.05 U/L). Creatinine remained unchanged. Renal congestion and necrosis was lower in group-C than group-B but was higher in group-D (p > 0.05). Mild cardiotoxicity occurred in groups B, C and D. Brain vascular congestion occurred at high doses of sunitinib administered alone or with paracetamol. Hepatic and renal biomarkers correlated with histopathology signs. Paracetamol and sunitinib coadministration may lead to dose dependent outcomes exhibiting mild hepatoprotective effect or increased hepatotoxicity. Sunitinib at high doses show renal, cardiac and brain toxicity. Liver and renal function monitoring is recommended.

Histopathology and biochemistry analysis of the interaction between sunitinib and paracetamol in mice

PubMed Central

2010-01-01

Background Sunitinib, a tyrosine kinase inhibitor to treat GIST and mRCC may interact with paracetamol as both undergo P450 mediated biotransformation and P-glycoprotein transport. This study evaluates the effects of sunitinib-paracetamol coadministration on liver and renal function biomarkers and liver, kidney, brain, heart and spleen histopathology. ICR male mice (n = 6 per group/dose) were administered saline (group-A) or paracetamol 500 mg/kg IP (group-B), or sunitinib at 25, 50, 80, 100, 140 mg/kg PO (group-C) or coadministered sunitinib at 25, 50, 80, 100, 140 mg/kg PO and paracetamol IP at fixed dose 500 mg/kg (group-D). Paracetamol was administered 15 min before sunitinib. Mice were sacrificed 4 h post sunitinib administration. Results Group-A serum ALT and AST levels were 14.29 ± 2.31 U/L and 160.37 ± 24.74 U/L respectively and increased to 249.6 ± 222.7 U/L and 377.1 ± 173.6 U/L respectively in group-B; group-C ALT and AST ranged 36.75-75.02 U/L and 204.4-290.3 U/L respectively. After paracetamol coadministration with low sunitinib doses (group-D), ALT and AST concentrations ranged 182.79-221.03 U/L and 259.7-264.4 U/L respectively, lower than group-B. Paracetamol coadministration with high sunitinib doses showed higher ALT and AST values (range 269.6-349.2 U/L and 430.2-540.3 U/L respectively), p < 0.05. Hepatic histopathology showed vascular congestion in group-B; mild congestion in group-C (but lesser than in group-B and D). In group-D, at low doses of sunitinib, lesser damage than in group-B occurred but larger changes including congestion were observed at high sunitinib doses. BUN levels were higher (p < 0.05) for group-B (33.81 ± 5.68 mg/dL) and group-D (range 35.01 ± 6.95 U/L to 52.85 ± 12.53 U/L) compared to group-A (15.60 ± 2.17 mg/dL) and group-C (range 17.50 ± 1.25 U/L to 26.68 ± 6.05 U/L). Creatinine remained unchanged. Renal congestion and necrosis was lower in group-C than group-B but was higher in group-D (p > 0.05). Mild cardiotoxicity occurred in groups B, C and D. Brain vascular congestion occurred at high doses of sunitinib administered alone or with paracetamol. Hepatic and renal biomarkers correlated with histopathology signs. Conclusions Paracetamol and sunitinib coadministration may lead to dose dependent outcomes exhibiting mild hepatoprotective effect or increased hepatotoxicity. Sunitinib at high doses show renal, cardiac and brain toxicity. Liver and renal function monitoring is recommended. PMID:20950441

Factors affecting the loss of MED12-mutated leiomyoma cells during in vitro growth.

PubMed

Bloch, Jeannine; Holzmann, Carsten; Koczan, Dirk; Helmke, Burkhard Maria; Bullerdiek, Jörn

2017-05-23

Uterine leiomyomas (UL) are the most prevalent symptomatic human tumors at all and somatic mutations of the gene encoding mediator subcomplex 12 (MED12) constitute the most frequent driver mutations in UL. Recently, a rapid loss of mutated cells during in vitro growth of UL-derived cell cultures was reported, resulting in doubts about the benefits of UL-derived cell cultures. To evaluate if the rapid loss of MED12-mutated cells in UL cell cultures depends on in vitro passaging, we set up cell cultures from nine UL from 40-50 year old Caucasian patients with at least one UL. Cultured UL cells were investigated for loss of MED12-mutated cells. Genetic characterization of native tumor samples and adjacent myometrium was done by array analysis. "Aged" primary cultures without passaging were compared to cells of three subsequent passages. Comparative analyses of the mutated/non-mutated ratios between native tissue, primary cells, and cultured tumor cells revealed a clear decrease of MED12-mutated cells. None of the tumors showed gross alterations of the array profiles, excluding the presence of gross genomic imbalances besides the MED12 mutations as a reason for the intertumoral variation in the loss of MED12-mutated cells. Albeit at a lesser rate, loss of MED12-mutated cells from cell cultures of UL occurs even without passaging thus indicating the requirement of soluble factors or matrix components lacking in vitro. Identification of these factors can help to understand the mechanisms of the growth of the most frequent type of uterine leiomyomas and to decipher novel drug targets.

Antiobesity effects of Undaria lipid capsules prepared with scallop phospholipids.

PubMed

Okada, Tomoko; Mizuno, Yasuyuki; Sibayama, Shinichi; Hosokawa, Masashi; Miyashita, Kazuo

2011-01-01

Based on previous research findings, a capsule was developed containing n-3 polyunsaturated fatty acid rich scallop phospholipids (PLs) with an incorporation of brown seaweed (Undaria pinnatifida) lipids (ULs) containing fucoxanthin. The antiobesity effects of the capsules were evaluated with an animal model using 3-wk-old male KK-A(y) mice. Each group received different combinations of lipid (UL, PL, UL + PL, or UL + PL capsule) either incorporated into the diet or into drinking water. Animals were sacrificed after a 4-wk experimental feeding period, and adipose tissues and organs were dissected and weighed. Blood samples were obtained to determine plasma lipid profiles. Uncoupling protein 1 (UCP1) mRNA expression levels were determined by real-time polymerase chain reaction analysis, and UCP1 expression was determined by western blotting analysis. Treatment with either UL alone or UL + PL (capsule) through drinking water resulted in a significant reduction in body weight, compared to the control group. The total white adipose tissue weight of mice fed the UL + PL capsule in drinking water was significantly reduced. Both UCP1 and UCP1 mRNA expression in epididymal fat from mice fed the capsule were significantly higher than in the control group. These results suggest that incorporation of UL into scallop-derived PL by means of capsulation may lead to an additive increase in the antiobesity properties of these bioactive lipids.

African Ancestry and Genetic Risk for Uterine Leiomyomata

PubMed Central

Wise, Lauren A.; Ruiz-Narvaez, Edward A.; Palmer, Julie R.; Cozier, Yvette C.; Tandon, Arti; Patterson, Nick; Radin, Rose G.; Rosenberg, Lynn; Reich, David

2012-01-01

Rates of uterine leiomyomata (UL) are 2–3 times higher in African Americans than in European Americans. It is unclear whether inherited factors explain the ethnic disparity. To investigate the presence of risk alleles for UL that are highly differentiated in frequency between African Americans and European Americans, the authors conducted an admixture-based genome-wide scan of 2,453 UL cases confirmed by ultrasound or surgery in the Black Women's Health Study (1997–2009), a national prospective cohort study. Controls (n = 2,102) were women who did not report a UL diagnosis through 2009. Mean percentage of European ancestry was significantly lower among cases (20.00%) than among controls (21.63%; age-adjusted mean difference = −1.76%, 95% confidence interval: −2.40, −1.12; P < 0.0001), and the association was stronger in younger cases. Admixture analyses showed suggestive evidence of association at chromosomes 2, 4, and 10. The authors also genotyped a dense set of tag single nucleotide polymorphisms at different loci associated with UL in Japanese women but failed to replicate the associations. This suggests that genetic variation for UL differs in populations with and without African ancestry. The admixture findings further indicate that no single highly differentiated locus is responsible for the ethnic disparity in UL, raising the possibility that multiple variants jointly contribute to the higher incidence of UL in African Americans. PMID:23161897

Progesterone is essential for maintenance and growth of uterine leiomyoma.

PubMed

Ishikawa, Hiroshi; Ishi, Kazutomo; Serna, Vanida Ann; Kakazu, Rafael; Bulun, Serdar E; Kurita, Takeshi

2010-06-01

Uterine leiomyomata (ULs) represent the most common tumor in women and can cause abnormal uterine bleeding, large pelvic masses, and recurrent pregnancy loss. Although the dependency of UL growth on ovarian steroids is well established, the relative contributions of 17beta-estradiol and progesterone are yet to be clarified. Conventionally, estradiol has been considered the primary stimulus for UL growth, and studies with cell culture and animal models support this concept. In contrast, no research model has clearly demonstrated a requirement of progesterone in UL growth despite accumulating clinical evidence for the essential role of progesterone in this tumor. To elucidate the functions of ovarian steroids in UL, we established a xenograft model reflecting characteristics of these tumors by grafting human UL tissue beneath the renal capsule of immunodeficient mice. Leiomyoma xenografts increased in size in response to estradiol plus progesterone through cell proliferation and volume increase in cellular and extracellular components. The xenograft growth induced by estradiol plus progesterone was blocked by the antiprogestin RU486. Furthermore, the volume of established UL xenografts decreased significantly after progesterone withdrawal. Surprisingly, treatment with estradiol alone neither increased nor maintained the tumor size. Although not mitogenic by itself, estradiol induced expression of progesterone receptor and supported progesterone action on leiomyoma xenografts. Taken together, our findings define that volume maintenance and growth of human UL are progesterone dependent.

Domain Interaction Studies of Herpes Simplex Virus 1 Tegument Protein UL16 Reveal Its Interaction with Mitochondria

PubMed Central

Chadha, Pooja; Sarfo, Akua; Zhang, Dan; Abraham, Thomas; Carmichael, Jillian

2016-01-01

ABSTRACT The UL16 tegument protein of herpes simplex virus 1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pairwise transfections, UL16 binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and the CTD. Here we report that the separated and coexpressed functional domains of UL16 are mutually responsive to each other in transfected cells and form complexes that are stable enough to be captured in coimmunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined. IMPORTANCE The HSV-1-encoded tegument protein UL16 is involved in multiple events of the virus replication cycle, ranging from virus assembly to cell-cell spread of the virus, and hence it can serve as an important drug target. Unfortunately, a lack of both structural and functional information limits our understanding of this protein. The discovery of domain interactions within UL16 and the novel ability of UL16 to interact with mitochondria in HSV-infected cells lays a foundational framework for future investigations aimed at deciphering the structure and function of not just UL16 of HSV-1 but also its homologs in other herpesviruses. PMID:27847362

The Product of the Herpes Simplex Virus Type 1 UL25 Gene Is Required for Encapsidation but Not for Cleavage of Replicated Viral DNA

PubMed Central

McNab, Alistair R.; Desai, Prashant; Person, Stan; Roof, Lori L.; Thomsen, Darrell R.; Newcomb, William W.; Brown, Jay C.; Homa, Fred L.

1998-01-01

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278–283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246–259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA. PMID:9445000

CHEMINFORMATICS TOOLS FOR TOXICANT CHARACTERIZATION

EPA Science Inventory

• Continued development of the Shape Signatures method is planed.  This effort will include further development of the Shape Signatures database of PDB-extracted ligands and of the clustering algorithm.

• In addition, we plan to develop an...

• 78 FR 70349 - Proposed Revision of Policy for Incorporating New Test Standards Into the List of Appropriate...

  Federal Register 2010, 2011, 2012, 2013, 2014

  2013-11-25

  ... Use with Low Energy Products. UL 6142 Small Wind Turbine Systems. UL 6420 Equipment Used for System... Seasonal-Use Cord-Connected Wiring Devices. UL 2560 Emergency Call Systems for Assisted Living and...

• Model-based iterative reconstruction and adaptive statistical iterative reconstruction: dose-reduced CT for detecting pancreatic calcification

  PubMed Central

  Katsura, Masaki; Akahane, Masaaki; Sato, Jiro; Matsuda, Izuru; Ohtomo, Kuni

  2016-01-01

  Background Iterative reconstruction methods have attracted attention for reducing radiation doses in computed tomography (CT). Purpose To investigate the detectability of pancreatic calcification using dose-reduced CT reconstructed with model-based iterative construction (MBIR) and adaptive statistical iterative reconstruction (ASIR). Material and Methods This prospective study approved by Institutional Review Board included 85 patients (57 men, 28 women; mean age, 69.9 years; mean body weight, 61.2 kg). Unenhanced CT was performed three times with different radiation doses (reference-dose CT [RDCT], low-dose CT [LDCT], ultralow-dose CT [ULDCT]). From RDCT, LDCT, and ULDCT, images were reconstructed with filtered-back projection (R-FBP, used for establishing reference standard), ASIR (L-ASIR), and MBIR and ASIR (UL-MBIR and UL-ASIR), respectively. A lesion (pancreatic calcification) detection test was performed by two blinded radiologists with a five-point certainty level scale. Results Dose-length products of RDCT, LDCT, and ULDCT were 410, 97, and 36 mGy-cm, respectively. Nine patients had pancreatic calcification. The sensitivity for detecting pancreatic calcification with UL-MBIR was high (0.67–0.89) compared to L-ASIR or UL-ASIR (0.11–0.44), and a significant difference was seen between UL-MBIR and UL-ASIR for one reader (P = 0.014). The area under the receiver-operating characteristic curve for UL-MBIR (0.818–0.860) was comparable to that for L-ASIR (0.696–0.844). The specificity was lower with UL-MBIR (0.79–0.92) than with L-ASIR or UL-ASIR (0.96–0.99), and a significant difference was seen for one reader (P < 0.01). Conclusion In UL-MBIR, pancreatic calcification can be detected with high sensitivity, however, we should pay attention to the slightly lower specificity. PMID:27110389

• Identification of Arabidopsis MYB56 as a novel substrate for CRL3BPM E3 ligases.

  PubMed

  Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

  2014-10-24

  Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies pointed out that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling the flowering time point in plants. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

• The non-essential UL50 gene of avian infectious laryngotracheitis virus encodes a functional dUTPase which is not a virulence factor.

  PubMed

  Fuchs, W; Ziemann, K; Teifke, J P; Werner, O; Mettenleiter, T C

  2000-03-01

  The DNA sequence of the infectious laryngotracheitis virus (ILTV) UL50, UL51 and UL52 gene homologues was determined. Although the deduced UL50 protein lacks the first of five conserved domains of the corresponding proteins of mammalian alphaherpesviruses, the ILTV gene product was also shown to possess dUTPase activity. The generation of UL50-negative ILTV mutants was facilitated by recombination plasmids encoding green fluorescent protein (GFP), and expression constructs of predicted transactivator proteins of ILTV (alphaTIF, ICP4) were successfully used to increase the infectivity of viral genomic DNA. A GFP-expressing UL50-deletion mutant of ILTV showed reduced cell-to-cell spread in vitro, and was attenuated in vivo. A similar deletion mutant without the foreign gene, however, propagated like wild-type ILTV in cell culture and was pathogenic in chickens. We conclude that the viral dUTPase is not required for efficient replication of ILTV in the respiratory tract of infected animals. The replication defect of the GFP-expressing ILTV recombinant is most likely caused by toxic effects of the reporter gene product, since spontaneously occurring inactivation mutants exhibited wild-type-like growth.

• Identification of structural protein-protein interactions of herpes simplex virus type 1.

  PubMed

  Lee, Jin H; Vittone, Valerio; Diefenbach, Eve; Cunningham, Anthony L; Diefenbach, Russell J

  2008-09-01

  In this study we have defined protein-protein interactions between the structural proteins of herpes simplex virus type 1 (HSV-1) using a LexA yeast two-hybrid system. The majority of the capsid, tegument and envelope proteins of HSV-1 were screened in a matrix approach. A total of 40 binary interactions were detected including 9 out of 10 previously identified tegument-tegument interactions (Vittone, V., Diefenbach, E., Triffett, D., Douglas, M.W., Cunningham, A.L., and Diefenbach, R.J., 2005. Determination of interactions between tegument proteins of herpes simplex virus type 1. J. Virol. 79, 9566-9571). A total of 12 interactions involving the capsid protein pUL35 (VP26) and 11 interactions involving the tegument protein pUL46 (VP11/12) were identified. The most significant novel interactions detected in this study, which are likely to play a role in viral assembly, include pUL35-pUL37 (capsid-tegument), pUL46-pUL37 (tegument-tegument) and pUL49 (VP22)-pUS9 (tegument-envelope). This information will provide further insights into the pathways of HSV-1 assembly and the identified interactions are potential targets for new antiviral drugs.

• The Tegument Protein UL71 of Human Cytomegalovirus Is Involved in Late Envelopment and Affects Multivesicular Bodies ▿

  PubMed Central

  Schauflinger, Martin; Fischer, Daniela; Schreiber, Andreas; Chevillotte, Meike; Walther, Paul; Mertens, Thomas; von Einem, Jens

  2011-01-01

  Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV. PMID:21289123

• Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

  DOE Office of Scientific and Technical Information (OSTI.GOV)

  Geiss, Brian J.; Cano, Gina L.; Tavis, John E.

  2004-12-05

  Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22.more » Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.« less

• Ultra Low Sulfur Home Heating Oil Demonstration Project

  DOE Office of Scientific and Technical Information (OSTI.GOV)

  Batey, John E.; McDonald, Roger

  2015-09-30

  This Ultra Low Sulfur (ULS) Home Heating Oil Demonstration Project was funded by the New York State Energy Research and Development Authority (NYSERDA) and has successfully quantified the environmental and economic benefits of switching to ULS (15 PPM sulfur) heating oil. It advances a prior field study of Low Sulfur (500 ppm sulfur) heating oil funded by NYSERDA and laboratory research conducted by Brookhaven National Laboratory (BNL) and Canadian researchers. The sulfur oxide and particulate matter (PM) emissions are greatly reduced as are boiler cleaning costs through extending cleaning intervals. Both the sulfur oxide and PM emission rates are directlymore » related to the fuel oil sulfur content. The sulfur oxide and PM emission rates approach near-zero levels by switching heating equipment to ULS fuel oil, and these emissions become comparable to heating equipment fired by natural gas. This demonstration project included an in-depth review and analysis of service records for both the ULS and control groups to determine any difference in the service needs for the two groups. The detailed service records for both groups were collected and analyzed and the results were entered into two spreadsheets that enabled a quantitative side-by-side comparison of equipment service for the entire duration of the ULS test project. The service frequency for the ULS and control group were very similar and did indicate increased service frequency for the ULS group. In fact, the service frequency with the ULS group was slightly less (7.5 percent) than the control group. The only exception was that three burner fuel pump required replacement for the ULS group and none were required for the control group.« less

• Optimized enzyme-linked immunosorbent assay for detecting cytomegalovirus infections during clinical trials of recombinant vaccines.

  PubMed

  Pagnon, Anke; Piras, Fabienne; Gimenez-Fourage, Sophie; Dubayle, Joseline; Arnaud-Barbe, Nadège; Hessler, Catherine; Caillet, Catherine

  2017-11-01

  In clinical trials of cytomegalovirus (CMV) glycoprotein B (gB) vaccines, CMV infection is detected by first depleting serum of anti-gB antibodies and then measuring anti-CMV antibodies with a commercially available enzyme-linked immunosorbent assay (ELISA) kit, with confirmation of positive findings by immunoblot. Identification of CMV immunoantigens for the development of an ELISA that detects specifically CMV infection in clinical samples from individuals immunized with gB vaccines. Sensitivity and specificity of ELISAs using antigenic regions of CMV proteins UL83/pp65, UL99/pp28, UL44/pp52, UL80a/pp38, UL57, and UL32/pp150 were measured. An IgG ELISA using a UL32/pp150 [862-1048] capture peptide was the most specific (93.7%) and sensitive (96.4%) for detecting CMV-specific antibodies in sera. The ELISA successfully detected CMV-specific antibodies in 22 of 22 sera of subjects who had been vaccinated with a gB vaccine but who had later been infected with CMV. The ELISA was linear over a wide range of CMV concentrations (57-16,814 ELISA units/mL) and was reproducible as indicated by a 5% intra-day and 7% inter-day coefficients of variation. The signal was specifically competed by UL32/pp150 [862-1048] peptide but not by CMV-gB or herpes simplex virus 2 glycoprotein D. Lipid and hemoglobin matrix did not interfere with the assay. The UL32/pp150 [862-1048] IgG ELISA can be used for the sensitive and specific detection of CMV infection in gB-vaccinated individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

• Identification of conserved amino acids in the herpes simplex virus type 1 UL8 protein required for DNA synthesis and UL52 primase interaction in the virus replisome.

  PubMed

  Muylaert, Isabella; Zhao, Zhiyuan; Andersson, Torbjörn; Elias, Per

  2012-09-28

  We have used oriS-dependent transient replication assays to search for species-specific interactions within the herpes simplex virus replisome. Hybrid replisomes derived from herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1) failed to support DNA replication in cells. Moreover, the replisomes showed a preference for their cognate origin of replication. The results demonstrate that the herpesvirus replisome behaves as a molecular machine relying on functionally important interactions. We then searched for functional interactions in the replisome context by subjecting HSV-1 UL8 protein to extensive mutagenesis. 52 mutants were made by replacing single or clustered charged amino acids with alanines. Four mutants showed severe replication defects. Mutant A23 exhibited a lethal phenotype, and mutants A49, A52 and A53 had temperature-sensitive phenotypes. Mutants A49 and A53 did not interact with UL52 primase as determined by co-immunoprecipitation experiments. Using GFP-tagged UL8, we demonstrate that all mutants were unable to support formation of ICP8-containing nuclear replication foci. Extended mutagenesis suggested that a highly conserved motif corresponding to mutant A49 serves an important role for establishing a physical contact between UL8 and UL52. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8.

• An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors.

  PubMed

  Nguyen, Thanh; Ho, Minh; Ghosh, Ambarnil; Kim, Truc; Yun, Sun Il; Lee, Seung Seo; Kim, Kyeong Kyu

  2016-10-07

  The ubiquitin pathway plays a critical role in regulating diverse biological processes, and its dysregulation is associated with various diseases. Therefore, it is important to have a tool that can control the ubiquitin pathway in order to improve understanding of this pathway and to develop therapeutics against relevant diseases. We found that Chicago Sky Blue 6B binds directly to the β-groove, a major interacting surface of ubiquitin. Hence, it could successfully inhibit the enzymatic activity of ubiquitin processing enzymes and the binding of ubiquitin to the CXCR4, a cell surface ubiquitin receptor. Furthermore, we demonstrated that this ubiquitin binding chemical could effectively suppress the ubiquitin induced cancer cell migration by blocking ubiquitin-CXCR4 interaction. Current results suggest that ubiquitin binding molecules can be developed as inhibitors of ubiquitin-protein interactions, which will have the value not only in unveiling the biological role of ubiquitin but also in treating related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

• CIDR

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  * Minimum # Experimental Samples DNA Volume (ul) Genomic DNA Concentration (ng/ul) Low Input DNA Volume (ul . **Please inquire about additional cost for low input option. Genotyping Minimum # Experimental Samples DNA sample quality. If you do submit WGA samples, you should anticipate a higher non-random missing data rate

• Novel versatile smart phone based Microplate readers for on-site diagnoses.

  PubMed

  Fu, Qiangqiang; Wu, Ze; Li, Xiuqing; Yao, Cuize; Yu, Shiting; Xiao, Wei; Tang, Yong

  2016-07-15

  Microplate readers are important diagnostic instruments, used intensively for various readout test kits (biochemical analysis kits and ELISA kits). However, due to their expensive and non-portability, commercial microplate readers are unavailable for home testing, community and rural hospitals, especially in developing countries. In this study, to provide a field-portable, cost-effective and versatile diagnostic tool, we reported a novel smart phone based microplate reader. The basic principle of this devise relies on a smart phone's optical sensor that measures transmitted light intensities of liquid samples. To prove the validity of these devises, developed smart phone based microplate readers were applied to readout results of various analytical targets. These targets included analanine aminotransferase (ALT; limit of detection (LOD) was 17.54 U/L), alkaline phosphatase (AKP; LOD was 15.56 U/L), creatinine (LOD was 1.35μM), bovine serum albumin (BSA; LOD was 0.0041mg/mL), prostate specific antigen (PSA; LOD was 0.76pg/mL), and ractopamine (Rac; LOD was 0.31ng/mL). The developed smart phone based microplate readers are versatile, portable, and inexpensive; they are unique because of their ability to perform under circumstances where resources and expertize are limited. Copyright © 2016 Elsevier B.V. All rights reserved.

• Pseudotumor cerebri secondary to subacute sclerosing panencephalitis.

  PubMed

  Ayçiçek, Ali; Işcan, Akin; Ceçe, Hasan

  2009-05-01

  Unusual presentations are not rare in subacute sclerosing panencephalitis. Five patients initially diagnosed with pseudotumor cerebri were ultimately determined to have pseudotumor cerebri secondary to subacute sclerosing panencephalitis. The present study retrospectively reviewed 56 cases history, neurologic symptoms, and clinical and laboratory data, as well as the outcomes. On admission, five patients (group 1) presenting with pseudotumor cerebri exhibited bilateral papilledema, and in each of them cranial magnetic resonance imaging revealed small lateral ventricles, effacement of the subarachnoid space, and no mass lesion. On admission, 51 patients (group 2) had no pseudotumor cerebri findings. The year of original measles infection, the interval between measles and onset of subacute sclerosing panencephalitis, and initial neurologic symptoms were similar, but length of symptoms before diagnosis of subacute sclerosing panencephalitis was shorter in group 1, and the clinical stage of subacute sclerosing panencephalitis on admission was more advanced in group 2. Cerebrospinal fluid mean open pressure was 378 +/- 22 H(2)O in group 1 and 146 +/- 28 H(2)O in group 2; cerebrospinal fluid antibody was 2038 +/- 768 U/L in group 1 and was 664 +/- 214 U/L in group 2. Only three of the five patients with pseudotumor cerebri had typical periodic discharges on electroencephalographic examination. These findings suggest that subacute sclerosing panencephalitis can cause pseudotumor cerebri.

• Ubiquitin enzymes in the regulation of immune responses.

  PubMed

  Ebner, Petra; Versteeg, Gijs A; Ikeda, Fumiyo

  2017-08-01

  Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.

• Biomarkers of Exposure to Toxic Substances Volume 7: Identification of Potential Serum Protein Biomarkers Indicative of Low Level Kidney Degradation in Response to Toxin Exposures

  DTIC Science & Technology

  2009-05-01

  equilibrated for 4 min with Buffer A with a flow rate of 1 mL/min at room temperature. Once the HPLC lines and MARS column were flushed and equilibrated...ul 4 ) FT mouse control HPLC 10 ul 9) E mouse control Spin Column 10 ul 5) E mouse control HPLC 10 ul 10) Blue MW Standard The distinct...of Low Level Kidney Degradation in Response to Toxin Exposures Christopher L. Woolard Camilla A. Mauzy Biosciences and Protection

• Impact of human cytomegalovirus infection UL55-nested polymerase chain reaction method in hematopoietic stem cell transplant donors and recipients.

  PubMed

  Banan, A A; Yaghobi, R; Ramzi, M; Mehrabani, D

  2009-09-01

  Human cytomegalovirus (HCMV) is one of the most important and critical viral causes of graft rejection among hematopoietic stem cell transplant (HSCT) recipients. Monitoring of this viral infection has a critical role in the management of HSCT clinical complications. In this retrospective cohort, blood (plasma and buffy coat) and urine samples were collected from 110 HSCT patients and 95 donors pretransplantation and weekly for 100 days posttransplantation. An HCMV-optimized UL55-nested polymerase chain reaction (PCR) method was used to detect HCMV infection. Genotyping of the HCMV UL55 gene was performed for all UL55-nested, PCR-positive samples. HSCT donor and recipient laboratory and clinical data were statistically analyzed using SPSS version 15 software. UL55-nested, PCR-positive results were obtained in 3540/4950 (71.5%), 3634/4950 (73.4%), and 3292/4950 (66.5%) of these plasma, buffy coat, and urine samples, respectively. Twenty-five percent of transplant donors were infected with HCMV. An increase in HCMV infection was observed from pre- to post-HSCT conditions. Detection of the gB2 UL55 genotype in most transplant patient samples suggested the need to examine the possible impact of HCMV UL55 genotypes and HCMV infections among stem cell transplant recipients.

• SLAP deficiency decreases dsDNA autoantibody production

  PubMed Central

  Peterson, Lisa K.; Pennington, Luke F.; Shaw, Laura A.; Brown, Meredith; Treacy, Eric C.; Friend, Samantha F.; Hatlevik, Øyvind; Rubtsova, Kira; Rubtsov, Anatoly V.; Dragone, Leonard L.

  2014-01-01

  Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP−/− mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP−/− 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection. PMID:24440645

SLAP deficiency decreases dsDNA autoantibody production.

PubMed

Peterson, Lisa K; Pennington, Luke F; Shaw, Laura A; Brown, Meredith; Treacy, Eric C; Friend, Samantha F; Hatlevik, Øyvind; Rubtsova, Kira; Rubtsov, Anatoly V; Dragone, Leonard L

2014-02-01

Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP(-/-) mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP(-/-) 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection. Copyright © 2013 Elsevier Inc. All rights reserved.

Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System*

PubMed Central

Singh, Rajesh K.; Zerath, Sylvia; Kleifeld, Oded; Scheffner, Martin; Glickman, Michael H.; Fushman, David

2012-01-01

Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of ubiquitin. We show via NMR that structurally, Rub1 and ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/Nedd8 and ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or ubiquitin or both, we examined whether ubiquitin receptors can differentiate between Rub1 and ubiquitin. Surprisingly, Rub1 interacts with proteasome ubiquitin-shuttle proteins comparably to ubiquitin but binds more weakly to a proteasomal ubiquitin receptor Rpn10. We identified Rub1-ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-ubiquitin heterodimers are recognized by various proteasomal ubiquitin shuttles and receptors comparably to K48-linked ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deubiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into ubiquitin pathways, it is recognized essentially like ubiquitin. PMID:23105008

Mmi1, the Yeast Homologue of Mammalian TCTP, Associates with Stress Granules in Heat-Shocked Cells and Modulates Proteasome Activity

PubMed Central

Grousl, Tomas; Stradalova, Vendula; Heeren, Gino; Richter, Klaus; Breitenbach-Koller, Lore; Malinsky, Jan; Hasek, Jiri; Breitenbach, Michael

2013-01-01

As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells. PMID:24204967

A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development*

PubMed Central

Franco, Maribel; Seyfried, Nicholas T.; Brand, Andrea H.; Peng, Junmin; Mayor, Ugo

2011-01-01

Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system. PMID:20861518

Clinical features and risk factors of acute hepatitis E with severe jaundice

PubMed Central

Xu, Bin; Yu, Hai-Bin; Hui, Wei; He, Jia-Li; Wei, Lin-Lin; Wang, Zheng; Guo, Xin-Hui

2012-01-01

AIM: To compares the clinical features of patients infected with hepatitis E virus (HEV) with or without severe jaundice. In addition, the risk factors for HEV infection with severe jaundice were investigated. METHODS: We enrolled 235 patients with HEV into a cross-sectional study using multi-stage sampling to select the study group. Patients with possible acute hepatitis E showing elevated liver enzyme levels were screened for HEV infection using serologic and molecular tools.HEV infection was documented by HEV antibodies and by the detection of HEV-RNA in serum. We used χ2 analysis, Fisher’s exact test, and Student’s t test where appropriate in this study. Significant predictors in the univariate analysis were then included in a forward, stepwise multiple logistic regression model. RESULTS: No significant differences in symptoms, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, or hepatitis B virus surface antigen between the two groups were observed. HEV infected patients with severe jaundice had significantly lower peak serum levels of γ-glutamyl-transpeptidase (GGT) (median: 170.31 U/L vs 237.96 U/L, P = 0.007), significantly lower ALB levels (33.84 g/L vs 36.89 g/L, P = 0.000), significantly lower acetylcholine esterase (CHE) levels (4500.93 U/L vs 5815.28 U/L, P = 0.000) and significantly higher total bile acid (TBA) levels (275.56 μmol/L vs 147.03 μmol/L, P = 0.000) than those without severe jaundice. The median of the lowest point time tended to be lower in patients with severe jaundice (81.64% vs 96.12%, P = 0.000). HEV infected patients with severe jaundice had a significantly higher viral load (median: 134 vs 112, P = 0.025) than those without severe jaundice. HEV infected patients with severe jaundice showed a trend toward longer median hospital stay (38.17 d vs 18.36 d, P = 0.073). Multivariate logistic regression indicated that there were significant differences in age, sex, viral load, GGT, albumin, TBA, CHE, prothrombin index, alcohol overconsumption, and duration of admission between patients infected with acute hepatitis E with and without severe jaundice. CONCLUSION: Acute hepatitis E patients may naturally present with severe jaundice. PMID:23326133

[The effect of urokinase on hepatic fibrogenesis in rats].

PubMed

Wu, Xi-run; Wang, Qi; Wang, Ling; Shi, Shui-sheng; Guo, Wen-dong

2009-12-01

To investigate the effect of urokinase on hepatic fibrogenesis in rats. Hepatic fibrosis was induced in rats by complex pathogenic factors including subcutaneous injections of carbon tetrachloride, alcohol and cholesterol feeding. Animals were randomly divided into 3 groups: normal control group, hepatic fibrosis group (complex pathogenic factors for 6 weeks), UK prevention group (complex pathogenic factors+UK for 6 weeks). The animals were sacrificed at the end of week 6. The expression of alpha-SMA, uPA, PAI-1, TGFb1, TIMP-1, collagen type I and type III proteins in hepatic fibrosis tissue was detected by immunohistochemistry, the expression of PAI-1 and TGFb1 mRNA in the hepatic fibrosis tissue was quantified by real time RT-PCR. The serum levels of hyaluronicacid (HA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (TBil) and the content of liver hydroxyproline (Hyp) were detected using ELISA kits. The serum ALT, AST, TBil, HA and the content of liver Hyp were (46.66+/-6.30) U/L, (126.26+/-31.65) U/L, (31.11+/-4.20) micromol/L, (109.70+/-18.81) microg/L and (0.98+/-0.09) mg/(g liver), respectively, in UK prevention group, which were significantly lower than those [(101.57+/-11.97) U/L, (205.89+/-56.26) U/L, (67.75+/-2.75) micromol/L, (184.43+/-32.36) microg/L and (1.65+/-0.16) mg/(g liver), respectively] in hepatic fibrosis group (q = 3.3801-20.0061, P < 0.01). The levels of a-SMA, collagen type I, type III, TIMP-1, PAI-1, TGFb1 proteins were (299.27+/-37.36), (210.05+/-27.17), (192.94+/-24.48), (213.70+/-32.21), (204.25+/-17.92), (205.97+/-23.81), respectively, in UK prevention group, which were significantly lower than those [(418.83+/-30.21), (323.77+/-21.53), (302.37+/-31.43), (376.63+/-25.19), (313.53+/-26.67) and (327.42+/-36.75), respectively] in hepatic fibrosis group. The level of uPA protein was increased, and the expression of PAI-1, TGFb1 mRNA in hepatic fibrosis tissue was decreased in UK prevention group. In the early stage of hepatic fibrogenesis, urokinase can attenuate the progression of rat hepatic fibrosis via upregulation of uPA, downregulation of TGFb1, and inhibition of HSC activation.

Ubiquitin enzymes in the regulation of immune responses

PubMed Central

Ebner, Petra; Versteeg, Gijs A.; Ikeda, Fumiyo

2017-01-01

Abstract Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses. PMID:28524749

Noncovalent Ubiquitin Interactions Regulate the Catalytic Activity of Ubiquitin Writers.

PubMed

Wright, Joshua D; Mace, Peter D; Day, Catherine L

2016-11-01

Covalent modification of substrate proteins with ubiquitin is the end result of an intricate network of protein-protein interactions. The inherent ability of the E1, E2, and E3 proteins of the ubiquitylation cascade (the ubiquitin writers) to interact with ubiquitin facilitates this process. Importantly, contact between ubiquitin and the E2/E3 writers is required for catalysis and the assembly of chains of a given linkage. However, ubiquitin is also an activator of ubiquitin-writing enzymes, with many recent studies highlighting the ability of ubiquitin to regulate activity and substrate modification. Here, we review the interactions between ubiquitin-writing enzymes and regulatory ubiquitin molecules that promote activity, and highlight the potential of these interactions to promote processive ubiquitin transfer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intake of fruit, vegetables, and carotenoids in relation to risk of uterine leiomyomata1234

PubMed Central

Radin, Rose G; Palmer, Julie R; Kumanyika, Shiriki K; Boggs, Deborah A; Rosenberg, Lynn

2011-01-01

Background: US black women have higher rates of uterine leiomyomata (UL) and lower intakes of fruit and vegetables than do white women. Whether fruit and vegetable intake is associated with UL in black women has not been studied. Objective: We assessed the association of dietary intake of fruit, vegetables, carotenoids, folate, fiber, and vitamins A, C, and E with UL in the Black Women's Health Study. Design: In this prospective cohort study, we followed 22,583 premenopausal women for incident UL (1997–2009). Diet was estimated by using food-frequency questionnaires in 1995 and 2001. Cox regression was used to derive incidence rate ratios (IRRs) and 95% CIs for the association between each dietary variable (in quintiles) and UL. Results: There were 6627 incident cases of UL diagnosed by ultrasonography (n = 4346) or surgery (n = 2281). Fruit and vegetable intake was inversely associated with UL (≥4 compared with <1 serving/d; IRR: 0.90; 95% CI: 0.82, 0.98; P-trend = 0.03). The association was stronger for fruit (≥2 servings/d compared with <2 servings/wk; IRR: 0.89; 95% CI: 0.81, 0.98; P-trend = 0.07) than for vegetables (≥2 servings/d compared with <4 servings/wk: IRR: 0.97; 95% CI: 0.89, 1.05; P-trend = 0.51). Citrus fruit intake was inversely associated with UL (≥3 servings/wk compared with <1 serving/mo: IRR: 0.92; 95% CI: 0.86, 1.00; P-trend = 0.01). The inverse association for dietary vitamin A (upper compared with lower quintiles: IRR: 0.89; 95% CI: 0.83, 0.97; P-trend = 0.01) appeared to be driven by preformed vitamin A (animal sources), not provitamin A (fruit and vegetable sources). UL was not materially associated with dietary intake of vitamins C and E, folate, fiber, or any of the carotenoids, including lycopene. Conclusion: These data suggest a reduced risk of UL among women with a greater dietary intake of fruit and preformed vitamin A. PMID:22071705

Dissecting the herpesvirus architecture by targeted proteolysis.

PubMed

Daniel, Gina R; Pegg, Caitlin E; Smith, Gregory A

2018-06-13

Herpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of varying amounts of twenty or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions of extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein: a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed in situ by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36 whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid. IMPORTANCE: Neuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals, but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument and with it, begin to tease apart the protein interactions that underlie this complex layer of the virion architecture. Copyright © 2018 American Society for Microbiology.

Comparative Assessment of Off-label and Unlicensed Drug Prescriptions in Children: FDA Versus ANSM Guidelines.

PubMed

Berdkan, Sandra; Rabbaa, Lara; Hajj, Aline; Eid, Bassam; Jabbour, Hicham; Osta, Nada El; Karam, Latife; Khabbaz, Lydia Rabbaa

2016-08-01

The main objectives of this study were to assess the incidence of off-label (OL) and/or unlicensed (UL) prescriptions in a sample of pediatric Lebanese patients by using US Food and Drug Administration (FDA) and the French Medical Regulatory Authority (ANSM) regulations. The goal was to analyze the divergences between regulations and to identify those drugs most commonly involved in OL-UL utilization. This study was a retrospective analysis (500 pediatric files) conducted in a Lebanese University hospital in 3 pediatric wards (chronic diseases, acute diseases, and the pediatric intensive care unit). The frequency of OL-UL drug use was significantly different between pediatric wards (P < 0.001), with the highest incidence occurring in the intensive care unit. The most frequent OL-UL prescriptions occurred with cancer (oncology) admissions. Age was significantly related to OL-UL frequency (highest incidence in children aged between 0 and 1 year). The number of drugs prescribed per patient ranged between 1 and 20 (mean [SD], 4.13 [2.6]). The incidence of OL-UL prescriptions was significantly higher in patients treated with a greater number of medicines (P < 0.001). Overall, 58.9% of drug prescriptions were authorized according to ANSM and 50.7% according to FDA regulations; 11.1% (ANSM) and 15.8% (FDA) were UL, and 30.2% (ANSM) and 33.5% (FDA), respectively, were OL use (where OL for the indication were the most common). The highest percentage of OL-UL prescriptions was seen with the following groups: blood and blood-forming organs, genitourinary system, and sex hormones. Divergence between FDA and ANSM was mainly observed for OL medicines. UL prescriptions assessed according to both regulations showed similar results. This study highlights the need for prescribers to continuously examine updates to official regulations to avoid using an OL-UL drug whenever possible. It also calls for better harmonization between worldwide official guidelines concerning drugs used in children to reduce risk factors for adverse drug reactions. Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.

Bovine herpesvirus type-1 glycoprotein K (gK) interacts with UL20 and is required for infectious virus production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haque, Muzammel; Stanfield, Brent; Kousoulas, Kons

We have previously shown that the HSV-1 gK and UL20 proteins interact and function in virion envelopment, membrane fusion, and neuronal entry. Alignment of the predicted secondary structures of gKs encoded by BoHV-1, HSV-1, HSV-2, EHV-1 and VZV indicated a high degree of domain conservation. Two BoHV-1 gK-null mutant viruses were created by either gK gene deletion or stop codon insertion. In addition, a V5 epitope-tag was inserted at the carboxyl terminus of gK gene to detect gK. The engineered gK-null mutant viruses failed to replicate and produce viral plaques. Co-immunoprecipitation of gK and UL20 expressed via different methods revealedmore » that gK and UL20 physically interacted in the presence or absence of other viral proteins. Confocal microscopy showed that gK and UL20 colocalized in infected cells. These results indicate that BoHV-1 gK and UL20 may function in a similar manner to other alphaherpesvirus orthologues specified by HSV-1, PRV and EHV-1. -- Highlights: •Glycoprotein K(gK) is conserved among alphaherpesviruses and serves similar functions. •The bovine herpesvirus-1 gK and UL20 proteins physically interact in a similar manner to herpes simplex virus type 1 and equine herpesvirus-1. •The bovine herpesvirus-1 (BoHV-1) gK interacts with UL20 and is essential for virus replication and spread.« less

[Inhibitory effects of silymarin on hepatic fibrosis induced by dimethylnitrosamine: experiment with rats].

PubMed

Zhao, Xin-yan; Wang, Bao-en; Wang, Tai-ling; Li, Xin-min

2006-09-26

To investigate the antifibrotic effects of silymarin on hepatic fibrosis. Sixty-one male Wistar rats were randomly divided into three groups: control group (15 rats); DMN model group (23 rats), injected intraperitoneally with dimethylnitrosamine (DMN) 10 mg/kg twice per week for 8 weeks to induce hepatic fibrosis; and silymarin group (23 rats), injected intraperitoneally with DMN and given silymarin 50 mg/kg by gastric gavage daily for 8 weeks. Eight weeks late all rats were sacrificed. Blood samples were collected to measure the alanine transaminase (ALT), aspirate aminotransferase (AST), albumin, and total bilirubin (TBIL). The hydroxyproline (Hyp) content in the liver tissue was measured. The histopathological changes as well as the fibrosis stages and score were examined by microscopy. The levels of ALT, AST, and TBIL of the silymarin groups were 59 U/L +/- 19 U/L, 159 U/L +/- 39 U/L, and mean rank 24 respectively, all significantly lower than those of the DMN model group (128 U/L +/- 25 U/L, 246 U/L +/- 61 U/L, and mean rank 37 respectively, P < 0.01, P = 0.001, and P = 0.003). Compared with DMN rats, the level of Hyp of the silymarin was lower by 42.6%, the hepatic score of the silymarin was 6.2 +/- 2.4, significantly than that of the DMN model group (12.8 +/- 4.4, P = 0.001), and more cases in the silymarin group were at the lower stages. Silymarin markedly inhibits and reverse the progression of hepatic fibrosis induced by dimethylnitrosamine.

An augmented reality system for upper-limb post-stroke motor rehabilitation: a feasibility study.

PubMed

Assis, Gilda Aparecida de; Corrêa, Ana Grasielle Dionísio; Martins, Maria Bernardete Rodrigues; Pedrozo, Wendel Goes; Lopes, Roseli de Deus

2016-08-01

To determine the clinical feasibility of a system based on augmented reality for upper-limb (UL) motor rehabilitation of stroke participants. A physiotherapist instructed the participants to accomplish tasks in augmented reality environment, where they could see themselves and their surroundings, as in a mirror. Two case studies were conducted. Participants were evaluated pre- and post-intervention. The first study evaluated the UL motor function using Fugl-Meyer scale. Data were compared using non-parametric sign tests and effect size. The second study used the gain of motion range of shoulder flexion and abduction assessed by computerized biophotogrammetry. At a significance level of 5%, Fugl-Meyer scores suggested a trend for greater UL motor improvement in the augmented reality group than in the other. Moreover, effect size value 0.86 suggested high practical significance for UL motor rehabilitation using the augmented reality system. System provided promising results for UL motor rehabilitation, since enhancements have been observed in the shoulder range of motion and speed. Implications for Rehabilitation Gain of range of motion of flexion and abduction of the shoulder of post-stroke patients can be achieved through an augmented reality system containing exercises to promote the mental practice. NeuroR system provides a mental practice method combined with visual feedback for motor rehabilitation of chronic stroke patients, giving the illusion of injured upper-limb (UL) movements while the affected UL is resting. Its application is feasible and safe. This system can be used to improve UL rehabilitation, an additional treatment past the traditional period of the stroke patient hospitalization and rehabilitation.

Characterising variation in five genetic loci of cytomegalovirus during treatment for congenital infection.

PubMed

Kadambari, Seilesh; Atkinson, Claire; Luck, Suzanne; Macartney, Malcolm; Conibear, Tim; Harrison, Ian; Booth, Clare; Sharland, Mike; Griffiths, Paul D

2017-03-01

Cytomegalovirus (CMV) is the most common congenital infection in humans and a leading cause of sensorineural hearing loss. Ganciclovir (6 mg/kg twice daily for 42 days) has been shown to reduce hearing deterioration and is used in clinical practice. Vaccines and passive administration of antibody are being evaluated in randomized controlled trials in allograft candidates, women of childbearing age, and pregnant women with primary CMV infection. To help define genetic variation in each of the targets of these therapeutic interventions, we amplified and sequenced genes UL97 (site utilised for ganciclovir phosphorylation), UL55 (glycoprotein B (gB) vaccine target) and UL128, UL130, and UL131a (specific monoclonal antibody targets). Serial blood, saliva, and urine samples (total 120) obtained from nine infants with symptomatic congenital CMV treated with 42 days' ganciclovir were analyzed. All samples tested were UL97 wild type at baseline and none developed mutations during treatment, showing no selection of resistance. The prevalences of UL55 genotypes were 28% gB1, 22% gB2, 1% gB3, and mixed in 20% samples. No mutations were noted in UL128-131a. Phylogenetic tree analysis showed that sequences with variations were found in multiple body sites of individual patients, so there was no evidence of body site compartmentalization of particular strains of CMV. The significance of these results for changes in diagnostic practices and therapeutic interventions against CMV are discussed. J. Med. Virol. 89:502-507, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Association of polymorphisms and haplotypes in the cytochrome P450 1B1 gene with uterine leiomyoma: A case control study

PubMed Central

SALIMI, SAEEDEH; KHODAMIAN, MARYAM; NAROOIE-NEJAD, MEHRNAZ; HAJIZADEH, AZAM; FAZELI, KIMIA; NAMAZI, LIDA; YAGHMAEI, MINOO

2015-01-01

Uterine leiomyoma (UL) is an estrogen-dependent neoplasm of the uterus and estrogen metabolizing enzymes affect its promotion and progression. The aim of the present study was to evaluate the association between four single-nucleotide polymorphisms (SNPs) of the cytochrome P450 1B1 (CYP1B1) gene and UL risk. Four SNPs of the CYP1B1 gene in 105 UL patients and 112 unrelated healthy controls were genotyped using a direct sequencing method. Haplotype analyses were performed with UNPHASED software and linkage disequilibrium (LD) was assessed by Haploview software. There were no associations between Leu432Val (rs1056836), Asp449Asp (rs1056837) and Asn453Ser (rs1800440) polymorphisms of the CYP1B1 gene and UL. Although the genotypic frequencies of the Arg368His (rs79204362) polymorphism did not differ between the two groups, the frequency of A (His) allele was significantly higher in UL females (P=0.02). In addition, the frequency of GTAA haplotype was significantly higher in the controls and played a protective role in UL susceptibility. A strong LD between the three common SNPs (rs1056836, rs1056837 and rs1800440) in the CYP1B1 gene was observed in the population. In conclusion, a higher frequency of the CYP1B1 368His (A) allele was observed in UL females. The frequency of the GTAA haplotype was significantly higher in healthy females and this haplotype played a protective role in UL susceptibility. PMID:26075073

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

PubMed

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have developed.

A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment.

PubMed

Dietz, Andrea N; Villinger, Clarissa; Becker, Stefan; Frick, Manfred; von Einem, Jens

2018-01-01

The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXXΦ) in pUL71. This led us to hypothesize a requirement of the YXXΦ motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXXΦ motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXXΦ mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXXΦ motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-β-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can lead to life-threatening infections in immunocompromised hosts. Current antiviral treatments target viral genome replication and are increasingly overcome by viral mutations. Therefore, identifying new targets for antiviral therapy is important for future development of novel treatment options. A detailed molecular understanding of the complex virus morphogenesis will identify potential viral as well as cellular targets for antiviral intervention. Secondary envelopment is an important viral process through which infectious virus particles are generated and which involves the action of several viral proteins, such as tegument protein pUL71. Targeting of pUL71 to the site of secondary envelopment appears to be crucial for its function during this process and is regulated by utilizing host trafficking mechanisms that are commonly exploited by viral glycoproteins. Thus, intracellular trafficking, if targeted, might present a novel target for antiviral therapy. Copyright © 2017 American Society for Microbiology.

[Construction and transfection of eucaryotic expression recombinant vector containing truncated region of UL83 gene of human cytomegalovirus and it's sheltered effect as DNA vaccine].

PubMed

Gao, Rong-Bao; Li, Yan-Qiu; Wang, Ming-Li

2006-06-01

To construct eucaryotic expression recombinant vector containing vivo truncated region of UL83 gene of human cytomegalovirus, realize its steady expression in Hep-2 cell, and study sheltered effect of the eucaryotic expression recombinant vector as DNA vaccine. A vivo truncated UL83 gene fragment encoding for truncated HCMV pp65 was obtained by PCR from human cytomegalovirus AD169 stock genome. By gene recombinant ways, the truncated UL83 gene fragment was cloned into eucaryotic expression vector pEGFP-C1 with reported gene coding GFP to construct recombinant vector pEGFP-C1-UL83. The recombinant vector pEGFP-C1-UL83 was tested by different methods including PCR, restriction digestion and gene sequencing. Test results showed the recombinant vector was constructed successfully. After pEGFP-C1-UL83 was transfected into Hep-2 cell by lipofectin mediation, expression of GFP and truncated pp65 fusion protein in Hep-2 cell was observed at different time points by fluorescence microscope. Results showed that quantity of fusion protein expression was the highest at 36h point. Then, Hep-2 cell was cultured selectively by RPMI-1640 containing G418 (200 microg/mL) to obtain a new cell stock of expressing truncated UL83 Gene fragment steadily. RT-PCR and Western blot results showed the truncated fragment of UL83 gene could be expressed steadily in Hep-2 cell. The result showed a new cell stock of expressing Tpp65 was established. This cell stock could be useful in some HCMV research fields, for example, it could be a tool in study of pp65 and HCMV infection, and it could provide a platform for the research into the therapy of HCMV infection. Immune sheltered effect of pEGFP-C1-UL83 as DNA vaccine was studied in vivo of HCMV congenital infection mouse model. The mouse model was immunized solely by pEGFP-C1-UL83, and was immunized jointly by pEGFP-C1-UL83 and its expression product. When the mouse was pregnant and brought to bed, differential antibody of anti-HCMV pp65 was tested by indirect ELISA in mother mouse, the infectious virus was separated with the method of virus separation, and pp65 antigen was checked up by indirect immunofluorescence staining in fetal mouse. Results showed differential antibody of anti-HCMV pp65 was produced in mouse model. Tilter of the antibody was from 1:2.51 to 1:50.79. Results of virus separation and pp65 checkup of fetal mouse brain tissue were negative. So the conclusion can be reached that pEGFP-C1-UL83 as DNA vaccine in vivo has sheltered effect which can prevent HCMV vertical transmission from mother mouse to her fetus.

Health Information in Modern Standard Arabic (al-ʻArabīyat ul-fuṣḥá)

MedlinePlus

... fuṣḥá (Modern Standard Arabic) MP4 Healthy Roads Media Tornadoes - English MP3 Tornadoes - al-ʻArabīyat ul-fuṣḥá (Modern Standard Arabic) MP3 Tornadoes - English MP4 Tornadoes - al-ʻArabīyat ul-fuṣḥá (Modern ...

Patients with Duchenne and Becker muscular dystrophies are not more asymmetrical than healthy controls on timed performance of upper limb tasks

PubMed Central

Artilheiro, M.C.; Sá, C.S.C.; Fávero, F.M.; Caromano, F.A.; Voos, M.C.

2017-01-01

This study aimed to investigate possible asymmetries and relationships between performance of dominant and non-dominant upper limbs (UL) in patients with Duchenne and Becker muscular dystrophies (DMD/BMD), to compare UL performance of patients and healthy subjects and to investigate the relationship between timed performance of UL and age, motor function and muscle strength in DMD/BMD patients. Sixteen patients with DMD and 3 with BMD were evaluated with Jebsen-Taylor Test (timed performance), Vignos scale and Dimension 3 of Motor Function Measure (motor function), and Medical Research Council scale (muscle strength) on a single session. ANOVA showed no asymmetry between dominant and non-dominant UL, except in the writing subtest, in patients and in healthy controls. There were relationships between dominant and non-dominant UL performances. Correlations between timed performance, motor function and muscle strength were found, but age was not correlated with these variables. These findings may reduce the assessment time, prevent fatigue and provide more accurate clinical reasoning involving UL in DMD/BMD treatment. PMID:28746422

Vaccination with a HSV-2 UL24 mutant induces a protective immune response in murine and guinea pig vaginal infection models.

PubMed

Visalli, Robert J; Natuk, Robert J; Kowalski, Jacek; Guo, Min; Blakeney, Susan; Gangolli, Seema; Cooper, David

2014-03-10

The rational design and development of genetically attenuated HSV-2 mutant viruses represent an attractive approach for developing both prophylactic and therapeutic vaccines for genital herpes. Previously, HSV-2 UL24 was shown to be a virulence determinant in both murine and guinea pig vaginal infection models. An UL24-βgluc insertion mutant produced syncytial plaques and replicated to nearly wild type levels in tissue culture, but induced little or no pathological effects in recipient mice or guinea pigs following vaginal infection. Here we report that immunization of mice or guinea pigs with high or low doses of UL24-βgluc elicited a highly protective immune response. UL24-βgluc immunization via the vaginal or intramuscular routes was demonstrated to protect mice from a lethal vaginal challenge with wild type HSV-2. Moreover, antigen re-stimulated splenic lymphocytes harvested from immunized mice exhibited both HSV-2 specific CTL activity and IFN-γ expression. Humoral anti-HSV-2 responses in serum were Th1-polarized (IgG2a>IgG1) and contained high-titer anti-HSV-2 neutralizing activity. Guinea pigs vaccinated subcutaneously with UL24-βgluc or the more virulent parental strain (186) were challenged with a heterologous HSV-2 strain (MS). Acute disease scores were nearly indistinguishable in guinea pigs immunized with either virus. Recurrent disease scores were reduced in UL24-βgluc immunized animals but not to the same extent as those immunized with strain 186. In addition, challenge virus was not detected in 75% of guinea pigs subcutaneously immunized with UL24-βgluc. In conclusion, disruption of the UL24 gene is a prime target for the development of a genetically attenuated live HSV-2 vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

PubMed Central

Webel, Rike; Hakki, Morgan; Prichard, Mark N.; Rawlinson, William D.; Marschall, Manfred

2014-01-01

ABSTRACT The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo. PMID:24522923

Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin

PubMed Central

Lee, Sora; Tumolo, Jessica M; Ehlinger, Aaron C; Jernigan, Kristin K; Qualls-Histed, Susan J; Hsu, Pi-Chiang; McDonald, W Hayes; Chazin, Walter J

2017-01-01

Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole. PMID:29130884

Effects of Multivitamin, Multimineral and Phytonutrient Supplementation on Nutrient Status and Biomarkers of Heart Health Risk in a Russian Population: A Randomized, Double Blind, Placebo Controlled Study.

PubMed

Isakov, Vasily A; Bogdanova, Alexandra A; Bessonov, Vladimir V; Sentsova, Tatiana B; Tutelyan, Victor A; Lin, Yumei; Kazlova, Valentina; Hong, Jina; Velliquette, Rodney A

2018-01-25

The primary objective of this clinical study was to evaluate the effect of a dietary multivitamin, multimineral and phytonutrient (VMP) supplement on blood nutrient status and biomarkers of heart health risk in a Russian population. One hundred twenty healthy adults (40-70 years) were recruited for a 56-day (eight-week) randomized, double blind, placebo controlled study with parallel design. Subjects were divided into two groups and received either a VMP or a placebo (PLA) supplement. Blood nutrient levels of β-carotene, α-tocopherol, vitamin C, B6, B12, red blood cell (RBC) folate, Zinc and Selenium were measured at baseline and on Days 28 and 56, and quercetin was measured at baseline and on Day 56. Blood biomarkers of heart health, i.e. homocysteine (Hcy), high-sensitivity C-reactive protein (hs-CRP), oxidized LDL (ox-LDL), gamma-glutamyl transferase (GGT), uric acid and blood lipid profile, were measured at baseline and Day 56. Dietary VMP supplementation for 56 days significantly increased circulating levels of quercetin, vitamin C, RBC folate and partially prevented the decline in vitamin B6 and B12 status. Both serum Hcy and GGT were significantly reduced (-3.97 ± 10.09 µmol/L; -1.68 ± 14.53 U/L, respectively) after VMP supplementation compared to baseline. Dietary VMP supplementation improved the nutrient status and reduced biomarkers of heart health risk in a Russian population.

Effects of Multivitamin, Multimineral and Phytonutrient Supplementation on Nutrient Status and Biomarkers of Heart Health Risk in a Russian Population: A Randomized, Double Blind, Placebo Controlled Study

PubMed Central

Bogdanova, Alexandra A.; Bessonov, Vladimir V.; Sentsova, Tatiana B.; Tutelyan, Victor A.; Lin, Yumei; Kazlova, Valentina; Velliquette, Rodney A.

2018-01-01

The primary objective of this clinical study was to evaluate the effect of a dietary multivitamin, multimineral and phytonutrient (VMP) supplement on blood nutrient status and biomarkers of heart health risk in a Russian population. One hundred twenty healthy adults (40–70 years) were recruited for a 56-day (eight-week) randomized, double blind, placebo controlled study with parallel design. Subjects were divided into two groups and received either a VMP or a placebo (PLA) supplement. Blood nutrient levels of β-carotene, α-tocopherol, vitamin C, B6, B12, red blood cell (RBC) folate, Zinc and Selenium were measured at baseline and on Days 28 and 56, and quercetin was measured at baseline and on Day 56. Blood biomarkers of heart health, i.e. homocysteine (Hcy), high-sensitivity C-reactive protein (hs-CRP), oxidized LDL (ox-LDL), gamma-glutamyl transferase (GGT), uric acid and blood lipid profile, were measured at baseline and Day 56. Dietary VMP supplementation for 56 days significantly increased circulating levels of quercetin, vitamin C, RBC folate and partially prevented the decline in vitamin B6 and B12 status. Both serum Hcy and GGT were significantly reduced (−3.97 ± 10.09 µmol/L; −1.68 ± 14.53 U/L, respectively) after VMP supplementation compared to baseline. Dietary VMP supplementation improved the nutrient status and reduced biomarkers of heart health risk in a Russian population. PMID:29370120

Anicteric hepatoxicity: a potential health risk of occupational exposures in Nigerian petroleum oil refining and distribution industry.

PubMed

Ezejiofor, Tobias I Ndubuisi; Ezejiofor, Anthonet N; Orisakwe, Orish E; Nwigwe, Hariet C; Osuala, Ferdinand Ou; Iwuala, Moses Oe

2014-01-22

Literature abounds linking one's job to certain unpalatable health outcomes. Since exposures to hazardous conditions in industrial environments often results in sundry health effects among workers, we embarked on this study to investigate the hepatic health effects of occupational activities in the petroleum refining and distribution industry. Biochemical markers of liver functions were assayed in plasma, using Reflotron dry chemistry spectrophotometric system. The study was conducted on randomly selected workers of Port Harcourt Refining Company (PHRC) and Pipelines and Petroleum Product Marketing Company (PPMC) both in Alesa-Eleme near Port Harcourt, Nigeria, as well as non-oil work civil servants serving as control subjects. Results showed that, bilirubin ranged 0.3-1.6 mg/dl with a mean of 0.66±0.20mg/dl among the oil workers as against 0.5-1.00mg/dl with a mean of 0.58±0.13mg/dl in non-oil workers, Alkaline phosphatase ranged 50.00-296.00u/l (mean: 126.21±39.49u/l) in oil workers as against 40.20-111u/l (mean: 66.83±18.54u/l) for non-oil workers, Aspartic transaminases (AST) ranged 5.80-140.20u/l (mean: 21.81±11.49u/l) in oil workers against 18.00-44.00u/l (mean: 26.89±6.99u/l) for non-oil workers, while Alanine transaminases (ALT) ranged 4.90-86.00u/l (mean: 22.14±11.28u/l) in oil workers as against 10.00-86.60u/l (mean: 22.30±10.22u/l) for the non-oil workers. A close study of the results revealed that although the mean values for all the studied parameters were still within the parametric reference ranges, however, relative to the referents, there were significant increases (P<0.05) in plasma bilirubin (though anicteric) and alkaline phosphatase that was not matched with a corresponding increase in the plasma transaminases, suggesting a possibility that toxic anicteric hepatoxicity is part of the potential health effects of sundry exposures in the Nigeria petroleum oil refining and distribution industry. Gender differentiation data showed that though the mean values for the parameters were higher in males than females, the increases were not significant in most cases (P>0.05), whereas data for age and exposure period classifications revealed that irrespective of the age of the worker, the effects are likely to start after the first five years, manifesting fully after the first decade of occupational exposures. Thus, an update of industrial/occupational health measures is necessary for a safer and healthier work environment.

Anicteric hepatoxicity: a potential health risk of occupational exposures in Nigerian petroleum oil refining and distribution industry

PubMed Central

2014-01-01

Background Literature abounds linking one’s job to certain unpalatable health outcomes. Since exposures to hazardous conditions in industrial environments often results in sundry health effects among workers, we embarked on this study to investigate the hepatic health effects of occupational activities in the petroleum refining and distribution industry. Method Biochemical markers of liver functions were assayed in plasma, using Reflotron dry chemistry spectrophotometric system. The study was conducted on randomly selected workers of Port Harcourt Refining Company (PHRC) and Pipelines and Petroleum Product Marketing Company (PPMC) both in Alesa-Eleme near Port Harcourt, Nigeria, as well as non-oil work civil servants serving as control subjects. Result and conclusion Results showed that, bilirubin ranged 0.3-1.6 mg/dl with a mean of 0.66±0.20mg/dl among the oil workers as against 0.5-1.00mg/dl with a mean of 0.58±0.13mg/dl in non-oil workers, Alkaline phosphatase ranged 50.00-296.00u/l (mean: 126.21±39.49u/l) in oil workers as against 40.20-111u/l (mean: 66.83±18.54u/l) for non-oil workers, Aspartic transaminases (AST) ranged 5.80-140.20u/l (mean: 21.81±11.49u/l) in oil workers against 18.00-44.00u/l (mean: 26.89±6.99u/l) for non-oil workers, while Alanine transaminases (ALT) ranged 4.90-86.00u/l (mean: 22.14±11.28u/l) in oil workers as against 10.00-86.60u/l (mean: 22.30±10.22u/l) for the non-oil workers. A close study of the results revealed that although the mean values for all the studied parameters were still within the parametric reference ranges, however, relative to the referents, there were significant increases (P<0.05) in plasma bilirubin (though anicteric) and alkaline phosphatase that was not matched with a corresponding increase in the plasma transaminases, suggesting a possibility that toxic anicteric hepatoxicity is part of the potential health effects of sundry exposures in the Nigeria petroleum oil refining and distribution industry. Gender differentiation data showed that though the mean values for the parameters were higher in males than females, the increases were not significant in most cases (P>0.05), whereas data for age and exposure period classifications revealed that irrespective of the age of the worker, the effects are likely to start after the first five years, manifesting fully after the first decade of occupational exposures. Thus, an update of industrial/occupational health measures is necessary for a safer and healthier work environment. PMID:24457023

Cell cultures in uterine leiomyomas: rapid disappearance of cells carrying MED12 mutations.

PubMed

Nadine Markowski, Dominique; Tadayyon, Mahboobeh; Bartnitzke, Sabine; Belge, Gazanfer; Maria Helmke, Burkhard; Bullerdiek, Jörn

2014-04-01

Uterine leiomyomas (UL) are the most frequent symptomatic human tumors. Nevertheless, their molecular pathogenesis is not yet fully understood. To learn more about the biology of these common neoplasms and their response to treatment, cell cultures derived from UL are a frequently used model system, but until recently appropriate genetic markers confirming their origin from the tumor cell population were lacking for most UL, i.e., those not displaying karyotypic abnormalities. The identification of MED12 mutations in the majority of UL makes it possible to trace the tumor cell population during in vitro passaging in the absence of cytogenetic abnormalities. The present study is addressing the in vitro survival of cells carrying MED12 mutations and its association with karyotypic alterations. The results challenge numerous in vitro studies into the biology and behavior of leiomyomas. Cells of one genetic subtype of UL, i.e., those with rearrangements of the high mobility AT-hook 2 protein gene (HMGA2), seem to be able to proliferate in vitro for many passages whereas tumor cells from the much more frequent MED12-mutated lesions barely survive even the first passages. Apparently, for the most frequent type of human UL no good in vitro model seems to exist because cells do not survive culturing. On the other hand, this inability may point to an Achilles' heel of this type of UL. Copyright © 2014 Wiley Periodicals, Inc.

Defect-Reduction Mechanism for Improving Radiative Efficiency in InGaN/GaN Light-Emitting Diodes using InGaN Underlayers

DOE PAGES

Armstrong, Andrew M.; Bryant, Benjamin N.; Crawford, Mary H.; ...

2015-04-01

The influence of a dilute In xGa 1-xN (x~0.03) underlayer (UL) grown below a single In 0.16Ga 0.84N quantum well (SQW), within a light-emitting diode(LED), on the radiative efficiency and deep level defect properties was studied using differential carrier lifetime (DCL) measurements and deep level optical spectroscopy (DLOS). DCL measurements found that inclusion of the UL significantly improved LED radiative efficiency. At low current densities, the non-radiative recombination rate of the LED with an UL was found to be 3.9 times lower than theLED without an UL, while the radiative recombination rates were nearly identical. This, then, suggests that themore » improved radiative efficiency resulted from reduced non-radiative defect concentration within the SQW. DLOS measurement found the same type of defects in the InGaN SQWs with and without ULs. However, lighted capacitance-voltage measurements of the LEDs revealed a 3.4 times reduction in a SQW-related near-mid-gap defect state for the LED with an UL. Furthermore, quantitative agreement in the reduction of both the non-radiative recombination rate (3.9×) and deep level density (3.4×) upon insertion of an UL corroborates deep level defect reduction as the mechanism for improved LED efficiency.« less

Model-based iterative reconstruction for reduction of radiation dose in abdominopelvic CT: comparison to adaptive statistical iterative reconstruction.

PubMed

Yasaka, Koichiro; Katsura, Masaki; Akahane, Masaaki; Sato, Jiro; Matsuda, Izuru; Ohtomo, Kuni

2013-12-01

To evaluate dose reduction and image quality of abdominopelvic computed tomography (CT) reconstructed with model-based iterative reconstruction (MBIR) compared to adaptive statistical iterative reconstruction (ASIR). In this prospective study, 85 patients underwent referential-, low-, and ultralow-dose unenhanced abdominopelvic CT. Images were reconstructed with ASIR for low-dose (L-ASIR) and ultralow-dose CT (UL-ASIR), and with MBIR for ultralow-dose CT (UL-MBIR). Image noise was measured in the abdominal aorta and iliopsoas muscle. Subjective image analyses and a lesion detection study (adrenal nodules) were conducted by two blinded radiologists. A reference standard was established by a consensus panel of two different radiologists using referential-dose CT reconstructed with filtered back projection. Compared to low-dose CT, there was a 63% decrease in dose-length product with ultralow-dose CT. UL-MBIR had significantly lower image noise than L-ASIR and UL-ASIR (all p<0.01). UL-MBIR was significantly better for subjective image noise and streak artifacts than L-ASIR and UL-ASIR (all p<0.01). There were no significant differences between UL-MBIR and L-ASIR in diagnostic acceptability (p>0.65), or diagnostic performance for adrenal nodules (p>0.87). MBIR significantly improves image noise and streak artifacts compared to ASIR, and can achieve radiation dose reduction without severely compromising image quality.

Arabidopsis thaliana is a susceptible host plant for the holoparasite Cuscuta spec.

PubMed

Birschwilks, Mandy; Sauer, Norbert; Scheel, Dierk; Neumann, Stefanie

2007-10-01

Arabidopsis thaliana and Cuscuta spec. represent a compatible host-parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host-parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP-ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP-ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP-ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.

Ubiquitin modifications

PubMed Central

Swatek, Kirby N; Komander, David

2016-01-01

Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modifications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the 'ubiquitin code'. Ubiquitin can be ubiquitinated on seven lysine (Lys) residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiquitin-like molecules (such as SUMO or NEDD8). Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiquitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation. PMID:27012465

Phosphorus losses from agricultural watersheds in the Mississippi Delta.

PubMed

Yuan, Yongping; Locke, Martin A; Bingner, Ronald L; Rebich, Richard A

2013-01-30

Phosphorus (P) loss from agricultural fields is of environmental concern because of its potential impact on water quality in streams and lakes. The Mississippi Delta has long been known for its fish productivity and recreational value, but high levels of P in fresh water can lead to algal blooms that have many detrimental effects on natural ecosystems. Algal blooms interfere with recreational and aesthetic water use. However, few studies have evaluated P losses from agricultural watersheds in the Mississippi Delta. To better understand the processes influencing P loss, rainfall, surface runoff, sediment, ortho-P (orthophosphate, PO(4)-P), and total P (TP) were measured (water years 1996-2000) for two subwatersheds (UL1 and UL2) of the Deep Hollow Lake Watershed and one subwatershed of the Beasley Lake Watershed (BL3) primarily in cotton production in the Mississippi Delta. Ortho-P concentrations ranged from 0.01 to 1.0 mg/L with a mean of 0.17 mg/L at UL1 (17.0 ha), 0.36 mg/L at UL2 (11.2 ha) and 0.12 mg/L at BL3 (7.2 ha). The TP concentrations ranged from 0.14 to 7.9 mg/L with a mean of 0.96 mg/L at UL1, 1.1 mg/L at UL2 and 1.29 mg/L at BL3. Among the three sites, UL1 and UL2 received P application in October 1998, and BL3 received P applications in the spring of 1998 and 1999. At UL1, ortho-P concentrations were 0.36, 0.25 and 0.16 for the first, second and third rainfall events after P application, respectively; At UL2, ortho-P concentrations were 1.0, 0.66 and 0.65 for the first, second and third rainfall events after P application, respectively; and at BL3, ortho-P concentrations were 0.11, 0.22 and 0.09 for the first, second and third rainfall events after P application, respectively. P fertilizer application did influence P losses, but high P concentrations observed in surface runoff were not always a direct result of P fertilizer application or high rainfall. Application of P in the fall (UL1 and UL2) resulted in more ortho-P losses, likely because high rainfall often occurred in the winter months soon after application. The mean ortho-P concentrations were higher at UL1 and UL2 than those at BL3, although BL3 received more P application during the monitoring period, because P was applied in spring at BL3. However, tillage associated with planting and incorporating applied P in the spring (BL3) may have resulted in more TP loss in sediment, thus the mean TP concentration was the highest at BL3. Ortho-P loss was correlated with surface runoff; and TP loss was correlated with sediment loss. These results indicate that applying P fertilizer in the spring may be recommended to reduce potential ortho-P loss during the fallow winter season; in addition, conservation practices may reduce potential TP loss associated with soil loss. Published by Elsevier Ltd.

Phosphorus losses from agricultural watersheds in the Mississippi Delta

USGS Publications Warehouse

Yuan, Yongping; Locke, Martin A.; Bingner, Ronald L.; Rebich, Richard A.

2013-01-01

Phosphorus (P) loss from agricultural fields is of environmental concern because of its potential impact on water quality in streams and lakes. The Mississippi Delta has long been known for its fish productivity and recreational value, but high levels of P in fresh water can lead to algal blooms that have many detrimental effects on natural ecosystems. Algal blooms interfere with recreational and aesthetic water use. However, few studies have evaluated P losses from agricultural watersheds in the Mississippi Delta. To better understand the processes influencing P loss, rainfall, surface runoff, sediment, ortho-P (orthophosphate, PO4–P), and total P (TP) were measured (water years 1996–2000) for two subwatersheds (UL1 and UL2) of the Deep Hollow Lake Watershed and one subwatershed of the Beasley Lake Watershed (BL3) primarily in cotton production in the Mississippi Delta. Ortho-P concentrations ranged from 0.01 to 1.0 mg/L with a mean of 0.17 mg/L at UL1 (17.0 ha), 0.36 mg/L at UL2 (11.2 ha) and 0.12 mg/L at BL3 (7.2 ha). The TP concentrations ranged from 0.14 to 7.9 mg/L with a mean of 0.96 mg/L at UL1, 1.1 mg/L at UL2 and 1.29 mg/L at BL3. Among the three sites, UL1 and UL2 received P application in October 1998, and BL3 received P applications in the spring of 1998 and 1999. At UL1, ortho-P concentrations were 0.36, 0.25 and 0.16 for the first, second and third rainfall events after P application, respectively; At UL2, ortho-P concentrations were 1.0, 0.66 and 0.65 for the first, second and third rainfall events after P application, respectively; and at BL3, ortho-P concentrations were 0.11, 0.22 and 0.09 for the first, second and third rainfall events after P application, respectively. P fertilizer application did influence P losses, but high P concentrations observed in surface runoff were not always a direct result of P fertilizer application or high rainfall. Application of P in the fall (UL1 and UL2) resulted in more ortho-P losses, likely because high rainfall often occurred in the winter months soon after application. The mean ortho-P concentrations were higher at UL1 and UL2 than those at BL3, although BL3 received more P application during the monitoring period, because P was applied in spring at BL3. However, tillage associated with planting and incorporating applied P in the spring (BL3) may have resulted in more TP loss in sediment, thus the mean TP concentration was the highest at BL3. Ortho-P loss was correlated with surface runoff; and TP loss was correlated with sediment loss. These results indicate that applying P fertilizer in the spring may be recommended to reduce potential ortho-P loss during the fallow winter season; in addition, conservation practices may reduce potential TP loss associated with soil loss.

Incidence of Ganciclovir Resistance in CMV-positive Renal Transplant Recipients and its Association with UL97 Gene Mutations.

PubMed

Aslani, Hamid Reza; Ziaie, Shadi; Salamzadeh, Jamshid; Zaheri, Sara; Samadian, Fariba; Mastoor-Tehrani, Shayan

2017-01-01

Human cytomegalovirus (CMV) remains the most common infection affecting organ transplant recipients. Despite advances in the prophylaxis and acute treatment of CMV, it remains an important pathogen affecting the short- and long-term clinical outcome of solid organ transplant recipient. The emergence of CMV resistance in a patient reduces the clinical efficacy of antiviral therapy, complicates therapeutic and clinical management decisions, and in some cases results in loss of the allograft and/or death of the patient. Common mechanisms of CMV resistance to ganciclovir have been described chiefly with the UL97 mutations. Here we evaluate Incidence of ganciclovir resistance in 144 CMV-positive renal transplant recipients and its association with UL97 gene mutations. Active CMV infection was monitored by viral DNA quantification in whole blood, and CMV resistance was assessed by UL97 gene sequencing. Six mutations in six patients were detected. Three patients (2.6%) of 112 patients with history of ganciclovir (GCV) treatment had clinical resistance with single UL97 mutations at loci known to be related to resistance (including mutations at codon 594, codon 460, and codon 520). three patients who were anti-CMV drug naïve had single UL97 mutations (D605E) without clinical resistance. Our results confirm and extend our earlier findings on the specific mutations in the UL97 phosphotransferase gene in loci that have established role in ganciclovir resistance and also indicate that clinical ganciclovir resistance due to UL97 gene mutations is an issue in subjects with history of with ganciclovir treatment. D605E mutations remains a controversial issue that needs further investigations.

Prevalence and clinical significance of mediator complex subunit 12 mutations in 362 Han Chinese samples with uterine leiomyoma.

PubMed

Wu, Juan; Zou, Yang; Luo, Yong; Guo, Jiu-Bai; Liu, Fa-Ying; Zhou, Jiang-Yan; Zhang, Zi-Yu; Wan, Lei; Huang, Ou-Ping

2017-07-01

Uterine leiomyomas (ULs) are the most common gynecological benign tumors originating from the myometrium. Prevalent mutations in the mediator complex subunit 12 (MED12) gene have been identified in ULs, and functional evidence has revealed that these mutations may promote the development of ULs. However, whether MED12 mutations are associated with certain clinical characteristics in ULs remains largely unknown. In the present study, the potential mutations of MED12 and its paralogous gene, mediator complex subunit 12-like (MED12L), were screened in 362 UL tumors from Han Chinese patients. A total of 158 out of 362 UL tumors (43.6%) were identified as harboring MED12 somatic mutations, and the majority of these mutations were restricted to the 44th residue. MED12 mutations were also observed in 2 out of 145 (1.4%) adjacent control myometrium. Furthermore, the mutation spectrum of MED12 in the concurrent leiomyomas was noticeably different. Correlation analysis of MED12 mutations with the available clinical features indicated that patients with mutated MED12 tended to have smaller cervical diameters. By contrast, no MED12L mutation was identified in the present samples. In summary, the present study demonstrated the presence of prevalent MED12 somatic mutations in UL samples, and the MED12 mutation was associated with smaller cervical diameters. The low mutation frequency of MED12 in adjacent control myometrium indicated that MED12 mutation may be an early event in the pathogenesis of ULs. Furthermore, MED12 mutation status in concurrent tumors from multiple leiomyomas supported several prior observations that the majority of these tumors arose independently.

Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

PubMed

Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

2017-10-01

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

PubMed Central

Ranaweera, Ruchira S.; Yang, Xiaolu

2013-01-01

The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation

PubMed Central

Han, Cong; Pao, Kuan-Chuan; Kazlauskaite, Agne; Muqit, Miratul M K; Virdee, Satpal

2015-01-01

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation. PMID:26010437

The Ubiquitin Code in the Ubiquitin-Proteasome System and Autophagy.

PubMed

Kwon, Yong Tae; Ciechanover, Aaron

2017-11-01

The conjugation of the 76 amino acid protein ubiquitin to other proteins can alter the metabolic stability or non-proteolytic functions of the substrate. Once attached to a substrate (monoubiquitination), ubiquitin can itself be ubiquitinated on any of its seven lysine (Lys) residues or its N-terminal methionine (Met1). A single ubiquitin polymer may contain mixed linkages and/or two or more branches. In addition, ubiquitin can be conjugated with ubiquitin-like modifiers such as SUMO or small molecules such as phosphate. The diverse ways to assemble ubiquitin chains provide countless means to modulate biological processes. We overview here the complexity of the ubiquitin code, with an emphasis on the emerging role of linkage-specific degradation signals (degrons) in the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (hereafter autophagy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Plant hormone signalling through the eye of the mass spectrometer.

PubMed

Walton, Alan; Stes, Elisabeth; De Smet, Ive; Goormachtig, Sofie; Gevaert, Kris

2015-03-01

Plant growth and development are regulated by hormones and the associated signalling pathways share several common steps, the first being the detection of the signal by receptor proteins. This typically leads to conformational changes in the receptor, thereby modifying its spectrum of interaction partners. Next, secondary signals are transmitted via rapid post-translational cascades, such as targeted phosphorylation or ubiquitination, resulting in the activation/deactivation, relocalization or degradation of target proteins. These events finally give rise to the signal-dependent read-out, such as changes in gene expression and regulation of protein activity. So far, the majority of studies aimed at unravelling hormone signalling pathways in plants relied on genetic or transcriptomic approaches. During the last decade however, MS-driven proteomic methods became increasingly popular tools in plant research as they reveal the specific mechanisms controlled by phytohormones, which for a large part occur on the level of the proteome. Here, we provide an up-to-date review on the growing body of work in these areas using MS-based techniques, with a focus on nonpeptide plant hormones. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains

PubMed Central

Liu, Chao; Liu, Weixiao; Ye, Yihong; Li, Wei

2017-01-01

Ubiquitination of a subset of proteins by ubiquitin chain elongation factors (E4), represented by Ufd2p in Saccharomyces cerevisiae, is a pivotal regulator for many biological processes. However, the mechanism of Ufd2p-mediated ubiquitination is largely unclear. Here, we show that Ufd2p catalyses K48-linked multi-monoubiquitination on K29-linked ubiquitin chains assembled by the ubiquitin ligase (Ufd4p), resulting in branched ubiquitin chains. This reaction depends on the interaction of K29-linked ubiquitin chains with two N-terminal loops of Ufd2p. Only following the addition of K48-linked ubiquitin to substrates modified with K29-linked ubiquitin chains, can the substrates be escorted to the proteasome for degradation. We demonstrate that this ubiquitin chain linkage switching reaction is essential for ERAD, oleic acid and acid pH resistance in yeast. Thus, our results suggest that Ufd2p functions by switching ubiquitin chain linkages to allow the degradation of proteins modified with a ubiquitin linkage, which is normally not targeted to the proteasome. PMID:28165462

A Perturbed Ubiquitin Landscape Distinguishes Between Ubiquitin in Trafficking and in Proteolysis*

PubMed Central

Ziv, Inbal; Matiuhin, Yulia; Kirkpatrick, Donald S.; Erpapazoglou, Zoi; Leon, Sebastien; Pantazopoulou, Marina; Kim, Woong; Gygi, Steven P.; Haguenauer-Tsapis, Rosine; Reis, Noa; Glickman, Michael H.; Kleifeld, Oded

2011-01-01

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery—the ubiquitin-proteasome system and the ubiquitin trafficking system—were unevenly perturbed by expression of K0 ubiquitin. PMID:21427232

E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin

PubMed Central

St-Cyr, Daniel J.; Ziemba, Amy; Garg, Pankaj; Plamondon, Serge; Auer, Manfred; Sidhu, Sachdev; Marinier, Anne; Kleiger, Gary; Tyers, Mike; Sicheri, Frank

2014-01-01

Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities. PMID:24316736

Effect of Food, Diet and Nutrition on Military Readiness and Preparedness of Army Personnel and Dependents in a Peacetime Environment

DTIC Science & Technology

1990-08-15

products or services of these organizations. For the protection of human subjects, the investigator(s) have adhered to policies of applicable Federal...CX5. Test Manufacturer Dilute Rqt D i 1 u t e with/ Enzyme (Compartmnt) Compartmnt C AMMO Sigma 9 ml H20/ 70 ul (B) + 700 ul 0. IM P0 4 buffer GLOL...Sigma 29 ml H20/ 100 ul + (A) 1.7 ml H20 LACT Sigma w o r k i n g 100 ul + buffer = Iml working 4ml H20 + buffer 2ml buffer reconst NAD with 5ml working

High conservation of herpes simplex virus UL5/UL52 helicase-primase complex in the era of new antiviral therapies.

PubMed

Collot, Marianne; Rouard, Caroline; Brunet, Christel; Agut, Henri; Boutolleau, David; Burrel, Sonia

2016-04-01

The emergence of herpes simplex virus (HSV) resistance to current antiviral drugs, that all target the viral DNA polymerase, constitutes a major obstacle to antiviral treatment effectiveness of HSV infections, especially in immunocompromised patients. A novel and promising class of inhibitors of the HSV UL5/UL52 helicase-primase (HP) complex has been reported to hinder viral replication with a high potency. In this study, we describe the low natural polymorphism (interstrain identity >99.1% at both nucleotide and amino acid levels) of HSV HP complex subunits pUL5 and pUL52 among 64 HSV (32 HSV-1 and 32 HSV-2) clinical isolates, and we show that the HSV resistance profile to the first-line antiviral drug acyclovir (ACV) does not impact on the natural polymorphism of HSV HP complex. Genotypic tools and polymorphism data concerning HSV HP complex provided herein will be useful to detect drug resistance mutations in a relevant time frame when HP inhibitors (HPIs), i.e., amenamevir and pritelivir, will be available in medical practice. Copyright © 2016 Elsevier B.V. All rights reserved.

History of uterine leiomyomata and incidence of breast cancer

PubMed Central

Wise, Lauren A.; Radin, Rose G.; Rosenberg, Lynn; Adams-Campbell, Lucile; Palmer, Julie R.

2015-01-01

Purpose Uterine leiomyomata (UL), benign tumors of the myometrium, are influenced by sex steroid hormones. A history of UL diagnosis has been associated with a higher risk of uterine malignancies. The relation between UL and breast cancer, another hormonally-responsive cancer, has not been studied. Methods We investigated the association between self-reported physician-diagnosed UL and incidence of breast cancer in the Black Women's Health Study, a prospective cohort study. We followed 57,747 participants without a history of breast cancer from 1995 to 2013. UL diagnoses were reported at baseline and biennially. Breast cancer was reported on biennial questionnaires and confirmed by pathology data from medical records or cancer registries. Cox regression was used to derive incidence rate ratios (IRRs) and 95% confidence intervals (CI) and adjust for potential confounders. Results There were 2,276 incident cases of breast cancer (1,699 invasive, 394 in situ, and 183 unknown) during 879,672 person-years of follow-up. The multivariable IRR for the overall association between history of UL and breast cancer incidence was 0.99 (95% CI: 0.90-1.08), with similar results for ER+ (IRR=1.03) and ER− breast cancer (IRR=1.05). IRRs for early diagnosis of UL (before age 30) were slightly above 1.0, with IRRs of 1.14 (95% CI: 0.99-1.31) for overall breast cancer, 1.14 (95% CI: 0.93-1.40) for ER+ breast cancer, and 1.20 (95% CI: 0.89-1.61) for ER− breast cancer. IRRs for early diagnosis of UL were elevated for breast cancer diagnosed before age 40 years (IRR=1.39, 95% CI: 0.97-1.99) and premenopausal breast cancer (IRR=1.26, 95% CI: 1.01-1.58). No consistent patterns in risk were observed across estrogen receptor subtypes, and IRRs did not differ appreciably within strata of BMI, female hormone use, mammography recency, or family history of breast cancer. Conclusions The present study of U.S. black women suggests that a history of UL diagnosis is unrelated to the incidence of breast cancer overall. The positive associations observed for early-diagnosed UL with breast cancer before age 40 and with premenopausal breast cancer require confirmation in future studies. PMID:26250515

History of uterine leiomyomata and incidence of breast cancer.

PubMed

Wise, Lauren A; Radin, Rose G; Rosenberg, Lynn; Adams-Campbell, Lucile; Palmer, Julie R

2015-10-01

Uterine leiomyomata (UL), benign tumors of the myometrium, are influenced by sex steroid hormones. A history of UL diagnosis has been associated with a higher risk of uterine malignancies. The relation between UL and breast cancer, another hormonally responsive cancer, has not been studied. We investigated the association between self-reported physician-diagnosed UL and incidence of breast cancer in the Black Women's Health Study, a prospective cohort study. We followed 57,747 participants without a history of breast cancer from 1995 to 2013. UL diagnoses were reported at baseline and biennially. Breast cancer was reported on biennial questionnaires and confirmed by pathology data from medical records or cancer registries. Cox regression was used to derive incidence rate ratios (IRRs) and 95 % confidence intervals (CI) and adjust for potential confounders. There were 2,276 incident cases of breast cancer (1,699 invasive, 394 in situ, and 183 unknown) during 879,672 person-years of follow-up. The multivariable IRR for the overall association between history of UL and breast cancer incidence was 0.99 (95 % CI 0.90-1.08), with similar results for ER + (IRR = 1.03) and ER - breast cancer (IRR = 1.05). IRRs for early diagnosis of UL (before age 30) were slightly above 1.0, with IRRs of 1.14 (95 % CI 0.99-1.31) for overall breast cancer, 1.14 (95 % CI 0.93-1.40) for ER + breast cancer, and 1.20 (95 % CI 0.89-1.61) for ER - breast cancer. IRRs for early diagnosis of UL were elevated for breast cancer diagnosed before 40 years of age (IRR = 1.39, 95 % CI 0.97-1.99) and premenopausal breast cancer (IRR = 1.26, 95 % CI 1.01-1.58). No consistent patterns in risk were observed across estrogen receptor subtypes, and IRRs did not differ appreciably within strata of BMI, female hormone use, mammography recency, or family history of breast cancer. The present study of US black women suggests that a history of UL diagnosis is unrelated to the incidence of breast cancer overall. The positive associations observed for early diagnosed UL with breast cancer before age 40 and with premenopausal breast cancer require confirmation in future studies.

A Review on Ubiquitination of Neurotrophin Receptors: Facts and Perspectives

PubMed Central

Sánchez-Sánchez, Julia; Arévalo, Juan Carlos

2017-01-01

Ubiquitination is a reversible post-translational modification involved in a plethora of different physiological functions. Among the substrates that are ubiquitinated, neurotrophin receptors (TrkA, TrkB, TrkC, and p75NTR) have been studied recently. TrkA is the most studied receptor in terms of its ubiquitination, and different E3 ubiquitin ligases and deubiquitinases have been implicated in its ubiquitination, whereas not much is known about the other neurotrophin receptors aside from their ubiquitination. Additional studies are needed that focus on the ubiquitination of TrkB, TrkC, and p75NTR in order to further understand the role of ubiquitination in their physiological and pathological functions. Here we review what is currently known regarding the ubiquitination of neurotrophin receptors and its physiological and pathological relevance. PMID:28335430

Review of Relocation of the System Program Office and Logistics Support for the F-117A Stealth Fighter

DTIC Science & Technology

1998-04-10

LMSW. Cost of Repair Number Avoided Nomenclature New Part Cost Repaired Cost Wing platypus panel $60,100 $4,500 10 $ 556,000 Lower rear door panel...72,200 7,000 48 3,129,600 Rear door trailing 76,000 6,000 2 140,000 edge panel Inboard platypus panel 56,000 4,200 46 2,410,400 Outboard platypus panel...Composites Shop’s contribution to resolving the problem consisted of the redesign of the wing and inboard and outboard platypus panels. The Advanced

Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

NASA Astrophysics Data System (ADS)

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-08-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

High Performance Liquid Chromatography Resolution of Ubiquitin Pathway Enzymes from Wheat Germ 1

PubMed Central

Sullivan, Michael L.; Callis, Judy; Vierstra, Richard D.

1990-01-01

The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with 125I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin 125I-lysozyme conjugates (ε-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (α-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16667769

Polymorphism in Human Cytomegalovirus UL40 Impacts on Recognition of Human Leukocyte Antigen-E (HLA-E) by Natural Killer Cells*

PubMed Central

Heatley, Susan L.; Pietra, Gabriella; Lin, Jie; Widjaja, Jacqueline M. L.; Harpur, Christopher M.; Lester, Sue; Rossjohn, Jamie; Szer, Jeff; Schwarer, Anthony; Bradstock, Kenneth; Bardy, Peter G.; Mingari, Maria Cristina; Moretta, Lorenzo; Sullivan, Lucy C.; Brooks, Andrew G.

2013-01-01

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a “mimic” of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a “polymorphic hot spot” within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors. PMID:23335510

The Presence of HLA-E-Restricted, CMV-Specific CD8+ T Cells in the Blood of Lung Transplant Recipients Correlates with Chronic Allograft Rejection.

PubMed

Sullivan, Lucy C; Westall, Glen P; Widjaja, Jacqueline M L; Mifsud, Nicole A; Nguyen, Thi H O; Meehan, Aislin C; Kotsimbos, Tom C; Brooks, Andrew G

2015-01-01

The human cytomegalovirus (CMV) immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA)-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC). UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8+ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS). Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation.

Polymorphism in human cytomegalovirus UL40 impacts on recognition of human leukocyte antigen-E (HLA-E) by natural killer cells.

PubMed

Heatley, Susan L; Pietra, Gabriella; Lin, Jie; Widjaja, Jacqueline M L; Harpur, Christopher M; Lester, Sue; Rossjohn, Jamie; Szer, Jeff; Schwarer, Anthony; Bradstock, Kenneth; Bardy, Peter G; Mingari, Maria Cristina; Moretta, Lorenzo; Sullivan, Lucy C; Brooks, Andrew G

2013-03-22

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a "mimic" of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a "polymorphic hot spot" within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.

Expression levels of glycoprotein O (gO) vary between strains of human cytomegalovirus, influencing the assembly of gH/gL complexes and virion infectivity.

PubMed

Zhang, Le; Zhou, Momei; Stanton, Richard; Kamil, Jeremy; Ryckman, Brent J

2018-05-09

Tropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the ER. This assembly process clearly differs among strains since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much less gH/gL/UL128-131 than ME. Remaining questions include 1) what are the mechanisms behind these assembly differences, and 2) do differences reflect in vitro culture adaptations or natural genetic variations? Since the UL74(gO) ORF differs by 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression was within 4-fold between TR and ME, but gO expression was 20-fold less by ME, and suggested differences in mRNA transcription, translation or rapid ER-associated degradation of gO. Trans-complementation of gO expression during ME replication gave 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in assembly of gH/gL complexes result from differences in expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than for higher gO expression. IMPORTANCE Specific genetic differences between independently isolated HCMV strains may result from purifying selection on de novo mutations arising during propagation in culture, or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability present in vivo This may have important implications for virus-host interactions such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains. Copyright © 2018 American Society for Microbiology.

Identification and characterization of a novel ISG15-ubiquitin mixed chain and its role in regulating protein homeostasis

PubMed Central

Fan, Jun-Bao; Arimoto, Kei-lchiro; Motamedchaboki, Khatereh; Yan, Ming; Wolf, Dieter A.; Zhang, Dong-Er

2015-01-01

As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site. Using a model substrate, we demonstrate that ISG15 can modify ubiquitin, which is immobilized on its substrate, to form ISG15-ubiquitin mixed chains. Furthermore, our results indicate that ISG15-ubiquitin mixed chains do not serve as degradation signals for a ubiquitin fusion degradation substrate. Accordingly, an ISG15-ubiquitin fusion protein, which mimics an ISG15-ubiquitin mixed chain, negatively regulates cellular turnover of ubiquitylated proteins. In addition, ISG15-ubiquitin mixed chains, which are detectable on endogenously ubiquitylated proteins, dampen cellular turnover of these proteins. Thus, our studies unveil an unanticipated interplay between two protein modification systems and highlight its role in coordinating protein homeostasis. PMID:26226047

The emerging complexity of ubiquitin architecture.

PubMed

Ohtake, Fumiaki; Tsuchiya, Hikaru

2017-02-01

Ubiquitylation is an essential post-translational modification (PTM) of proteins with diverse cellular functions. Polyubiquitin chains with different topologies have different cellular roles, and are referred to as a 'ubiquitin code'. Recent studies have begun to reveal that more complex ubiquitin architectures function as important signals in several biological pathways. These include PTMs of ubiquitin itself, such as acetylated ubiquitin and phospho-ubiquitin. Moreover, important roles for heterogeneous polyubiquitin chains, such as mixed or branched chains, have been reported, which significantly increase the diversity of the ubiquitin code. In this review, we describe mass spectrometry-based methods to characterize the ubiquitin signal. We also describe recent advances in our understanding of complex ubiquitin architectures, including our own findings concerning ubiquitin acetylation and branching within polyubiquitin chains. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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Science.gov Websites

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[Clinical values of single or repeated triptorelin stimulating test in the differential diagnosis between idiopathic hypogonadotropic hypogonadism and constitutional delayed puberty].

PubMed

Mao, Jiang-Feng; Wu, Xue-Yan; Lu, Shuang-Yu; Nie, Min

2011-10-01

To investigate the values of single or repeated luteinizing hormone (LH) releasing hormone analogue (triptorelin) stimulating test in the differential diagnosis between idiopathic hypogonadotropic hypogonadism (IHH) and constitutional delayed puberty (CDP). Male patients (n = 133) without puberty onset after the age of 14 were recruited for triptorelin stimulating test and were followed up for 24 - 48 months until the diagnosis were confirmed: 86 were IHH and the other 47 were CDP. Repeated triptorelin stimulating tests were conducted in 9 IHH patients and 13 CDP patients one year after the first stimulating tests with an attempt to evaluate the dynamic change of hypothalamus-pituitary-testis axis function. The relationship between the final diagnosis and the peak LH value (LH(max)), and the changes of repeated LH(max) were investigated. In the single triptorelin stimulating test, LH(max) was (1.9 +/- 1.2) U/L in IHH group, which was significantly lower than that in CDP group [(13.7 +/- 8.3) U/L] (P < 0.01); 75 IHH patients (87.2%) had a LH(max) lower than 4 U/L, while only 2 CDP patients (4.3%) had a LH(max) lower than 4 U/L. When LH(max) < 4U/L was used as a criteria for the diagnosis of IHH, the single triptorelin stimulating test had a sensitivity of 87.2%, a specificity of 95.7%, and a positive predictive value of 97.4%. The repeated triptorelin stimulating tests performed one year later showed that the LH(max) in the 9 IHH patients increased from (4.7 +/- 2.5) U/L to (5.1 +/- 3.3) U/L (P = 0.78), while that in the 13 CDP patients increased from (10.7 +/- 3.3) U/L to (24.5 +/- 5.7) U/L (P < 0.05). A single triptorelin stimulating test is highly effective in differentiating IHH from CDP. For some patients without definitive diagnosis, a repeated triptorelin stimulating test performed one year later may provide more valuable information on the dynamic change of the hypothalamus-pituitary-testis axis function.

Evaluation of selected binding domains for the analysis of ubiquitinated proteomes

PubMed Central

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-01-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising, but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ∼200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle. PMID:23649778

Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25

PubMed Central

Kawaguchi, Kohei; Uo, Kazune; Tanaka, Toshiaki; Komada, Masayuki

2017-01-01

Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core. PMID:28327663

News and Announcements

NASA Astrophysics Data System (ADS)

1999-06-01

1999 EAS Awards The Eastern Analytical Symposium (EAS) announces the winners of their 1999 awards, which will be presented during their annual meeting, to be held November 14-19, 1999, at the Garden State Convention Center in Somerset, NJ. ACS Analytical Chemistry Division, Findeis Young Investigator Award

• David Clemmer, Indiana University

• Milton L. Lee, Brigham Young University

• Phil Williams, Grain Research Laboratory, Winnipeg, Canada

• Frank A. L. Anet, University of California, Los Angeles (Emeritus)

• Catherine Fenselau, University of Maryland at College Park

• Harald Martens, Norwegian University of Science and Technology

• Course, Curriculum, and Laboratory Improvement (CCLI) June 7, 1999
• NSF Collaboratives for Excellence in Teacher Preparation (CETP)
• Preliminary proposals, Track 1 May 1, 1999
• Formal proposals, Track 1 September 1, 1999
• DUE online 1999 guidelines, NSF 99-53 available at http://www.nsf.gov/cgi-bin/getpub?nsf9953

The Camille and Henry Dreyfus Foundation, Inc.

• Camille Dreyfus Teacher-Scholar Awards Program: November 16, 1998
• Henry Dreyfus Teacher-Scholar Awards Program: July 1, 1999
• New Faculty Awards Program: May 14, 1999
• Faculty Start-up Grants for Undergraduate Institutions: May 14, 1999
• Scholar/Fellow Program for Undergraduate Institutions: July 1, 1999
• Special Grant Program in the Chemical Sciences: July 15, 1999
• Postdoctoral Program in Environmental Chemistry: February 26, 1999

• Cottrell College Science Awards: May 15 and November 15
• Cottrell Scholars: First regular business day in September
• Partners in Science: December 1 (the final opportunity for this program is summer 1999)
• Research Opportunity Awards: May 1 and October 1
• Research Innovation Awards: May 1

• Andrew Sinclair, Royal Melbourne Institute of Technology-University of Australia

• Edwin N. Frankel, University of California at Davis

• High School Chemistry (May 19-21)
• Physical Chemistry (June 9-11)
• General Chemistry (June 16-18)
• Advanced Organic Chemistry (June 23-25)
• Environmental Chemistry (June 30-July 2)
• Biochemistry (July 7-9)
• Inorganic Chemistry (Aug. 9-11)
• Electrochemistry (Nov. 10-12)

• Arlene Russell, UCLA, Los Angeles, CA
• Conrad Stanitski, University of Central Arkansas, Conway, AR

• Frank Torre, Springfield College, Springfield, MA
• Anna Wilson, Purdue University, West Lafayette, IN

• Craig Bowen, Clemson University, Clemson, SC
• Mark Freilich, University of Memphis, Memphis, TN
• Marcy Towns, Ball State University, Muncie, IN
• Carol White, Athens Area Technical Institute, Athens, GA

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

PubMed Central

Ivanova, Lyudmila; Buch, Anna; Döhner, Katinka; Pohlmann, Anja; Binz, Anne; Prank, Ute; Sandbaumhüter, Malte

2016-01-01

ABSTRACT Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766WD1767 and 1862WE1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17+)Lox-pUL36-WD/AA-WE/AA and HSV-1(17+)Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread. PMID:27009950

Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex

PubMed Central

Das, Subhendu; Ortiz, Daniel A.; Gurczynski, Stephen J.; Khan, Fatin

2014-01-01

ABSTRACT Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis. IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development. PMID:24899189

Recognition of Poly-Ubiquitins by the Proteasome through Protein Refolding Guided by Electrostatic and Hydrophobic Interactions.

PubMed

Zhang, Yi; Vuković, Lela; Rudack, Till; Han, Wei; Schulten, Klaus

2016-08-25

Specificity of protein degradation by cellular proteasomes comes from tetra-ubiquitin recognition. We carry out molecular dynamics simulations to characterize how the ubiquitin receptor Rpn10 recognizes in the 26S proteasome K48-linked tetra-ubiquitin. In the binding pose, ubiquitin and Rpn10 interact primarily through hydrophobic patches. However, K48-linked tetra-ubiquitin mostly assumes a closed form in solution prior to binding, in which its hydrophobic patches are not exposed to solvent. Likewise, the hydrophobic ubiquitin interacting motifs (UIMs) of Rpn10 are mostly protected prior to binding. As a result, ubiquitin recognition in the proteasome requires refolding of both K48-linked tetra-ubiquitin and Rpn10. Simulations suggest that conserved complementary electrostatic patterns of Rpn10 and ubiquitins guide protein association (stage 1 in the recognition process), which induces refolding (stage 2), and then facilitates formation of hydrophobic contacts (stage 3). The simulations also explain why Rpn10 has a higher affinity for K48-linked tetra-ubiquitin than for mono-ubiquitin and K48-linked di- and tri-ubiquitins. Simulation results expand on the current view that the flexible arm of Rpn10 acts as an extended fragment of α-helices and flexible coils in the recognition process.

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension

PubMed Central

Smit, Judith J; Monteferrario, Davide; Noordermeer, Sylvie M; van Dijk, Willem J; van der Reijden, Bert A; Sixma, Titia K

2012-01-01

Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains. PMID:22863777

Qualitative ubiquitome unveils the potential significances of protein lysine ubiquitination in hyphal growth of Aspergillus nidulans.

PubMed

Chu, Xin-Ling; Feng, Ming-Guang; Ying, Sheng-Hua

2016-02-01

Protein ubiquitination is an evolutionarily conserved post-translational modification process in eukaryotes, and it plays an important role in many biological processes. Aspergillus nidulans, a model filamentous fungus, contributes to our understanding of cellular physiology, metabolism and genetics, but its ubiquitination is not completely revealed. In this study, the ubiquitination sites in the proteome of A. nidulans were identified using a highly sensitive mass spectrometry combined with immuno-affinity enrichment of the ubiquitinated peptides. The 4816 ubiquitination sites were identified in 1913 ubiquitinated proteins, accounting for 18.1% of total proteins in A. nidulans. Bioinformatic analysis suggested that the ubiquitinated proteins associated with a number of biological functions and displayed various sub-cellular localisations. Meanwhile, seven motifs were revealed from the ubiquitinated peptides, and significantly over-presented in the different pathways. Comparison of the enriched functional catalogues indicated that the ubiquitination functions divergently during growth of A. nidulans and Saccharomyces cerevisiae. Additionally, the proteins in A. nidulans-specific sub-category (cell growth/morphogenesis) were subjected to the protein interaction analysis which demonstrated that ubiquitination is involved in the comprehensive protein interactions. This study presents a first proteomic view of ubiquitination in the filamentous fungus, and provides an initial framework for exploring the physiological roles of ubiquitination in A. nidulans.

Ubiquitin Chains Modified by the Bacterial Ligase SdeA Are Protected from Deubiquitinase Hydrolysis.

PubMed

Puvar, Kedar; Zhou, Yiyang; Qiu, Jiazhang; Luo, Zhao-Qing; Wirth, Mary J; Das, Chittaranjan

2017-09-12

The SidE family of Legionella pneumophila effectors is a unique group of ubiquitin-modifying enzymes. Along with catalyzing NAD + -dependent ubiquitination of certain host proteins independent of the canonical E1/E2/E3 pathway, they have also been shown to produce phosphoribosylated free ubiquitin. This modified ubiquitin product is incompatible with conventional E1/E2/E3 ubiquitination processes, with the potential to lock down various cellular functions that are dependent on ubiquitin signaling. Here, we show that in addition to free ubiquitin, Lys63-, Lys48-, Lys11-, and Met1-linked diubiquitin chains are also modified by SdeA in a similar fashion. Both the proximal and distal ubiquitin moieties are targeted in the phosphoribosylation reaction. Furthermore, this renders the ubiquitin chains unable to be processed by a variety of deubiquitinating enzymes. These observations broaden the scope of SdeA's modulatory functions during Legionella infection.

Proteasome subunit Rpn13 is a novel ubiquitin receptor

PubMed Central

Husnjak, Koraljka; Elsasser, Suzanne; Zhang, Naixia; Chen, Xiang; Randles, Leah; Shi, Yuan; Hofmann, Kay; Walters, Kylie; Finley, Daniel; Dikic, Ivan

2010-01-01

Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. Here we report the identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the proteasome. Rpn13 binds ubiquitin via a conserved N-terminal region termed the Pru domain (Pleckstrin-like receptor for ubiquitin), which binds K48-linked diubiquitin with an affinity of ∼90 nM. Like proteasomal ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like domains of the UBL/UBA family of ubiquitin receptors. A synthetic phenotype results in yeast when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are combined, indicating functional linkage between these ubiquitin receptors. Since Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating enzyme, our findings suggest a coupling of chain recognition and disassembly at the proteasome. PMID:18497817

Validating the Test Procedures Described in UL 1741 SA and IEEE P1547.1: Preprint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahmud, Rasel; Hoke, Anderson F; Narang, David J

This paper investigates the test procedures specified in UL 1741 SA and the upcoming revision to IEEE P1547.1. A 550 kVA photovoltaic inverter was chosen for the tests. This research reveals some of key the components to consider while doing certification tests for UL 1741 SA and IEEE P1547.1. This paper also identifies some issues requiring consideration for future releases of the standard, i.e. IEEE P1547.1. This paper investigates the test procedures specified in UL 1741 SA and the upcoming revision to IEEE P1547.1. A 550 kVA photovoltaic inverter was chosen for the tests. This research reveals some of keymore » the components to consider while doing certification tests for UL 1741 SA and IEEE P1547.1. This paper also identifies some issues requiring consideration for future releases of the standard, i.e. IEEE P1547.1.« less

Slab Geometry and Deformation in the Northern Nazca Subduction Zone Inferred From The Relocation and Focal mechanisms of Intermediate-Depth Earthquakes

NASA Astrophysics Data System (ADS)

Chang, Y.; Warren, L. M.; Prieto, G. A.

2015-12-01

In the northern Nazca subduction zone, the Nazca plate is subducting to the east beneath the South American Plate. At ~5.6ºN, the subducting plate has a 240-km east-west offset associated with a slab tear, called the Caldas tear, that separates the northern and southern segments. Our study seeks to better define the slab geometry and deformation in the southern segment, which has a high rate of intermediate-depth earthquakes (50-300 km) between 3.6ºN and 5.2ºN in the Cauca cluster. From Jan 2010 to Mar 2014, 228 intermediate-depth earthquakes in the Cauca cluster with local magnitude Ml 2.5-4.7 were recorded by 65 seismic stations of the Colombian National Seismic Network. We review and, if necessary, adjust the catalog P and S wave arrival picks. We use the travel times to relocate the earthquakes using a double difference relocation method. For earthquakes with Ml ≥3.8, we also use waveform modeling to compute moment tensors . The distribution of earthquake relocations shows an ~15-km-thick slab dipping to the SE. The dip angle increases from 20º at the northern edge of the cluster to 38º at the southern edge. Two concentrated groups of earthquakes extend ~40 km vertically above the general downdip trend, with a 20 km quiet gap between them at ~100 km depth. The earthquakes in the general downdip seismic zone have downdip compressional axes, while earthquakes close to the quiet gap and in the concentrated groups have an oblique component. The general decrease in slab dip angle to the north may be caused by mantle flow through the Caldas tear. The seismicity gap in the slab may be associated with an active deformation zone and the concentrated groups of earthquakes with oblique focal mechanisms could be due to a slab fold.

OTUB1 Co-opts Lys48-Linked Ubiquitin Recognition to Suppress E2 Enzyme Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Juang, Yu-Chi; Landry, Marie-Claude; Sanches, Mario

2012-03-26

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibitedmore » E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.« less

Mass spectrometry techniques for studying the ubiquitin system.

PubMed

Heap, Rachel E; Gant, Megan S; Lamoliatte, Frederic; Peltier, Julien; Trost, Matthias

2017-10-15

Post-translational control of proteins through covalent attachment of ubiquitin plays important roles in all eukaryotic cell functions. The ubiquitin system in humans consists of 2 E1, 35 E2 and >600 E3 ubiquitin ligases as well as hundreds of deubiquitylases, which reverse ubiquitin attachment. Moreover, there are hundreds of proteins with ubiquitin-binding domains that bind one of the eight possible polyubiquitin chains. Dysfunction of the ubiquitin system is associated with many diseases such as cancer, autoimmunity and neurodegeneration, demonstrating the importance of ubiquitylation. Therefore, enzymes of the ubiquitin system are considered highly attractive drug targets. In recent years, mass spectrometry (MS)-based techniques have become increasingly important in the deciphering of the ubiquitin system. This short review addresses the state-of-the-art MS techniques for the identification of ubiquitylated proteins and their ubiquitylation sites. We also discuss the identification and quantitation of ubiquitin chain topologies and highlight how the activity of enzymes in the ubiquitin pathway can be measured. Finally, we present current MS tools that can be used for drug discovery in the ubiquitin space. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation

PubMed Central

Hanada, Kazuharu; Ohsawa, Noboru

2017-01-01

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP–RING–ZfUBP–CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. PMID:28768733

The decoy Fcγ receptor encoded by the cytomegalovirus UL119-UL118 gene has differential affinity to IgG proteins expressing different GM allotypes.

PubMed

Pandey, Janardan P; Namboodiri, Aryan M; Radwan, Faisal F; Nietert, Paul J

2015-08-01

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in many diseases. However, there is significant divergence between HCMV seroprevalence and the prevalence of HCMV-associated diseases, implying the presence of host genetic factors that might modulate immunity to this virus. HCMV deploys many sophisticated strategies to evade host immunosurveillance. One strategy involves encoding for proteins that have functional properties of the Fcγ receptor (FcγR). The aim of the present investigation was to determine whether the UL119-UL118-encoded recombinant FcγR ectodomain binds differentially to genetically disparate IgG1 proteins. Results show that mean absorbance values for binding of HCMV UL119-UL118-encoded Fcγ receptor to the immunoglobulin GM (γ marker) 1,17-expressing IgG1 were significantly higher than to the IgG1 expressing the allelic GM 3 allotype (0.225 vs. 0.151; p=0.039). These findings suggest possible mechanisms underlying the maintenance of immunoglobulin GM gene polymorphism and its putative role in the etiology of HCMV-associated diseases. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Modulated discharge of Purkinje and stellate cells persists after unilateral loss of vestibular primary afferent mossy fibers in mice

PubMed Central

Yakhnitsa, V.

2013-01-01

Cerebellar Purkinje cells are excited by two afferent pathways: climbing and mossy fibers. Climbing fibers evoke large “complex spikes” (CSs) that discharge at low frequencies. Mossy fibers synapse on granule cells whose parallel fibers excite Purkinje cells and may contribute to the genesis of “simple spikes” (SSs). Both afferent systems convey vestibular information to folia 9c–10. After making a unilateral labyrinthectomy (UL) in mice, we tested how the discharge of CSs and SSs was changed by the loss of primary vestibular afferent mossy fibers during sinusoidal roll tilt. We recorded from cells identified by juxtacellular neurobiotin labeling. The UL preferentially reduced vestibular modulation of CSs and SSs in folia 8–10 contralateral to the UL. The effects of a UL on Purkinje cell discharge were similar in folia 9c–10, to which vestibular primary afferents project, and in folia 8–9a, to which they do not project, suggesting that vestibular primary afferent mossy fibers were not responsible for the UL-induced alteration of SS discharge. UL also induced reduced vestibular modulation of stellate cell discharge contralateral to the UL. We attribute the decreased modulation to reduced vestibular modulation of climbing fibers. In summary, climbing fibers modulate CSs directly and SSs indirectly through activation of stellate cells. Whereas vestibular primary afferent mossy fibers cannot account for the modulated discharge of SSs or stellate cells, the nonspecific excitation of Purkinje cells by parallel fibers may set an operating point about which the discharges of SSs are sculpted by climbing fibers. PMID:23966673

Correlation between systemic lupus erythematosus and cytomegalovirus infection detected by different methods.

PubMed

Chen, Jing; Zhang, Huidi; Chen, Peirong; Lin, Qiaoai; Zhu, Xiaochun; Zhang, Lifang; Xue, Xiangyang

2015-04-01

Human cytomegalovirus (HCMV), a β-herpes virus subfamily member, leads to a lifelong, latent infection in most humans, but the correlation between HCMV infection and systemic lupus erythematosus (SLE) remains controversial. We analyzed the relevance of HCMV infection in SLE by analyzing the peripheral blood leukocytes (PBLs) and serum samples of 60 patients with SLE and 111 healthy individuals. HCMV genes UL55 and UL138 were detected in PBLs by polymerase chain reaction (PCR), and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. The relationship between cellular HCMV infection in PBLs and common clinical indicators of SLE was further explored. Data indicated that the frequency of positive IgG and IgM anti-CMV antibodies was not significantly different in SLE patients and controls. However, compared to the healthy controls, the titers of IgG and IgM anti-CMV antibodies in SLE patients were significantly higher. The detection of cellular HCMV infection showed that almost all subjects were positive for UL138 gene in PBLs, but the positivity for UL55 gene was lower in PBLs. HCMV UL138 detection in PBLs was highly consistent with the frequency of the HCMV-specific IgG test and did not show significant difference in SLE patients and healthy controls. However, compared with that in healthy people, the positivity rate for cellular HCMV UL55 detection was significantly higher in SLE patients (P < 0.001). In addition, cellular HCMV UL55 with positive detection in PBLs was associated with significantly different clinical characteristics of SLE than that with negative detection. In conclusion, our data confirmed that the HCMV infection was related to the development of SLE. Especially, some clinical strains or substrains of HCMV, such as containing the UL55 gene in HCMV's genome, might play a vital role in the development of SLE.

Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes.

PubMed

Tencer, Adam H; Liang, Qin; Zhuang, Zhihao

2016-08-23

Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest (>50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin's interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double-mutant cycle analysis targeting the ubiquitin residues located in the tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists.

Cellular homeoproteins, SATB1 and CDP, bind to the unique region between the human cytomegalovirus UL127 and major immediate-early genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee Jialing; Klase, Zachary; Gao Xiaoqi

An AT-rich region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region (UR). It has been shown that the UR represses activation of transcription from the UL127 promoter and functions as a boundary between the divergent UL127 and MIE genes during human CMV infection [Angulo, A., Kerry, D., Huang, H., Borst, E.M., Razinsky, A., Wu, J., Hobom, U., Messerle, M., Ghazal, P., 2000. Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter. J.more » Virol. 74 (6), 2826-2839; Lundquist, C.A., Meier, J.L., Stinski, M.F., 1999. A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer. J. Virol. 73 (11), 9039-9052]. A putative forkhead box-like (FOX-like) site, AAATCAATATT, was identified in the UR and found to play a key role in repression of the UL127 promoter in recombinant virus-infected cells [Lashmit, P.E., Lundquist, C.A., Meier, J.L., Stinski, M.F., 2004. Cellular repressor inhibits human cytomegalovirus transcription from the UL127 promoter. J. Virol. 78 (10), 5113-5123]. However, the cellular factors which associate with the UR and FOX-like region remain to be determined. We reported previously that pancreatic-duodenal homeobox factor-1 (PDX1) bound to a 45-bp element located within the UR [Chao, S.H., Harada, J.N., Hyndman, F., Gao, X., Nelson, C.G., Chanda, S.K., Caldwell, J.S., 2004. PDX1, a Cellular Homeoprotein, Binds to and Regulates the Activity of Human Cytomegalovirus Immediate Early Promoter. J. Biol. Chem. 279 (16), 16111-16120]. Here we demonstrate that two additional cellular homeoproteins, special AT-rich sequence binding protein 1 (SATB1) and CCAAT displacement protein (CDP), bind to the human CMV UR in vitro and in vivo. Furthermore, CDP is identified as a FOX-like binding protein and a repressor of the UL127 promoter, while SATB1 has no effect on UL127 expression. Since CDP is known as a transcription repressor and a nuclear matrix-associated region binding protein, CDP may have a role in the regulation of human CMV transcription.« less

47 CFR 27.1182 - Reimbursement under the Cost-Sharing Plan.

Code of Federal Regulations, 2011 CFR

2011-10-01

... reimbursement, an AWS relocator must submit documentation of the relocation agreement to the clearinghouse... involuntary relocation, an AWS relocator must submit documentation of the relocated system within 30 calendar... above ground level height of the system's receiving antenna centerline. (3) The AWS relocator must also...

47 CFR 27.1182 - Reimbursement under the Cost-Sharing Plan.

Code of Federal Regulations, 2010 CFR

2010-10-01

... reimbursement, an AWS relocator must submit documentation of the relocation agreement to the clearinghouse... involuntary relocation, an AWS relocator must submit documentation of the relocated system within 30 calendar... above ground level height of the system's receiving antenna centerline. (3) The AWS relocator must also...

25 CFR 700.93 - Relocation plan.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 25 Indians 2 2010-04-01 2010-04-01 false Relocation plan. 700.93 Section 700.93 Indians THE OFFICE OF NAVAJO AND HOPI INDIAN RELOCATION COMMISSION OPERATIONS AND RELOCATION PROCEDURES General Policies and Instructions Definitions § 700.93 Relocation plan. The relocation plan shall be the plan prepared...

Ramjet and Ramrocket Propulsion Systems for Missiles Held at Monterey, California, London (United Kingdom), and Neubiberg (Germany F. R.) on 5-6; 10-11 and 13-14 September 1984

DTIC Science & Technology

1984-10-01

rement ablat6 ou clramisfi, 18 conductivitA du rfasdu est la seule propriat6 qui agisse sur le gradient thermique dans la paroi. C’ost pourquoi, un...SPEAKERS Mr J.G.Bendot Mr P.Cazin The Marquardt Company O.N.E.R.A. 16555 Saticoy Street 29, Avcnue de la Division Leclerc Van Nuys, California 91409 92320...A cM rk a t *rc 2-16 - LA SRM FLIGHT TEST VEHICLE (U.S. 1965) ALVRJFIH;ES3EIL FIGURE 6. MULTIPLE AFT &mOUIVT D l L TRA J T P O UL i N S T M TERMINAL

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB*

PubMed Central

Kensche, Tobias; Tokunaga, Fuminori; Ikeda, Fumiyo; Goto, Eiji; Iwai, Kazuhiro; Dikic, Ivan

2012-01-01

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli. PMID:22605335

Ubiquitin proteasome system in Parkinson's disease: a keeper or a witness?

PubMed

Martins-Branco, Diogo; Esteves, Ana R; Santos, Daniel; Arduino, Daniela M; Swerdlow, Russell H; Oliveira, Catarina R; Januario, Cristina; Cardoso, Sandra M

2012-12-01

The aim of this work was to evaluate the role of ubiquitin-proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models. We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations. We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. aSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. Copyright © 2012 Elsevier Inc. All rights reserved.

Ubiquitin Proteasome System in Parkinson Disease: a keeper or a witness?

PubMed Central

Martins-Branco, Diogo; Esteves, Ana R.; Santos, Daniel; Arduino, Daniela M.; Swerdlow, Russell H.; Oliveira, Catarina R.; Januario, Cristina; Cardoso, Sandra M.

2014-01-01

Objective The aim of this work was to evaluate the role of Ubiquitin-Proteasome System (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson disease (PD) cellular models. Method We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patients population we evaluated aSN levels in plasma and the influence of several demographic characteristics in the above mentioned determinations. Results We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a down regulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomers levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. Interpretation aSN oligomers are ubiquitinated and we identified an ubiquitin-dependent clearance insufficiency with accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. PMID:22921536

Ubiquitin in Influenza Virus Entry and Innate Immunity.

PubMed

Rudnicka, Alina; Yamauchi, Yohei

2016-10-24

Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle.

Ubiquitin in Influenza Virus Entry and Innate Immunity

PubMed Central

Rudnicka, Alina; Yamauchi, Yohei

2016-01-01

Viruses are obligatory cellular parasites. Their mission is to enter a host cell, to transfer the viral genome, and to replicate progeny whilst diverting cellular immunity. The role of ubiquitin is to regulate fundamental cellular processes such as endocytosis, protein degradation, and immune signaling. Many viruses including influenza A virus (IAV) usurp ubiquitination and ubiquitin-like modifications to establish infection. In this focused review, we discuss how ubiquitin and unanchored ubiquitin regulate IAV host cell entry, and how histone deacetylase 6 (HDAC6), a cytoplasmic deacetylase with ubiquitin-binding activity, mediates IAV capsid uncoating. We also discuss the roles of ubiquitin in innate immunity and its implications in the IAV life cycle. PMID:27783058

Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles

NASA Technical Reports Server (NTRS)

Riley, Danny A.; Bain, James L. W.; Haas, Arthur L.; Ellis, Stanley

1988-01-01

Solid-phase immunochemical methods were employed to probe the dynamics of ubiquitin pools within selected rat skeletal muscles. The total ubiquitin content of red muscles was greater than that of white muscles, even though the fractional conjugation was similar for both types of muscles. The specificity for conjugated ubiquitin in solid-phase applications, previously demonstrated for an affinity-purified antibody against SDS-denatured ubiquitin, was retained when used as a probe for ubiquitin-protein adducts in tissue sections. Immunohistochemical localization revealed that differences in ubiquitin pools derived from the relative content of red (oxidative) vs white (glycolytic) fibers, with the former exhibiting a higher content of ubiquitin conjugates. Subsequent immunogold labeling demonstrated statistically significant enhanced localization of ubiquitin conjugates to the Z-lines in both red and white muscle fiber types.

Ubiquitination in Periodontal Disease: A Review.

PubMed

Tsuchida, Sachio; Satoh, Mamoru; Takiwaki, Masaki; Nomura, Fumio

2017-07-10

Periodontal disease (periodontitis) is a chronic inflammatory condition initiated by microbial infection that leads to gingival tissue destruction and alveolar bone resorption. The periodontal tissue's response to dental plaque is characterized by the accumulation of polymorphonuclear leukocytes, macrophages, and lymphocytes, all of which release inflammatory mediators and cytokines to orchestrate the immunopathogenesis of periodontal disease. Ubiquitination is achieved by a mechanism that involves a number of factors, including an ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase. Ubiquitination is a post-translational modification restricted to eukaryotes that are involved in essential host processes. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Increasing numbers of recent reports have provided evidence that many approaches are delivering promising reports for discovering the relationship between ubiquitination and periodontal disease. The scope of this review was to investigate recent progress in the discovery of ubiquitinated protein in diseased periodontium and to discuss the ubiquitination process in periodontal diseases.

Synthetic and semi-synthetic strategies to study ubiquitin signaling.

PubMed

van Tilburg, Gabriëlle Ba; Elhebieshy, Angela F; Ovaa, Huib

2016-06-01

The post-translational modification ubiquitin can be attached to the ɛ-amino group of lysine residues or to a protein's N-terminus as a mono ubiquitin moiety. Via its seven intrinsic lysine residues and its N-terminus, it can also form ubiquitin chains on substrates in many possible ways. To study ubiquitin signals, many synthetic and semi-synthetic routes have been developed for generation of ubiquitin-derived tools and conjugates. The strength of these methods lies in their ability to introduce chemo-selective ligation handles at sites that currently cannot be enzymatically modified. Here, we review the different synthetic and semi-synthetic methods available for ubiquitin conjugate synthesis and their contribution to how they have helped investigating conformational diversity of diubiquitin signals. Next, we discuss how these methods help understanding the ubiquitin conjugation-deconjugation system by recent advances in ubiquitin ligase probes and diubiquitin-based DUB probes. Lastly, we discuss how these methods help studying post-translational modification of ubiquitin itself. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ubiquitination dynamics in the early-branching eukaryote Giardia intestinalis

PubMed Central

Niño, Carlos A; Chaparro, Jenny; Soffientini, Paolo; Polo, Simona; Wasserman, Moises

2013-01-01

Ubiquitination is a highly dynamic and versatile posttranslational modification that regulates protein function, stability, and interactions. To investigate the roles of ubiquitination in a primitive eukaryotic lineage, we utilized the early-branching eukaryote Giardia intestinalis. Using a combination of biochemical, immunofluorescence-based, and proteomics approaches, we assessed the ubiquitination status during the process of differentiation in Giardia. We observed that different types of ubiquitin modifications present specific cellular and temporal distribution throughout the Giardia life cycle from trophozoites to cyst maturation. Ubiquitin signal was detected in the wall of mature cysts, and enzymes implicated in cyst wall biogenesis were identified as substrates for ubiquitination. Interestingly, inhibition of proteasome activity did not affect trophozoite replication and differentiation, while it caused a decrease in cyst viability, arguing for proteasome involvement in cyst wall maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation. Our results indicate that ubiquitination is critical for several cellular processes in this primitive eukaryote. PMID:23613346

Target Specificity of the E3 Ligase LUBAC for Ubiquitin and NEMO Relies on Different Minimal Requirements*

PubMed Central

Smit, Judith J.; van Dijk, Willem J.; El Atmioui, Dris; Merkx, Remco; Ovaa, Huib; Sixma, Titia K.

2013-01-01

The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin. PMID:24030825

75 FR 77002 - Expansion of the Scope of NRTL Recognition of Underwriters Laboratories Inc.; Modification to the...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-10

...): IEEE C37.20.4 Indoor AC Switches (1 kV-38 kV) for Use in Metal-Enclosed Switchgear \\a\\ IEEE C37.20.6 4.76 kV to 38 kV Rated Grounding and Testing Devices Used in Enclosures \\a\\ IEEE C37.23 Metal-Enclosed... Sprinkler Pipe for Fire Protection Service UL 962 Household and Commercial Furnishings \\c\\ UL 1340 Hoists UL...

Looking for Gold Nuggets in the Melting Pot: Language, Cultural Awareness, and the Fourth Generation Warrior

DTIC Science & Technology

2006-04-01

and actively engage in the larger society and its political processes.108 Dr. Qamar -ul Huda, assistant professor of Islamic Studies and Comparative...August 2003, http://mediaguidetoislam.sfsu.edu/intheus/03d_experience. htm. 109 Qamar -ul Huda, “Forging an American Muslim Identity: Time for...Dictionary of Cultural Literacy, Third Edition. Houghton Mifflin Company, 2002, http://www.bartleby.com/59/6/givemeyourti.html. Huda, Qamar -ul

Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases.

PubMed

Crowe, Sean O; Pham, Grace H; Ziegler, Jacob C; Deol, Kirandeep K; Guenette, Robert G; Ge, Ying; Strieter, Eric R

2016-08-17

Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin-specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of ubiquitin chains when studying how DUBs process these chains. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Crystal Structure and Conformations of an Unbranched Mixed Tri-Ubiquitin Chain Containing K48 and K63 Linkages.

PubMed

Padala, Prasanth; Soudah, Nadine; Giladi, Moshe; Haitin, Yoni; Isupov, Michail N; Wiener, Reuven

2017-12-08

The ability of ubiquitin to function in a wide range of cellular processes is ascribed to its capacity to cause a diverse spectrum of modifications. While a target protein can be modified with monoubiquitin, it can also be modified with ubiquitin chains. The latter include seven types of homotypic chains as well as mixed ubiquitin chains. In a mixed chain, not all the isopeptide bonds are restricted to a specific lysine of ubiquitin, resulting in a chain possessing more than one type of linkage. While structural characterization of homotypic chains has been well elucidated, less is known about mixed chains. Here we present the crystal structure of a mixed tri-ubiquitin chain at 3.1-Å resolution. In the structure, the proximal ubiquitin is connected to the middle ubiquitin via K48 and these two ubiquitins adopt a compact structure as observed in K48 di-ubiquitin. The middle ubiquitin links to the distal ubiquitin via its K63 and these ubiquitins adopt two conformations, suggesting a flexible structure. Using small-angle X-ray scattering, we unexpectedly found differences between the conformational ensembles of the above tri-ubiquitin chains and chains possessing the same linkages but in the reverse order. In addition, cleavage of the K48 linkage by DUB is faster if this linkage is at the distal end. Taken together, our results suggest that in mixed chains, not only the type of the linkages but also their sequence determine the structural and functional properties of the chain. Copyright © 2017 Elsevier Ltd. All rights reserved.

Non-degradative Ubiquitination of Protein Kinases

PubMed Central

Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

2016-01-01

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

Diggin’ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

PubMed Central

Rubel, Carrie E.; Schisler, Jonathan C.; Hamlett, Eric D.; DeKroon, Robert M.; Gautel, Mathias; Alzate, Oscar; Patterson, Cam

2013-01-01

The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing Tandem Ubiquitin Binding Entities (TUBE) technology, two-dimensional differential in gel electrophoresis (2-D DIGE), and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets. PMID:23695782

Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

PubMed Central

Lechtenberg, Bernhard C.; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K.; Ware, Carl F.; Mace, Peter D.; Riedl, Stefan J.

2015-01-01

Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

41 CFR 302-12.101 - May we enter into a contract with a relocation services company for the company to provide...

Code of Federal Regulations, 2010 CFR

2010-07-01

... contract with a relocation services company for the company to provide relocation services? 302-12.101... the company to provide relocation services? Yes, you may enter into a contract with a relocation services company for the company to provide relocation services. ...

Selective autophagy: ubiquitin-mediated recognition and beyond.

PubMed

Kraft, Claudine; Peter, Matthias; Hofmann, Kay

2010-09-01

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Whereas the ubiquitin-proteasome system is involved in the rapid degradation of proteins, autophagy pathways can selectively remove protein aggregates and damaged or excess organelles. Proteasome-mediated degradation requires previous ubiquitylation of the cargo, which is then recognized by ubiquitin receptors directing it to 26S proteasomes. Although autophagy has long been viewed as a random cytoplasmic degradation system, the involvement of ubiquitin as a specificity factor for selective autophagy is rapidly emerging. Recent evidence also suggests active crosstalk between proteasome-mediated degradation and selective autophagy. Here, we discuss the molecular mechanisms that link autophagy and the proteasome system, as well as the emerging roles of ubiquitin and ubiquitin-binding proteins in selective autophagy. On the basis of the evolutionary history of autophagic ubiquitin receptors, we propose a common origin for metazoan ubiquitin-dependent autophagy and the cytoplasm-to-vacuole targeting pathway of yeast.

The mechanism of linkage-specific ubiquitin chain elongation by a single-subunit E2

PubMed Central

Wickliffe, Katherine E.; Lorenz, Sonja; Wemmer, David E.; Kuriyan, John; Rape, Michael

2011-01-01

Ubiquitin chains of different topologies trigger distinct functional consequences, including protein degradation and reorganization of complexes. The assembly of most ubiquitin chains is promoted by E2s, yet how these enzymes achieve linkage specificity is poorly understood. We have discovered that the K11-specific Ube2S orients the donor ubiquitin through an essential non-covalent interaction that occurs in addition to the thioester bond at the E2 active site. The E2-donor ubiquitin complex transiently recognizes the acceptor ubiquitin, primarily through electrostatic interactions. The recognition of the acceptor ubiquitin surface around Lys11, but not around other lysines, generates a catalytically competent active site, which is composed of residues of both Ube2S and ubiquitin. Our studies suggest that monomeric E2s promote linkage-specific ubiquitin chain formation through substrate-assisted catalysis. PMID:21376237

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation.

PubMed

Shoji, Shisako; Hanada, Kazuharu; Ohsawa, Noboru; Shirouzu, Mikako

2017-09-07

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP-RING-ZfUBP-CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Gemfibrozil-induced myositis in a patient with normal renal function.

PubMed

Hahn, Martin; Sriharan, Kalavally; McFarland, M Shawn

2010-01-01

To describe a case of gemfibrozil monotherapy-induced myositis in a patient with normal renal function A 68-year-old white man presented to his primary care clinic complaining of a 6-month history of total body pain. His past medical history was significant for hypertension, diabetes mellitus, hyperlipidemia, gastroesophageal reflux disease, benign prostatic hypertrophy, arthritis, impotence, and pancreatic cancer that required excision of part of his pancreas. His home drug regimen included bupropion 75 mg twice daily, gemfibrozil 600 mg twice daily for the past 8 months, glimiperide 1 mg daily, insulin glargine 5 units at bedtime, insulin aspart 5 units in the evening, lisinopril 10 mg daily, omeprazole 40 mg daily, pregabalin 100 mg daily, and sildenafil 100 mg as needed. Laboratory test results were significant for elevated aspartate aminotransferase (AST) 78 U/L (reference range 15-46 U/L), alanine aminotransferase (ALT) 83 U/L (13-69 U/L), and creatine kinase (CK) 3495 U/L (55-170 U/L). Serum creatinine was normal at 1.19 mg/dL. The physician determined that the elevated CK indicated myositis secondary to gemfibrozil use, and gemfibrozil was subsequently discontinued. The patient returned 1 week later to repeat the laboratory tests. Results were CK 220 U/L, AST 26 U/L, ALT 43 U/L, and serum creatinine 1.28 mg/dL. The patient was asked to return in 3 weeks to repeat the laboratory tests. At that time, CK had continued to decrease to 142 U/L, and the AST and ALT had returned to normal, at 22 and 29 U/L, respectively. The patient reported complete resolution of total body pain 3 weeks after discontinuation of gemfibrozil. Follow-up 5 weeks after discontinuation revealed no change compared to the 3-week follow-up. Myositis most often produces weakness and elevated CK levels more than 10 times the upper limit of normal. The risk of developing myositis, myopathy, or rhabdomyolysis is low (1%) when fibrates such as gemfibrozil are used as monotherapy. Evaluation of the literature revealed one case of gemfibrozil-related myositis in a patient with chronic renal failure. There is also one report of myopathy associated with gemfibrozil monotherapy in a patient with normal renal function. The present case is the first documented case of gemfibrozil monotherapy-induced myositis in a patient with normal renal function. The Naranjo probability scale indicated a probable relationship between gemfibrozil treatment and the onset of myositis in our patient. Other potential causes of myositis were ruled out by patient interview and chart review. Although the risk of myositis appears to be low with gemfibrozil monotherapy, clinicians should be aware of the potential for this adverse event. For patients taking gemfibrozil monotherapy who present with myalgia, discontinuation of the medication may be necessary for the alleviation of pain.

Negative Influence of Motor Impairments on Upper Limb Movement Patterns in Children with Unilateral Cerebral Palsy. A Statistical Parametric Mapping Study

PubMed Central

Simon-Martinez, Cristina; Jaspers, Ellen; Mailleux, Lisa; Desloovere, Kaat; Vanrenterghem, Jos; Ortibus, Els; Molenaers, Guy; Feys, Hilde; Klingels, Katrijn

2017-01-01

Upper limb three-dimensional movement analysis (UL-3DMA) offers a reliable and valid tool to evaluate movement patterns in children with unilateral cerebral palsy (uCP). However, it remains unknown to what extent the underlying motor impairments explain deviant movement patterns. Such understanding is key to develop efficient rehabilitation programs. Although UL-3DMA has been shown to be a useful tool to assess movement patterns, it results in a multitude of data, challenging the clinical interpretation and consequently its implementation. UL-3DMA reports are often reduced to summary metrics, such as average or peak values per joint. However, these metrics do not take into account the continuous nature of the data or the interdependency between UL joints, and do not provide phase-specific information of the movement pattern. Moreover, summary metrics may not be sensitive enough to estimate the impact of motor impairments. Recently, Statistical Parametric Mapping (SPM) was proposed to overcome these problems. We collected UL-3DMA of 60 children with uCP and 60 typically developing children during eight functional tasks and evaluated the impact of spasticity and muscle weakness on UL movement patterns. SPM vector field analysis was used to analyze movement patterns at the level of five joints (wrist, elbow, shoulder, scapula, and trunk). Children with uCP showed deviant movement patterns in all joints during a large percentage of the movement cycle. Spasticity and muscle weakness negatively impacted on UL movement patterns during all tasks, which resulted in increased wrist flexion, elbow pronation and flexion, increased shoulder external rotation, decreased shoulder elevation with a preference for movement in the frontal plane and increased trunk internal rotation. Scapular position was altered during movement initiation, although scapular movements were not affected by muscle weakness or spasticity. In conclusion, we identified pathological movement patterns in children with uCP and additionally mapped the negative impact of spasticity and muscle weakness on these movement patterns, providing useful insights that will contribute to treatment planning. Last, we also identified a subset of the most relevant tasks for studying UL movements in children with uCP, which will facilitate the interpretation of UL-3DMA data and undoubtedly contribute to its clinical implementation. PMID:29051729

Depressive Symptoms and Risk of Uterine Leiomyomata

PubMed Central

Wise, Lauren A.; Se, Li; Palmer, Julie R.; Rosenberg, Lynn

2014-01-01

Objective Uterine leiomyomata (UL) are a major source of gynecologic morbidity and the primary indication for hysterectomy. Depression can cause dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, which may affect the synthesis of reproductive hormones involved in UL pathogenesis. We assessed the association between depressive symptoms and UL among 15,963 premenopausal women. Study Design Data were derived from the Black Women’s Health Study, a prospective cohort study. In 1999 and 2005, the Center for Epidemiologic Studies Depression Scale (CES-D) was used to ascertain depressive symptoms. On biennial follow-up questionnaires from 1999 through 2011, women reported physician-diagnosed depression, antidepressant use, and UL diagnoses. Incidence rate ratios (IRR) and 95% confidence intervals (CI) were estimated using multivariable Cox regression. Results There were 4,722 incident UL cases diagnosed by ultrasound (n=3,793) or surgery (n=929) during 131,262 person-years of follow-up. Relative to baseline CES-D scores <16, IRRs were 1.05 (95% CI, 0.98–1.13) for CES-D scores 16–24 and 1.16 (95% CI, 1.06–1.27) for CES-D scores ≥25 (P-trend=0.001). IRRs for current and past physician-diagnosed depression relative to no depression were 1.15 (95% CI: 0.98, 1.34) and 1.25 (95% CI: 1.13, 1.39), respectively. Results persisted after further control for antidepressant use. IRRs for current and past use of antidepressants (any indication) relative to never use were 1.11 (95% CI: 0.97, 1.28) and 1.32 (95% CI: 1.14, 1.52), respectively. Conclusions In this cohort of black women, greater depressive symptoms were associated with UL, independent of antidepressant use, supporting the hypothesis that dysregulation of the HPA axis increases UL risk. PMID:25514762

Cyclin D1 G870A polymorphism: Association with uterine leiomyoma risk and in silico analysis

PubMed Central

Salimi, Saeedeh; Shahrakipour, Mahnaz; Hajizadeh, Azam; Mokhtari, Mojgan; Mousavi, Mahdieh; Teimoori, Batool; Yaghmaei, Minoo

2017-01-01

Uterine leiomyoma (UL) is the most common benign tumor causing considerable morbidity during the reproductive years in women. Cyclin D1 (CCND1) is a cell cycle regulatory protein that is required for the G1 phase, and increased expression levels of this protein may affect tumorigenesis. The present study aimed to assess the possible effect of the CCND1 G870A polymorphism on UL susceptibility. A total of 154 women with UL and 197 healthy women who were age-, body mass index (BMI)- and ethnicity-matched were genotyped for the CCND1 G870A (rs9344) polymorphism using the polymerase chain reaction-restriction fragment length polymorphism method. The effects of G870A transition on the structure of mRNA and proteins of CCND1 was evaluated using bioinformatics tools. The frequency of the CCND1 870AA genotype was significantly higher in women with UL compared with the control subjects, and the risk of UL was 1.4-fold higher in women with the AA genotype when compared with the GG genotype before and after adjusting for age, BMI, and ethnicity [odds ratio (OR), 1.4; 95% confidence interval (CI), 1.1–2 (P=0.02)]. The frequency of CCND1 870GA genotype was not significantly different between the two groups. The frequency of the CCND1 870A allele was significantly higher in the women with UL when compared with the control subjects (57 vs. 48%; P=0.02). The in silico analysis revealed that the G870A transition may fundamentally alter the structure of the CCND1-mRNA. Thus, the CCND1 870AA genotype was associated with UL susceptibility in a sample of women from the southeast of Iran. PMID:28357079

Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered.

PubMed

Rieder, Franz J J; Kastner, Marie-Theres; Hartl, Markus; Puchinger, Martin G; Schneider, Martina; Majdic, Otto; Britt, William J; Djinović-Carugo, Kristina; Steininger, Christoph

2017-03-01

Protein phosphorylation has important regulatory functions in cell homeostasis and is tightly regulated by kinases and phosphatases. The tegument of human cytomegalovirus (CMV) contains not only several proteins reported to be extensively phosphorylated but also cellular protein phosphatases (PP1 and PP2A). To investigate this apparent inconsistency, we evaluated the phosphorylation status of the tegument proteins pUL32 and pp65 by enzymatic dephosphorylation and MS. Enzymatic dephosphorylation with bacterial λ phosphatase, but not with PP1, shifted the pUL32-specific signal on reducing SDS-PAGE from ~150 to ~148 kDa, a mass still much larger than the ~118 kDa obtained from our diffusion studies and from the calculated protein mass of ~113 kDa. Remarkably, inhibition of phosphatases through treatment with the phosphatase inhibitors calyculin A and okadaic acid resulted in a shift to ~190 or ~180 kDa, respectively, indicating that a considerable number of potential phosphorylated residues on pUL32 are not phosphorylated under normal conditions. MS revealed a general state of hypophosphorylation of CMV phosphoproteins with only 17 phosphorylated residues detected on pUL32 and 19 on pp65, respectively. Moreover, bioinformatics analysis shows that the C-terminal two-thirds of pUL32 are intrinsically disordered and that most phosphorylations map to this region. In conclusion, we show that important CMV tegument proteins are indeed phosphorylated, though to a lesser extent than previously reported, and the difference in mobility on SDS-PAGE and calculated mass of pUL32 may not be attributed to phosphorylation but more likely due to the partially intrinsically disordered nature of pUL32.

Effects of resistance training using known vs unknown loads on eccentric-phase adaptations and concentric velocity.

PubMed

Hernández-Davó, J L; Sabido, R; Behm, D G; Blazevich, A J

2018-02-01

The aims of this study were to compare both eccentric- and concentric-phase adaptations in highly trained handball players to 4 weeks of twice-weekly rebound bench press throw training with varying loads (30%, 50% and 70% of one-repetition maximum [1-RM]) using either known (KL) or unknown (UL) loads and to examine the relationship between changes in eccentric- and concentric-phase performance. Twenty-eight junior team handball players were divided into two experimental groups (KL or UL) and a control group. KL subjects were told the load prior each repetition, while UL were blinded. For each repetition, the load was dropped and then a rebound bench press at maximum velocity was immediately performed. Both concentric and eccentric velocity as well as eccentric kinetic energy and musculo-articular stiffness prior to the eccentric-concentric transition were measured. Results showed similar increases in both eccentric velocity and kinetic energy under the 30% 1-RM but greater improvements under 50% and 70% 1-RM loads for UL than KL. UL increased stiffness under all loads (with greater magnitude of changes). KL improved concentric velocity only under the 30% 1-RM load while UL also improved under 50% and 70% 1-RM loads. Improvements in concentric movement velocity were moderately explained by changes in eccentric velocity (R 2 =.23-.62). Thus, UL led to greater improvements in concentric velocity, and the improvement is potentially explained by increases in the speed (as well as stiffness and kinetic energy) of the eccentric phase. Unknown load training appears to have significant practical use for the improvement of multijoint stretch-shortening cycle movements. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of dietary intake data using the tolerable upper intake levels.

PubMed

Carriquiry, Alicia L; Camaño-Garcia, Gabriel

2006-02-01

We discuss the problem of assessing nutrient intake relative to the tolerable upper intake levels (UL) for the nutrient proposed by the Institute of Medicine and focus on 2 important topics: the estimation of usual nutrient intake distributions and the extent to which intakes above the UL can be considered risky. With the information that is currently available for most nutrients, it is not possible to estimate the proportion of individuals in a group with intakes that place them at risk. This is because the shape of the dose-response curve needed to carry out a risk assessment is unknown for most nutrients. Thus, intakes above UL cannot be declared to be unsafe. Intakes below the UL, however, are likely to pose no risk to individuals in the group. Because determining the proportion of individuals with intakes below the UL requires estimation of an upper-tail percentile of the intake distribution, the use of 1-d intake data or otherwise unadjusted intake data are likely to lead to severely biased estimates. It is important to remove within-individual variance in intakes from daily intakes so that the tails of the usual intake distribution are accurately estimated. Underreporting of the amount of nutrients consumed will tend to shift the estimated usual nutrient intake distribution downwards. In this case, the true proportion of individuals with intakes below the UL is likely to be overestimated.

Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress.

PubMed

Hamirally, Sofia; Kamil, Jeremy P; Ndassa-Colday, Yasmine M; Lin, Alison J; Jahng, Wan Jin; Baek, Moon-Chang; Noton, Sarah; Silva, Laurie A; Simpson-Holley, Martha; Knipe, David M; Golan, David E; Marto, Jarrod A; Coen, Donald M

2009-01-01

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser(22)). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser(22) is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Feasibility and effectiveness of adding object-related bilateral symmetrical training to mirror therapy in chronic stroke: A randomized controlled pilot study.

PubMed

Rodrigues, Letícia Cardoso; Farias, Nayara Correa; Gomes, Raquel Pinheiro; Michaelsen, Stella Maris

2016-01-01

To evaluate the feasibility and effectiveness of adding object-related bilateral symmetrical training to mirror therapy (MT) to improve upper limb (UL) activity in chronic stroke patients. Sixteen patients with moderate UL impairment were randomly allocated to either the experimental (EG) or control (CG) group. Both groups performed 1 hour sessions, 3 days/week for 4 weeks, involving object-related bilateral symmetrical training. EG performed the tasks observing their nonparetic UL reflected in the mirror, while CG observed the paretic UL directly. The primary outcome measure was unilateral and bilateral UL activity according to the Test d'Évaluation des Membres Supérieurs de Personnes Âgées (TEMPA). All measurements were taken at baseline, post-training, and follow-up (2 weeks). TEMPA total score showed the main effect of time. Significant improvement was found for bilateral but not unilateral tasks. Both groups showed gains after training, with no differences between them. This study showed the feasibility of adding object-related bilateral training to MT. Both types of training improved UL bilateral activity; however, a larger sample is required for a definitive study. Other studies need to be carried out to evaluate the effectiveness of combining more distal-oriented movements and object-related unilateral training to improve these effects in chronic stroke patients.

Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

PubMed

Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

2017-11-10

Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

USP7 small-molecule inhibitors interfere with ubiquitin binding.

PubMed

Kategaya, Lorna; Di Lello, Paola; Rougé, Lionel; Pastor, Richard; Clark, Kevin R; Drummond, Jason; Kleinheinz, Tracy; Lin, Eva; Upton, John-Paul; Prakash, Sumit; Heideker, Johanna; McCleland, Mark; Ritorto, Maria Stella; Alessi, Dario R; Trost, Matthias; Bainbridge, Travis W; Kwok, Michael C M; Ma, Taylur P; Stiffler, Zachary; Brasher, Bradley; Tang, Yinyan; Jaishankar, Priyadarshini; Hearn, Brian R; Renslo, Adam R; Arkin, Michelle R; Cohen, Frederick; Yu, Kebing; Peale, Frank; Gnad, Florian; Chang, Matthew T; Klijn, Christiaan; Blackwood, Elizabeth; Martin, Scott E; Forrest, William F; Ernst, James A; Ndubaku, Chudi; Wang, Xiaojing; Beresini, Maureen H; Tsui, Vickie; Schwerdtfeger, Carsten; Blake, Robert A; Murray, Jeremy; Maurer, Till; Wertz, Ingrid E

2017-10-26

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

PubMed Central

Lin, Yu-Chen; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication. PMID:28536288

Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin

USDA-ARS?s Scientific Manuscript database

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. T...

Does nonylphenol promote the growth of uterine fibroids?

PubMed

Shen, Yang; Ren, Mu-Lan; Feng, Xu; Gao, Yong-Xing; Xu, Qian; Cai, Yun-Lang

2014-07-01

To study the effect and mechanism of action of nonylphenol (NP), an environmental oestrogen, on uterine leiomyoma (UL) cells. Primary culture and subculture of human UL cells, identified as smooth muscle cells by immunocytochemical staining with a monoclonal anti-α-smooth muscle actin antibody, were performed. The viability of cells treated with various concentrations of NP for 24, 48 and 72h was determined by CCK-8 assay. mRNA expression of oestrogen receptor α (ERα), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) was detected using real-time quantitative polymerase chain reaction, and protein expression was detected using Western blot analysis for all groups. NP promoted the growth of UL cells and expression of ERα, IGF-1 and VEGF; this was positively correlated with the concentration and duration of NP treatment. NP promotes the growth of UL cells. The mechanism of action appears to be over-expression of IGF-1 and VEGF, up-regulated by ERα, resulting in the growth of UL cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Genomic variation of the fibropapilloma-associated marine turtle herpes virus across seven geographic areas and three host species

USGS Publications Warehouse

Greenblatt, R.J.; Quackenbush, S.L.; Casey, R.N.; Rovnak, J.; Balazs, G.H.; Work, Thierry M.; Casey, J.W.; Sutton, C.A.

2005-01-01

Fibropapillomatosis (FP) of marine turtles is an emerging neoplastic disease associated with infection by a novel turtle herpesvirus, fibropapilloma-associated turtle herpesvirus (FPTHV). This report presents 23 kb of the genome of an FPTHV infecting a Hawaiian green turtle (Chelonia mydas). By sequence homology, the open reading frames in this contig correspond to herpes simplex virus genes UL23 through UL36. The order, orientation, and homology of these putative genes indicate that FPTHV is a member of the Alphaherpesvirinae. The UL27-, UL30-, and UL34-homologous open reading frames from FPTHVs infecting nine FP-affected marine turtles from seven geographic areas and three turtle species (C. mydas, Caretta caretta, and Lepidochelys olivacea) were compared. A high degree of nucleotide sequence conservation was found among these virus variants. However, geographic variations were also found: the FPTHVs examined here form four groups, corresponding to the Atlantic Ocean, West pacific, mid-Pacific, and east Pacific. Our results indicate that FPTHV was established in marine turtle populations prior to the emergence of FP as it is currently known.

A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

PubMed Central

Stringer, Daniel K.

2011-01-01

ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination. PMID:21242292

The IDA/BPT Crisis Relocation Planning Model: Description, Documentation and User’s Guide to the Computer Program.

DTIC Science & Technology

1982-12-22

Columbia 3 39 Ohio 5 12 Florida 4 40 Oklahoma 6 13 Georgia 4 41 Oregon 10 14 Guam 9 42 Pennsylvania 3 15 Hawaii 9 43 Puerto Rico 2 16 Idaho 10 44 Rhode...TY 22627. ESMOND PROVIDENCE PENNINGTON BENNINGTON ? 15. . 575"N. BOSTON SUFFEL.K P%-D BATH S2.4AHOC 4 134. " . 13644. BOSTON SUFFOLK 1>3D LEWISTON ...YORK LEWISTON ANDROSCOCGIN 1. 15 53. 500. O23 290220. SPRINGFIELD HAMPDEN PITTSFIELD BERKS HIRE 1. 10 56. 462. 1 24 59567. NEWF4)RT NEWPORT DANIELSON

76 FR 46830 - Notice of Proposed Information Collection: Comment Request; Application for Displacement...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-03

... Information Collection: Comment Request; Application for Displacement/Relocation/Temporary Relocation... Proposal: Application for Displacement/Relocation/ Temporary Relocation Assistance for Person. OMB Control...: Application for displacement/relocation assistance for persons (families, individuals, businesses, nonprofit...

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

PubMed

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

PubMed Central

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

The importance of regulatory ubiquitination in cancer and metastasis

PubMed Central

Gallo, L. H.; Ko, J.; Donoghue, D. J.

2017-01-01

ABSTRACT Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis. PMID:28166483

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen

Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or asmore » enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.« less

Ubiquitin-mediated modulation of the cytoplasmic viral RNA sensor RIG-I.

PubMed

Oshiumi, Hiroyuki; Matsumoto, Misako; Seya, Tsukasa

2012-01-01

RIG-I-like receptors, including RIG-I, MDA5 and LGP2, recognize cytoplasmic viral RNA. The RIG-I protein consists of N-terminal CARDs, central RNA helicase and C-terminal domains. RIG-I activation is regulated by ubiquitination. Three ubiquitin ligases target the RIG-I protein. TRIM25 and Riplet ubiquitin ligases are positive regulators of RIG-I and deliver the K63-linked polyubiquitin moiety to RIG-I CARDs and the C-terminal domain. RNF125, another ubiquitin ligase, is a negative regulator of RIG-I and mediates K48-linked polyubiquitination of RIG-I, leading to the degradation of the RIG-I protein by proteasomes. The K63-linked polyubiquitin chains of RIG-I are removed by a deubiquitin enzyme, CYLD. Thus, CYLD is a negative regulator of RIG-I. Furthermore, TRIM25 itself is regulated by ubiquitination. HOIP and HOIL proteins are ubiquitin ligases and are also known as linear ubiquitin assembly complexes (LUBACs). The TRIM25 protein is ubiquitinated by LUBAC and then degraded by proteasomes. The splice variant of RIG-I encodes a protein that lacks the first CARD of RIG-I, and the variant RIG-I protein is not ubiquitinated by TRIM25. Therefore, ubiquitin is the key regulator of the cytoplasmic viral RNA sensor RIG-I.

[Assessment of high sensitivity C-reactive protein (HS-CRP) as a marker of liver inflammation in patients with metabolic syndrome].

PubMed

Rodríguez-Leal, Gustavo Arturo; Morán, Segundo; Gallardo, Irazu; Milke, Pilar; Guevara-González, Luis

2006-01-01

C-reactive protein (CRP) plays an important role on inflammatory processes associated to the metabolic syndrome (MS), alike of insulin sensitivity, endothelial dysfunction and fibrinolysis insufficiency. Alanine aminotransferase (ALT) may be a sensible marker for the diagnosis of hepatic damage and has therefore been used as an alternative method for the noninvasive diagnosis of non-alcoholic fatty liver disease (NAFLD), especially in epidemiological studies. At the present time, the possible utility of high sensitivity CRP (hsCRP) as a simple measure to detect the degree of hepatic inflammatory response during the development NAFLD in MS has not been explored. To evaluate the measurement of serologic hsCRP for the identification of hepatic inflammatory response in patients with MS. Seven hundred and forty persons (526 men and 214 women), mean age 45 +/- 11 years who were asymptomatic and otherwise seeming healthy in whom a medical questionnaire was applied underwent physical examination, laboratory testing, hepatic ultrasound and measurement of hsCRP by the immuno-turbidimetric method. Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of all possible hsCRP for detecting different degrees of hepatic inflammation (ALT > 44 U/L and ALT > 88 U/L). Patients were stratified according to the presence of metabolic syndrome (MS) and ALT concentration in three groups: Group I, having MS and ALT > 44 U/L (n = 39); Group II, having ALT > 44 U/L without MS (n = 105) and Group III, having ALT < or = 44 U/L without MS (n = 596). The optimal hsCRP cut-off for detecting patients with ALT 44 U/L was 2.5 mg/L (sensibility 66%; specificity 50%) and for detecting patients with ALT > 88 U/L was 2.35 (sensibility 72%; specificity 59%). hsCRP serum concentrations in Group I were significantly higher than in Group II and Group III (p < 0.05) but no difference was found between Group II and Group III (Group I = 6.0 +/- 6.7 mg/L vs. Group II = 2.8 +/- 3.1 mg/L, vs. Group III = 2.9 +/- 4.1 mg/L). ALT concentrations were also significantly higher in Group I than in Group II and Group III, (p < 0.05) and a difference between Group II and Group III (p < 0.05) was also found (Group I = 72 +/- 31 U/L vs. Group II = 64 +/- 29 U/L vs. Group III = 24 +/- 8 U/L). These results suggest that the measurement of hsCRP for the identification of hepatic inflammatory response in patients with MS with NAFLD is limited because of its low sensibility and specificity observed on identifying different degrees of hepatic inflammation.

Vapb/Amyotrophic lateral sclerosis 8 knock-in mice display slowly progressive motor behavior defects accompanying ER stress and autophagic response.

PubMed

Larroquette, Frédérique; Seto, Lesley; Gaub, Perrine L; Kamal, Brishna; Wallis, Deeann; Larivière, Roxanne; Vallée, Joanne; Robitaille, Richard; Tsuda, Hiroshi

2015-11-15

Missense mutations (P56S) in Vapb are associated with autosomal dominant motor neuron diseases: amyotrophic lateral sclerosis and lower motor neuron disease. Although transgenic mice overexpressing the mutant vesicle-associated membrane protein-associated protein B (VAPB) protein with neuron-specific promoters have provided some insight into the toxic properties of the mutant proteins, their role in pathogenesis remains unclear. To identify pathological defects in animals expressing the P56S mutant VAPB protein at physiological levels in the appropriate tissues, we have generated Vapb knock-in mice replacing wild-type Vapb gene with P56S mutant Vapb gene and analyzed the resulting pathological phenotypes. Heterozygous P56S Vapb knock-in mice show mild age-dependent defects in motor behaviors as characteristic features of the disease. The homozygous P56S Vapb knock-in mice show more severe defects compared with heterozygous mice reflecting the dominant and dose-dependent effects of P56S mutation. Significantly, the knock-in mice demonstrate accumulation of P56S VAPB protein and ubiquitinated proteins in cytoplasmic inclusions, selectively in motor neurons. The mutant mice demonstrate induction of ER stress and autophagic response in motor neurons before obvious onset of behavioral defects, suggesting that these cellular biological defects might contribute to the initiation of the disease. The P56S Vapb knock-in mice could be a valuable tool to gain a better understanding of the mechanisms by which the disease arises. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mesolimbic and Nigrostriatal Dopaminergic Systems: Behavioral Neuropharmacology.

DTIC Science & Technology

1985-08-01

presented in Table Table III List of drugs D ru gVeh i c l e Intracerebral infusions Dopamine agonist~s Apomorphine hydrochloride 0.1% Na metabisulfite...saline GABA 0.9% saline Picrotoxin 0 .9%saline Systemic injections Dopamine agents d-Amphetamine sulfate 0.9% saline Aponiorphine hydrochloride 0.9...3H)methionine (15 Ci/mmole, lmCi/ml. 16 Amersham), 122 ul of freshly prepared pargyline hydrochloride (10.2 mM), 326 ul of I M Tris pH 10.8, 246 ul

Novel Interventions for Heat/Exercise Induced Sudden Death and Fatigue

DTIC Science & Technology

2014-12-01

T., Ji, R., Hanna,A., Joshi, A. Long, C., Oakes, J., Tran,T., Corona ,B., Lorca,S., Ingalls, C., Narkar, V., Lanner,J.,Bayle,J., Durham, W. and...hydration and discharged home. His CK levels remained increased (>2000 U/L) for 2 months before gradually decreasing to the 1000 U/L range. After...followed, and the patient was discharged home still in pain with a CK of 3800 U/L after a total of 5 hospital days wherein he received only IV hydration

Ubiquitination in Periodontal Disease: A Review

PubMed Central

Tsuchida, Sachio; Satoh, Mamoru; Takiwaki, Masaki; Nomura, Fumio

2017-01-01

Periodontal disease (periodontitis) is a chronic inflammatory condition initiated by microbial infection that leads to gingival tissue destruction and alveolar bone resorption. The periodontal tissue’s response to dental plaque is characterized by the accumulation of polymorphonuclear leukocytes, macrophages, and lymphocytes, all of which release inflammatory mediators and cytokines to orchestrate the immunopathogenesis of periodontal disease. Ubiquitination is achieved by a mechanism that involves a number of factors, including an ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin–protein ligase. Ubiquitination is a post-translational modification restricted to eukaryotes that are involved in essential host processes. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Increasing numbers of recent reports have provided evidence that many approaches are delivering promising reports for discovering the relationship between ubiquitination and periodontal disease. The scope of this review was to investigate recent progress in the discovery of ubiquitinated protein in diseased periodontium and to discuss the ubiquitination process in periodontal diseases. PMID:28698506

Ubiquitinated Sirtuin 1 (SIRT1) Function Is Modulated during DNA Damage-induced Cell Death and Survival*

PubMed Central

Peng, Lirong; Yuan, Zhigang; Li, Yixuan; Ling, Hongbo; Izumi, Victoria; Fang, Bin; Fukasawa, Kenji; Koomen, John; Chen, Jiandong; Seto, Edward

2015-01-01

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR. PMID:25670865

Phospho-ubiquitin: upending the PINK–Parkin–ubiquitin cascade

PubMed Central

Matsuda, Noriyuki

2016-01-01

Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson’s disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1–Parkin–ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling. PMID:26839319

Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?

PubMed

Middleton, Adam J; Wright, Joshua D; Day, Catherine L

2017-11-10

Attachment of ubiquitin to proteins relies on a sophisticated enzyme cascade that is tightly regulated. The machinery of ubiquitylation responds to a range of signals, which remarkably includes ubiquitin itself. Thus, ubiquitin is not only the central player in the ubiquitylation cascade but also a key regulator. The ubiquitin E3 ligases provide specificity to the cascade and often bind the substrate, while the ubiquitin-conjugating enzymes (E2s) have a pivotal role in determining chain linkage and length. Interaction of ubiquitin with the E2 is important for activity, but the weak nature of these contacts has made them hard to identify and study. By reviewing available crystal structures, we identify putative ubiquitin binding sites on E2s, which may enhance E2 processivity and the assembly of chains of a defined linkage. The implications of these new sites are discussed in the context of known E2-ubiquitin interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Functions of perch relocations in a communal night roost of wintering bald eagles

USGS Publications Warehouse

Yackel Adams, A.A.; Skagen, S.K.; Knight, R.L.

2000-01-01

We investigated the functions of perch relocations within a communal night roost of wintering bald eagles (Haliaeetus leucocephalus) along the Nooksack River, Washington, during two winters. We tested seven predictions of two nonexclusive hypotheses: (1) bald eagles relocate within roosts to assess foraging success of conspecifics and (2) bald eagles relocate to obtain thermoregulatory benefits from an improved microclimate. Additionally, we gathered descriptive information to allow refinement of further alternative hypotheses. We rejected the hypothesis that relocations are a means of assessing foraging success. Contrary to our expectations, immature eagles did not relocate to be closer to adults, and relocations were less frequent when food was less abundant. Our data support the hypothesis that eagles relocate within night roosts to obtain a favorable microclimate during winters when they are subjected to cold stress and food stress. In both winters, relocations were more frequent in the evening than in the morning. In both winters, most evening relocations were to the center of the roost rather than to its edge, and the frequency of relocation to the center was greater when temperatures were low. The microclimate hypothesis, however, explains only a limited number of relocations. Based on our findings, it is likely that relocation has multiple functions, including establishing and (or) maintaining foraging associations, establishing and (or) maintaining social-dominance hierarchies when food is less abundant, and nonsocial activities.

Decoding the Ubiquitin-Mediated Pathway of Arthropod Disease Vectors

PubMed Central

Choy, Anthony; Severo, Maiara S.; Sun, Ruobai; Girke, Thomas; Gillespie, Joseph J.; Pedra, Joao H. F.

2013-01-01

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector. PMID:24205097

46 CFR 25.01-3 - Incorporation by reference.

Code of Federal Regulations, 2013 CFR

2013-10-01

..., Devices Providing Backfire Flame Control for Gasoline Engines in Marine Applications, June 1989 25.35-1 Underwriter's Laboratories (UL) 12 Laboratory Drive, Research Triangle Park, NC 27709 UL 1111, Marine...

46 CFR 25.01-3 - Incorporation by reference.

Code of Federal Regulations, 2014 CFR

2014-10-01

..., Devices Providing Backfire Flame Control for Gasoline Engines in Marine Applications, June 1989 25.35-1 Underwriter's Laboratories (UL) 12 Laboratory Drive, Research Triangle Park, NC 27709 UL 1111, Marine...

Defining the optimal cut-off values for liver enzymes in diagnosing blunt liver injury.

PubMed

Koyama, Tomohide; Hamada, Hirohisa; Nishida, Masamichi; Naess, Paal A; Gaarder, Christine; Sakamoto, Tetsuya

2016-01-25

Patients with blunt trauma to the liver have elevated levels of liver enzymes within a short time post injury, potentially useful in screening patients for computed tomography (CT). This study was performed to define the optimal cut-off values for serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in patients with blunt liver injury diagnosed with contrast enhanced multi detector-row CT (CE-MDCT). All patients admitted from May 2006 to July 2013 to Teikyo University Hospital Trauma and Critical Care Center, and who underwent abdominal CE-MDCT within 3 h after blunt trauma, were retrospectively enrolled. Using receiver operating characteristic (ROC) curve analysis, the optimal cut-off values for AST and ALT were defined, and sensitivity and specificity were calculated. Of a total of 676 blunt trauma patients 64 patients were diagnosed with liver injury (Group LI+) and 612 patients without liver injury (Group LI-). Group LI+ and LI- were comparable for age, Revised Trauma Score, and Probability of survival. The groups differed in Injury Severity Score [median 21 (interquartile range 9-33) vs. 17 (9-26) (p < 0.01)]. Group LI+ had higher AST than LI- [276 (48-503) vs. 44 (16-73); p < 0.001] and higher ALT [240 (92-388) vs. 32 (16-49); p < 0.001]. Using ROC curve analysis, the optimal cut-off values for AST and ALT were set at 109 U/l and 97 U/l, respectively. Based on these values, AST ≥ 109 U/l had a sensitivity of 81%, a specificity of 82%, a positive predictive value of 32%, and a negative predictive value of 98%. The corresponding values for ALT ≥ 97 U/l were 78, 88, 41 and 98%, respectively, and for the combination of AST ≥ 109 U/l and/or ALT ≥ 97 U/l were 84, 81, 32, 98%, respectively. We have identified AST ≥ 109 U/l and ALT ≥ 97 U/l as optimal cut-off values in predicting the presence of liver injury, potentially useful as a screening tool for CT scan in patients otherwise eligible for observation only or as a transfer criterion to a facility with CT scan capability.

Upper Limb Isokinetic Strengthening Versus Passive Mobilization in Patients With Chronic Stroke: A Randomized Controlled Trial.

PubMed

Coroian, Flavia; Jourdan, Claire; Bakhti, Karima; Palayer, Claire; Jaussent, Audrey; Picot, Marie-Christine; Mottet, Denis; Julia, Marc; Bonnin, Huey-Yune; Laffont, Isabelle

2018-02-01

To assess the benefit of isokinetic strengthening of the upper limb (UL) in patients with chronic stroke as compared to passive mobilization. Randomized blinded assessor controlled trial. Physical Medicine and Rehabilitation departments of 2 university hospitals. Patients (N=20) with incomplete hemiplegia (16 men; mean age, 64y; median time since stroke, 32mo). A 6-week comprehensive rehabilitation program, 3d/wk, 3 sessions/d. In addition, a 45-minute session per day was performed using an isokinetic dynamometer, with either isokinetic strengthening of elbow and wrist flexors/extensors (isokinetic strengthening group) or passive joint mobilization (control group). The primary endpoint was the increase in Upper Limb Fugl-Meyer Assessment (UL-FMA) score at day 45 (t1). Secondary endpoints were increases in UL-FMA scores, Box and Block Test scores, muscle strength, spasticity, and Barthel Index at t1, t2 (3mo), and t3 (6mo). Recruitment was stopped early because of excessive fatigue in the isokinetic strengthening group. The increase in UL-FMA score at t1 was 3.5±4.4 in the isokinetic strengthening group versus 6.0±4.5 in the control group (P=.2). Gains in distal UL-FMA scores were larger (3.1±2.8) in the control group versus 0.6±2.5 in the isokinetic strengthening group (P=.05). No significant group difference was observed in secondary endpoints. Mixed models confirmed those results. Regarding the whole sample, gains from baseline were significant for the UL-FMA at t1 (+4.8; P<.001), t2, and t3 and for the Box and Block Test at t1 (+3; P=.013) and t2. In a comprehensive rehabilitation program, isokinetic strengthening did not show superiority to passive mobilization for UL rehabilitation. Findings also suggest a sustained benefit in impairments and function of late UL rehabilitation programs for patients with stroke. Copyright © 2017 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin

PubMed Central

Nowicka, Urszula; Zhang, Daoning; Walker, Olivier; Krutauz, Daria; Castañeda, Carlos A.; Chaturvedi, Apurva; Chen, Tony Y.; Reis, Noa; Glickman, Michael H.; Fushman, David

2015-01-01

SUMMARY Ddi1 belongs to a family of shuttle proteins targeting polyubiquitinated substrates for proteasomal degradation. Unlike the other proteasomal shuttles, Rad23 and Dsk2, Ddi1 remains an enigma: its function is not fully understood and structural properties are poorly characterized. We determined the structure and binding properties of the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of Ddi1 from Saccharomyces cerevisiae. We found that, while Ddi1UBA forms a characteristic UBA:ubiquitin complex, Ddi1UBL has entirely uncharacteristic binding preferences. Despite having a ubiquitin-like fold, Ddi1UBL does not interact with typical UBL-receptors but, unexpectedly, binds ubiquitin, forming a unique interface mediated by hydrophobic contacts and by salt-bridges between oppositely-charged residues of Ddi1UBL and ubiquitin. In stark contrast with ubiquitin and other UBLs, the β-sheet surface of Ddi1UBL is negatively charged and, therefore, is recognized in a completely different way. The dual functionality of Ddi1UBL, capable of binding both ubiquitin and proteasome, suggests a novel mechanism for Ddi1 as a proteasomal shuttle. PMID:25703377

Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications

PubMed Central

Ortega Roldan, Jose L.; Casares, Salvador; Ringkjøbing Jensen, Malene; Cárdenes, Nayra; Bravo, Jerónimo; Blackledge, Martin; Azuaga, Ana I.; van Nuland, Nico A. J.

2013-01-01

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. PMID:24039852

RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage.

PubMed

Paul, Atanu; Wang, Bin

2017-05-18

Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response. Copyright © 2017 Elsevier Inc. All rights reserved.

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

PubMed Central

Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

2016-01-01

Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and complexed with monoubiquitin or K48-linked diubiquitin were solved, revealing that three neighboring outer helices from the T1 toroid engage two ubiquitins. This binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between deubiquitination and substrate degradation. We find that proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition, but rather in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Importantly, the UBL interactors at T1 and T2 are distinct, assigning substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, T1 and T2. The Rpn1 toroid represents a novel class of binding domains for ubiquitin and UBL proteins. This study thus defines a novel two-site recognition domain intrinsic to the proteasome that uses homologous ubiquitin/UBL-class ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity. A ligand-binding hotspot in the proteasome for assembling substrates and cofactors Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome to illustrate the Rpn1 T1 and T2 sites bound to a ubiquitin chain (yellow) and deubiquitinating enzyme Ubp6 (green), respectively. PDB 4CR2 and 2B9R were used for this figure. Hundreds of pathways for degradation converge at ubiquitin recognition by proteasome. Here we found that the five known proteasomal ubiquitin receptors are collectively nonessential for ubiquitin recognition, and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or K48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for K48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus a two-site recognition domain intrinsic to the proteasome uses homologous ubiquitin/UBL-class ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme. PMID:26912900

Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

NASA Technical Reports Server (NTRS)

Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.

1998-01-01

The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

46 CFR 160.077-5 - Incorporation by reference.

Code of Federal Regulations, 2010 CFR

2010-10-01

... (NBS) “The Universal Color Language” and “The Color Names Dictionary” in Color: Universal Language and Dictionary of Names, National Bureau of Standards Special Publication 440. Underwriters Laboratories (UL) UL...

Dynamic survey of mitochondria by ubiquitin

PubMed Central

Escobar-Henriques, Mafalda; Langer, Thomas

2014-01-01

Ubiquitin is a post-translational modifier with proteolytic and non-proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria-ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin-dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin-dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria. PMID:24569520

76 FR 39117 - Notice of Proposed Information Collection: Relocation and Real Property Acquisition...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-05

... Information Collection: Relocation and Real Property Acquisition, Recordkeeping Requirements Under the Uniform Relocation Assistance and Real Property Acquisition Policies Act of 1970, as Amended (URA) Comment Request..., DC 20410. FOR FURTHER INFORMATION CONTACT: Bryan O'Neill, Relocation Specialist, Relocation and Real...

Determinants of Swe1p Degradation in Saccharomyces cerevisiae

PubMed Central

McMillan, John N.; Theesfeld, Chandra L.; Harrison, Jacob C.; Bardes, Elaine S. G.; Lew, Daniel J.

2002-01-01

Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCFMet30 acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCFMet30) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist. PMID:12388757

Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth*

PubMed Central

MacDiarmid, Colin W.; Taggart, Janet; Jeong, Jeeyon; Kerdsomboon, Kittikhun; Eide, David J.

2016-01-01

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2E301K) suppressed the ubi4Δ growth defect. The rpt2E301K mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2E301K mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells. PMID:27432887

Crystal Structure of the Ubiquitin-associated (UBA) Domain of p62 and Its Interaction with Ubiquitin*

PubMed Central

Isogai, Shin; Morimoto, Daichi; Arita, Kyohei; Unzai, Satoru; Tenno, Takeshi; Hasegawa, Jun; Sou, Yu-shin; Komatsu, Masaaki; Tanaka, Keiji; Shirakawa, Masahiro; Tochio, Hidehito

2011-01-01

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62. PMID:21715324

The validity of proxy-based data on loneliness in suicide research: a case-control psychological autopsy study in rural China.

PubMed

Niu, Lu; Jia, Cunxian; Ma, Zhenyu; Wang, Guojun; Yu, Zhenjun; Zhou, Liang

2018-05-01

There is a lack of evidence for the role of loneliness on suicide using psychological autopsy method, and the validity of proxy informants' reports on loneliness is not well established. This study aimed to investigate the validity of proxy respondent reports on loneliness, and the reliability and validity of the University of California Los Angeles Loneliness Scale-6 (ULS-6) as used in psychological autopsy method with rural elderly people in China. Two hundred forty-two suicide cases and 242 normal community controls were selected, and the psychological autopsy method was utilized to collect information. Data from proxy respondents of the living controls were compared with data reported by the targets (gold standards). Subject-proxy concordance for ULS-6 was fair (ICC = 0.447) in the living controls. The suicide cases were more likely to have a higher score of ULS-6 than the living controls. Additionally, our data supported that ULS-6 had adequate psychometric properties in both suicide and control groups: factor analyses yielded one-factor component solution; Cronbach's alpha (both > 0.90) demonstrated excellent internal consistency; the Spearman correlation analysis indicated that the ULS-6 score was positively correlated with depression; and negatively correlated with QOL and social support. Results support proxy-based data on loneliness in research of suicide in older adults in rural China, and the ULS-6 is a psychometrically sound instrument for measuring loneliness in psychological autopsy studies.

Randomized comparison of ultra-brief bifrontal and unilateral electroconvulsive therapy for major depression: cognitive side-effects.

PubMed

Sienaert, P; Vansteelandt, K; Demyttenaere, K; Peuskens, J

2010-04-01

The cognitive side-effects of bifrontal (BF) and right unilateral (UL) ultra-brief pulse (0.3 ms) electroconvulsive therapy (ECT) were compared, in the treatment of patients with a depressive episode. Neuropsychological functioning in patients with a medication refractory depressive episode, that were treated with a course of BF ultra-brief ECT at 1.5 times seizure threshold (ST) or UL ultra-brief ECT at 6 times ST, by random assignment, was assessed before treatment, and 1 and 6 weeks after the treatment course, by a blinded rater. Of the 64 patients that were included, 32 (50%) received BF ECT, and 32 (50%) received UL ECT, by random assignment. Neuropsychological testing 1 and 6 weeks after treatment was performed by 30 (93.75%) and 19 (59.37%) patients, respectively, in the BF-group and 29 (90.62%) and 20 (62.50%), respectively, in the UL-group. There was no deterioration in any of the neuropsychological measures. Patients rated their memory as clearly improved after treatment. There were no significant differences between the patients given BF ECT and those given UL ECT. Ultrabrief pulse ECT, used either in combination with a UL electrode position and a stimulus of 6 times ST, or a BF electrode position with a stimulus of 1.5 times ST, are effective antidepressant techniques, that do not have a deleterious effect on cognitive function. Copyright 2009 Elsevier B.V. All rights reserved.

Simulating Various Terrestrial and Uav LIDAR Scanning Configurations for Understory Forest Structure Modelling

NASA Astrophysics Data System (ADS)

Hämmerle, M.; Lukač, N.; Chen, K.-C.; Koma, Zs.; Wang, C.-K.; Anders, K.; Höfle, B.

2017-09-01

Information about the 3D structure of understory vegetation is of high relevance in forestry research and management (e.g., for complete biomass estimations). However, it has been hardly investigated systematically with state-of-the-art methods such as static terrestrial laser scanning (TLS) or laser scanning from unmanned aerial vehicle platforms (ULS). A prominent challenge for scanning forests is posed by occlusion, calling for proper TLS scan position or ULS flight line configurations in order to achieve an accurate representation of understory vegetation. The aim of our study is to examine the effect of TLS or ULS scanning strategies on (1) the height of individual understory trees and (2) understory canopy height raster models. We simulate full-waveform TLS and ULS point clouds of a virtual forest plot captured from various combinations of max. 12 TLS scan positions or 3 ULS flight lines. The accuracy of the respective datasets is evaluated with reference values given by the virtually scanned 3D triangle mesh tree models. TLS tree height underestimations range up to 1.84 m (15.30 % of tree height) for single TLS scan positions, but combining three scan positions reduces the underestimation to maximum 0.31 m (2.41 %). Combining ULS flight lines also results in improved tree height representation, with a maximum underestimation of 0.24 m (2.15 %). The presented simulation approach offers a complementary source of information for efficient planning of field campaigns aiming at understory vegetation modelling.

Ubiquitin Ligases: Structure, Function, and Regulation.

PubMed

Zheng, Ning; Shabek, Nitzan

2017-06-20

Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover

PubMed Central

Swaney, Danielle L; Rodríguez-Mias, Ricard A; Villén, Judit

2015-01-01

Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin can itself be phosphorylated at nearly every serine, threonine, and tyrosine residue. However, the effect of this modification on ubiquitin function is largely unknown. Here, we characterized the effects of phosphorylation of yeast ubiquitin at serine 65 in vivo and in vitro. We find this post-translational modification to be regulated under oxidative stress, occurring concomitantly with the restructuring of the ubiquitin landscape into a highly polymeric state. Phosphomimetic mutation of S65 recapitulates the oxidative stress phenotype, causing a dramatic accumulation of ubiquitylated proteins and a proteome-wide reduction of protein turnover rates. Importantly, this mutation impacts ubiquitin chain disassembly, chain linkage distribution, ubiquitin interactions, and substrate targeting. These results demonstrate that phosphorylation is an additional mode of ubiquitin regulation with broad implications in cellular physiology. PMID:26142280

MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

PubMed

Nezich, Catherine L; Wang, Chunxin; Fogel, Adam I; Youle, Richard J

2015-08-03

The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

41 CFR 302-2.6 - May I be reimbursed for relocation expenses if I relocate to a new official station that does not...

Code of Federal Regulations, 2014 CFR

2014-07-01

... relocation expenses if I relocate to a new official station that does not meet the 50-mile distance test? 302... reimbursed for relocation expenses if I relocate to a new official station that does not meet the 50-mile... official station that does not meet the 50-mile distance test. (a) The distance test is met when the new...

41 CFR 302-2.6 - May I be reimbursed for relocation expenses if I relocate to a new official station that does not...

Code of Federal Regulations, 2012 CFR

2012-07-01

... relocation expenses if I relocate to a new official station that does not meet the 50-mile distance test? 302... reimbursed for relocation expenses if I relocate to a new official station that does not meet the 50-mile... official station that does not meet the 50-mile distance test. (a) The distance test is met when the new...

41 CFR 302-2.6 - May I be reimbursed for relocation expenses if I relocate to a new official station that is less...

Code of Federal Regulations, 2011 CFR

2011-07-01

... relocation expenses if I relocate to a new official station that is less than 50 miles from my old official... reimbursed for relocation expenses if I relocate to a new official station that is less than 50 miles from my... to a new official station that is less than 50 miles from your old official station, unless the head...

41 CFR 302-2.6 - May I be reimbursed for relocation expenses if I relocate to a new official station that does not...

Code of Federal Regulations, 2013 CFR

2013-07-01

... relocation expenses if I relocate to a new official station that does not meet the 50-mile distance test? 302... reimbursed for relocation expenses if I relocate to a new official station that does not meet the 50-mile... official station that does not meet the 50-mile distance test. (a) The distance test is met when the new...

Evaluation of freshwater mussel relocation as a conservation and management strategy

USGS Publications Warehouse

Cope, W. Gregory; Waller, Diane L.

1995-01-01

The relocation of unionacean mussels is commonly used as a conservation and management tool in large rivers and streams. Relocation has been used to recolonize areas where mussel populations have been eliminated by prior pollution events, to remove mussels from construction zones and to re-establish populations of endangered species. More recently, relocation has been used to protect native freshwater mussels from colonization by the exotic zebra mussel Dreissena polymorpha. We conducted a literature review of mussel relocations and evaluated their relative success as a conservation and management strategy. We found that 43% of all relocations were conducted because of construction projects that were forced to comply with the Endangered Species Act 1973 and that only 16% were monitored for five or more consecutive years. Most (43%) relocation projects were conducted from July to September, presumably a period when reproductive stress is relatively low for most species and the metabolic rate is sufficient for reburrowing in the substrate. The mortality of relocated mussels was unreported in 27% of projects; reported mortality varied widely among projects and species and was difficult to assess. The mean mortality of relocated mussels was 49% based on an average recovery rate of 43%. There is little guidance on the methods for relocation or for monitoring the subsequent long-term status of relocated mussels. Based on this evaluation, research is needed to develop criteria for selecting a suitable relocation site and to establish appropriate methods and guidelines for conducting relocation projects.

Global analysis of ubiquitome in PRRSV-infected pulmonary alveolar macrophages.

PubMed

Zhang, Huan; Fang, Liurong; Zhu, Xinyu; Wang, Dang; Xiao, Shaobo

2018-06-18

Protein lysine ubiquitination is a dynamic reversible post-translational modification that plays key roles in modulating different cellular processes. Porcine reproductive and respiratory syndrome virus (PRRSV) is a notorious pathogen, causing tremendous economic losses for the global swine industry. The possible involvement of ubiquitination in PRRSV infection is unclear. So anti-ubiquitination-based enrichment and LC-MS were performed to investigate the global ubiquitination events triggered by PRRSV infection in pulmonary alveolar macrophages. We totally identified 4044 lysine ubiquitination sites on 1580 cellular proteins, of which 983 sites on 717 proteins were significantly altered at 36 h postinfection. A systematic, intensive bioinformatic analysis of the ubiquitome data suggested that PRRSV suppresses the host immune responses by manipulating the ubiquitination of important adaptors and effectors, including TRAF6, JAK1, STAT1, and ISGs. Ubiquitination was also observed on 15 PRRSV proteins, including important virus proteases and structural proteins that function in virus infectivity and neutralizing antibody elicitation. The efficient replication of PRRSV requires an intact ubiquitin-proteasome system. Our study is the first to analyze the global ubiquitination events in pulmonary alveolar macrophages during PRRSV infection. It provides insight into the molecular mechanisms of PRRSV pathogenesis, promoting the development of antiviral drugs. PRRSV is a notorious pathogen which has been resulting in huge economic losses in the swine industry since the first outbreak. Therefore, more in-depth knowledge of the PRRSV immunoregulatory mechanisms and valid control methods to combat the virus are urgently needed. Ubiquitination is an important post-translational modification regulating various cellular processes. However, information about the possible involvement of ubiquitination responses to PRRSV infection is limited. In this study, a quantitative proteomic approach was first used to analyze ubiquitination level alteration in PRRSV-infected PAMs. We demonstrate that PRRSV can suppresses the host immune responses by manipulating the ubiquitination of important effectors that include TRAF6, JAK1, STAT1, and ISGs. Furthermore, 15 PRRSV proteins undergo ubiquitination and efficient replication of PRRSV requires an intact ubiquitin-proteasome system. Our study will significantly expand our knowledge about the molecular mechanisms of PRRSV pathogenesis and provides novel insights into the development of antiviral drugs. Copyright © 2018. Published by Elsevier B.V.

Changes in biochemical markers after lower limb fractures.

PubMed

Stoffel, Karl; Engler, Hanna; Kuster, Markus; Riesen, Walter

2007-01-01

The bone remodeling sequence after bone fracture changes the concentrations of biochemical bone markers, but the relationships of fracture size and of healing time to changes in biomarkers are unclear. The present pilot study was undertaken to determine the changes found in serum bone markers after plate osteosynthesis of closed distal tibial and malleolar fractures during a study period of 24 weeks. We measured tatrate-resistant acid phosphatase (TRACP 5b), collagen type I C-terminal telopeptide (ICTP), bone-specific alkaline phosphatase (bone ALP), osteocalcin (OC), procollagen type I C-terminal propeptide (PICP), procollagen type III N-terminal propeptide (PIIINP), and human cartilage glycoprotein 39 (YKL-40) in 20 patients with lower limb fractures (10 malleolar, 10 tibia). A physical examination and radiographs were completed to assess evidence of union. All malleolar fractures healed within 6 weeks, whereas 2 tibial fractures did not show complete bone healing after 24 weeks. Changes were comparable but more pronounced in the tibia group, and marker concentrations remained increased at the end of study (bone ALP, 86 vs 74 U/L; OC, 14.9 vs 7.7 microg/L; ICTP: 5.6 vs 3.3 microg/L at day 84 after osteosynthesis, P <0.05 in tibia; 80 vs 70 U/L, 8 vs 5.2 microg/L, and 3.5 vs 3.2 microg/L, respectively, in the malleolar fracture group). In normal bone healing, changes in bone turnover markers were primarily dependent on the fracture size. Delayed tibia fracture healing may involve a disturbance in bone remodeling.

The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsuchiya, Hikaru; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

2013-06-28

Highlights: •The parallel reaction monitoring method was applied to ubiquitin quantification. •The ubiquitin PRM method is highly sensitive even in biological samples. •Using the method, we revealed that Ufd4 assembles the K29-linked ubiquitin chain. -- Abstract: Ubiquitylation is an essential posttranslational protein modification that is implicated in a diverse array of cellular functions. Although cells contain eight structurally distinct types of polyubiquitin chains, detailed function of several chain types including K29-linked chains has remained largely unclear. Current mass spectrometry (MS)-based quantification methods are highly inefficient for low abundant atypical chains, such as K29- and M1-linked chains, in complex mixtures thatmore » typically contain highly abundant proteins. In this study, we applied parallel reaction monitoring (PRM), a quantitative, high-resolution MS method, to quantify ubiquitin chains. The ubiquitin PRM method allows us to quantify 100 attomole amounts of all possible ubiquitin chains in cell extracts. Furthermore, we quantified ubiquitylation levels of ubiquitin-proline-β-galactosidase (Ub-P-βgal), a historically known model substrate of the ubiquitin fusion degradation (UFD) pathway. In wild-type cells, Ub-P-βgal is modified with ubiquitin chains consisting of 21% K29- and 78% K48-linked chains. In contrast, K29-linked chains are not detected in UFD4 knockout cells, suggesting that Ufd4 assembles the K29-linked ubiquitin chain(s) on Ub-P-βgal in vivo. Thus, the ubiquitin PRM is a novel, useful, quantitative method for analyzing the highly complicated ubiquitin system.« less

Comparative Analysis of gO Isoforms Reveals that Strains of Human Cytomegalovirus Differ in the Ratio of gH/gL/gO and gH/gL/UL128-131 in the Virion Envelope

PubMed Central

Zhou, Momei; Yu, Qin; Wechsler, Anya

2013-01-01

Herpesvirus glycoprotein complex gH/gL provides a core entry function through interactions with the fusion protein gB and can also influence tropism through receptor interactions. The Epstein-Barr virus gH/gL and gH/gL/gp42 serve both functions for entry into epithelial and B cells, respectively. Human cytomegalovirus (HCMV) gH/gL can be bound by the UL128-131 proteins or gO. The phenotypes of gO and UL128-131 mutants suggest that gO-gH/gL interactions are necessary for the core entry function on all cell types, whereas the binding of UL128-131 to gH/gL likely relates to a distinct receptor-binding function for entry into some specific cell types (e.g., epithelial) but not others (e.g., fibroblasts and neurons). There are at least eight isoforms of gO that differ by 10 to 30% of amino acids, and previous analysis of two HCMV strains suggested that some isoforms of gO function like chaperones, disassociating during assembly to leave unbound gH/gL in the virion envelope, while others remain bound to gH/gL. For the current report, we analyzed the gH/gL complexes present in the virion envelope of several HCMV strains, each of which encodes a distinct gO isoform. Results indicate that all strains of HCMV contain stable gH/gL/gO trimers and gH/gL/UL128-131 pentamers and little, if any, unbound gH/gL. TR, TB40/e, AD169, and PH virions contained vastly more gH/gL/gO than gH/gL/UL128-131, whereas Merlin virions contained mostly gH/gL/UL128-131, despite abundant unbound gO remaining in the infected cells. Suppression of UL128-131 expression during Merlin replication dramatically shifted the ratio toward gH/gL/gO. These data suggest that Merlin gO is less efficient than other gO isoforms at competing with UL128-131 for binding to gH/gL. Thus, gO diversity may influence the pathogenesis of HCMV through effects on the assembly of the core versus tropism gH/gL complexes. PMID:23804643

ICP22 and the UL13 Protein Kinase Are both Required for Herpes Simplex Virus-Induced Modification of the Large Subunit of RNA Polymerase II

PubMed Central

Long, Melissa C.; Leong, Vivian; Schaffer, Priscilla A.; Spencer, Charlotte A.; Rice, Stephen A.

1999-01-01

Herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the large subunit of RNA polymerase II (RNAP II), resulting in the depletion of the hypophosphorylated and hyperphosphorylated forms of this polypeptide (known as IIa and IIo, respectively) and induction of a novel, alternatively phosphorylated form (designated IIi). We previously showed that the HSV-1 immediate-early protein ICP22 is involved in this phenomenon, since induction of IIi and depletion of IIa are deficient in cells infected with 22/n199, an HSV-1 ICP22 nonsense mutant (S. A. Rice, M. C. Long, V. Lam, P. A. Schaffer, and C. A. Spencer, J. Virol. 69:5550–5559, 1995). However, depletion of IIo still occurs in 22/n199-infected cells. This suggests either that another viral gene product affects the RNAP II large subunit or that the truncated ICP22 polypeptide encoded by 22/n199 retains residual activity which leads to IIo depletion. To distinguish between these possibilities, we engineered an HSV-1 ICP22 null mutant, d22-lacZ, and compared it to 22/n199. The two mutants are indistinguishable in their effects on the RNAP II large subunit, suggesting that an additional viral gene product is involved in altering RNAP II. Two candidates are UL13, a protein kinase which has been implicated in ICP22 phosphorylation, and the virion host shutoff (Vhs) factor, the expression of which is positively regulated by ICP22 and UL13. To test whether UL13 is involved, a UL13-deficient viral mutant, d13-lacZ, was engineered. This mutant was defective in IIi induction and IIa depletion, displaying a phenotype very similar to that of d22-lacZ. In contrast, a Vhs mutant had effects that were indistinguishable from wild-type HSV-1. Therefore, UL13 but not the Vhs function plays a role in modifying the RNAP II large subunit. To study the potential role of UL13 in viral transcription, we carried out nuclear run-on transcription analyses in infected human embryonic lung cells. Infections with either UL13 or ICP22 mutants led to significantly reduced amounts of viral genome transcription at late times after infection. Together, our results suggest that ICP22 and UL13 are involved in a common pathway that alters RNAP II phosphorylation and that in some cell lines this change promotes viral late transcription. PMID:10364308

Ubiquitin is part of the retrovirus budding machinery

NASA Astrophysics Data System (ADS)

Patnaik, Akash; Chau, Vincent; Wills, John W.

2000-11-01

Retroviruses contain relatively large amounts of ubiquitin, but the significance of this finding has been unknown. Here, we show that drugs that are known to reduce the level of free ubiquitin in the cell dramatically reduced the release of Rous sarcoma virus, an avian retrovirus. This effect was suppressed by overexpressing ubiquitin and also by directly fusing ubiquitin to the C terminus of Gag, the viral protein that directs budding and particle release. The block to budding was found to be at the plasma membrane, and electron microscopy revealed that the reduced level of ubiquitin results in a failure of mature virus particles to separate from each other and from the plasma membrane during budding. These data indicate that ubiquitin is actually part of the budding machinery.

[Ubiquitin-proteasome system and sperm DNA repair: An update].

PubMed

Zhang, Guo-Wei; Cai, Hong-Cai; Shang, Xue-Jun

2016-09-01

The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.

Identification of primary and secondary UBA footprints on the surface of ubiquitin in cell-mimicking crowded solution.

PubMed

Munari, Francesca; Bortot, Andrea; Zanzoni, Serena; D'Onofrio, Mariapina; Fushman, David; Assfalg, Michael

2017-04-01

Despite significant advancements in our understanding of ubiquitin-mediated signaling, the influence of the intracellular environment on the formation of transient ubiquitin-partner complexes remains poorly explored. In our work, we introduce macromolecular crowding as a first level of complexity toward the imitation of a cellular environment in the study of such interactions. Using NMR spectroscopy, we find that the stereospecific complex of ubiquitin and the ubiquitin-associated domain (UBA) is minimally perturbed by the crowding agent Ficoll. However, in addition to the primary canonical recognition patch on ubiquitin, secondary patches are identified, indicating that in cell-mimicking crowded solution, UBA contacts ubiquitin at multiple sites. © 2017 Federation of European Biochemical Societies.

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

PubMed Central

Kazlauskaite, Agne; Kondapalli, Chandana; Gourlay, Robert; Campbell, David G.; Ritorto, Maria Stella; Hofmann, Kay; Alessi, Dario R.; Knebel, Axel; Trost, Matthias; Muqit, Miratul M. K.

2014-01-01

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease. PMID:24660806

Ubiquitin--conserved protein or selfish gene?

PubMed

Catic, André; Ploegh, Hidde L

2005-11-01

The posttranslational modifier ubiquitin is encoded by a multigene family containing three primary members, which yield the precursor protein polyubiquitin and two ubiquitin moieties, Ub(L40) and Ub(S27), that are fused to the ribosomal proteins L40 and S27, respectively. The gene encoding polyubiquitin is highly conserved and, until now, those encoding Ub(L40) and Ub(S27) have been generally considered to be equally invariant. The evolution of the ribosomal ubiquitin moieties is, however, proving to be more dynamic. It seems that the genes encoding Ub(L40) and Ub(S27) are actively maintained by homologous recombination with the invariant polyubiquitin locus. Failure to recombine leads to deterioration of the sequence of the ribosomal ubiquitin moieties in several phyla, although this deterioration is evidently constrained by the structural requirements of the ubiquitin fold. Only a few amino acids in ubiquitin are vital for its function, and we propose that conservation of all three ubiquitin genes is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as a 'selfish gene'.

Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

PubMed Central

Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

2016-01-01

Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

The ubiquitin-proteasome pathway an emerging anticancer strategy for therapeutics: a patent analysis.

PubMed

Jain, Chakresh K; Arora, Shivam; Khanna, Aparna; Gupta, Money; Wadhwa, Gulshan; Sharma, Sanjeev K

2015-01-01

The degradation of intracellular proteins is targeted by ubiquitin via non-lysosomal proteolytic pathway in the cell system. These ubiquitin molecules have been found to be conserved from yeast to humans. Ubiquitin proteasome machinery utilises ATP and other mechanisms for degrading proteins of cytosol as well as nucleus. This process of ubiquitination is regulated by activating the E3 enzyme ligase, involved in phosphorylation. In humans, proteins which regulate the cell cycle are controlled by ubiquitin; therefore the ubiquitin-proteasome pathway can be targeted for novel anti-cancer strategies. Dysregulation of the components of the ubiquitin system has been linked to many diseases like cancer and inflammation. The primary triggering mechanism (apoptosis) of these diseases can also be induced when TNF-related apoptosis-inducing ligand (TRAIL) binds to its specific receptor DR4 and DR5. In this review, the emerging prospects and importance of ubiquitin proteasome pathway as an evolving anticancer strategy have been discussed. Current challenges in the field of drug discovery have also been discussed on the basis of recent patents on cancer diagnosis and therapeutics.

Phospho-ubiquitin: upending the PINK-Parkin-ubiquitin cascade.

PubMed

Matsuda, Noriyuki

2016-04-01

Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson's disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1-Parkin-ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)*

PubMed Central

Hendriks, Ivo A.; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V.; Vertegaal, Alfred C. O.

2015-01-01

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

Evaluation of recombinant adenovirus vaccines based on glycoprotein D and truncated UL25 against herpes simplex virus type 2 in mice.

PubMed

Liu, Wei; Zhou, Yan; Wang, Ziyan; Zhang, Zeqiang; Wang, Qizhi; Su, Weiheng; Chen, Yan; Zhang, Yan; Gao, Feng; Jiang, Chunlai; Kong, Wei

2017-05-01

The high prevalence of herpes simplex virus 2 (HSV-2) infections in humans necessitates the development of a safe and effective vaccine that will need to induce vigorous T-cell responses to control viral infection and transmission. We designed rAd-gD2, rAd-gD2ΔUL25, and rAd-ΔUL25 to investigate whether recombinant replication-defective adenoviruses vaccine could induce specific T-cell responses and protect mice against intravaginal HSV-2 challenge compared with FI-HSV-2. In the present study, recombinant adenovirus-based HSV-2 showed higher reductions in mortality and stronger antigen-specific T-cell responses compared with FI-HSV-2 and the severity of genital lesions in mice immunized with rAd-gD2ΔUL25 was significantly decreased by eliciting IFN-γ-secreting T-cell responses compared with rAd-gD2 and rAd-ΔUL25 groups. Our results demonstrated the immunogenicity and protective efficacy of recombinant adenovirus vaccines in acute HSV-2 infection following intravaginal challenge in mice. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

46 CFR 169.115 - Incorporation by reference.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Exhaust Systems for Propulsion and Auxiliary Engines” (1973) H-24.9 (g) and (h)—“Fuel Strainers and Fuel...) Underwriters Laboratories, Inc. (UL), 12 Laboratory Drive, Research Triangle Park, NC 27709-3995 UL 19-78...

46 CFR 120.410 - Lighting fixtures.

Code of Federal Regulations, 2010 CFR

2010-10-01

...) Each table lamp, desk lamp, floor lamp, or similar equipment must be secured in place so that it cannot... Fixtures,” UL 1571, “Incandescent Lighting Fixtures,” UL 1572, “High Intensity Discharge Lighting Fixtures...

Cure width potential for MOD resin composite molar restorations.

PubMed

Palin, William M; Senyilmaz, Dilek P; Marquis, Peter M; Shortall, Adrian C

2008-08-01

To investigate the capability of modern light-curing units exhibiting differences in emission spectra and light source exit window dimensions, for "one-shot" full-width curing of extensive (molar MOD) resin composite restorations. Specimens of Tetric (TT), Tetric Ceram HB (TC), Tetric Evoceram (TE) and Tetric Ceram Bleach (TB) resin composites containing varying ratios of Lucirin (TPO) and/or camphorquinone (CQ) photoinitiators were packed into a bar-shaped mould (12 mm length x 2 mm width x 2 mm thickness). Each product was irradiated using a halogen (Optilux 401; QTH), a conventional LED (LEDemetron; LED) and two so-called "third generation" oval-footprint LED light-curing units (LCUs) of the same model. The latter featured bimodal emission spectra (blue and ultraviolet diodes) with either high (unmodified output) and approximately 50% (modified output) blue light intensity (UltraLume-5; ULs, ULm, respectively). Vickers hardness number was obtained across the lateral extent of the bar at 1mm increments from the centre point on both upper and lower surfaces of the specimens. Significant linear relationships (R(2)=0.71-0.98) for each distance from the central position of all LCUs were identified between measured light intensity and corresponding upper and lower surface hardness values for each product (P<0.05). No significant differences (P>0.05) were recorded in total upper surface hardness of TC or TE cured with LED (68.7+/-3.2 and 70.5+/-2.5) or ULm (56.8+/-2.0 and 57.7+/-2.0). However, upper surface hardness of TT (CQ only) cured with ULm was significantly decreased (P<0.05) compared with other LCUs. When the ratio of hardness at the edge to central positions of the bar-shaped specimens for either surface was calculated, no significant difference (P>0.05) was identified for TB (containing TPO and decreased CQ) cured with either ULs or ULm (P>0.05) and was significantly increased (P<0.05) when cured with ULs compared with LED and QTH. Variability in light intensity across the curing-tip face, spectral output of dental light-curing units and differences in product photoinitiator chemistry all influence curing efficiency significantly across the width of extensive resin composite geometries.

A patient with thyrotropinoma cosecreting growth hormone and follicle-stimulating hormone with low alpha-glycoprotein: a new subentity?

PubMed

Elhadd, Tarik A; Ghosh, Sujoy; Teoh, Wei Leng; Trevethick, Katy Ann; Hanzely, Zoltan; Dunn, Laurence T; Malik, Iqbal A; Collier, Andrew

2009-08-01

Thyrotropinomas are rare pituitary tumors. In 25 percent of cases there is autonomous secretion of a second pituitary hormone, adding to the clinical complexity. We report a patient with thyrotropin (TSH)-dependant hyperthyroidism along with growth hormone (GH) and follicle-stimulating hormone (FSH) hypersecretion but low alpha-glycoprotein (alpha-subunit) concentrations, a hitherto unique constellation of findings. A 67-year-old Scottish lady presented with longstanding ankle edema, paroxysmal atrial fibrillation, uncontrolled hypertension, fine tremors, warm peripheries, and agitation. Initial findings were a small goiter, elevated serum TSH of 7.37 mU/L (normal range, 0.30-6.0 mU/L), a free-thyroxine concentration of 34.9 pmol/L (normal range, 9.0-24.0 pmol/L), a flat TSH response to TSH-releasing hormone, and serum alpha-subunit of 3.1 IU/L (normal, <3.0 IU/L). There was no evidence of an abnormal thyroid hormone beta receptor by genotyping. Serum FSH was 56.8 U/L, but the luteinizing hormone (LH) was 23.6 U/L (postmenopausal FSH and LH reference ranges both >30 U/L) Basal insulin-like growth factor I was elevated to 487 microg/L with the concomitant serum GH being 14.1 mU/L, and subsequent serum GH values 30 minutes after 75 g oral glucose being 19.1 mU/L and 150 minutes later being 13.7 mU/L. An magnetic resonance imaging pituitary revealed a macroadenoma. Pituitary adenomectomy was performed with the histology confirming a pituitary adenoma, and the immunohistochemistry staining showed positive reactivity for FSH with scattered cells staining for GH and TSH. Staining for other anterior pituitary hormones was negative. After pituitary surgery she became clinically and biochemically euthyroid, the serum IFG-1 became normal, but the pattern of serum FSH and LH did not change. This case of plurihormonal thyrotropinoma is unique in having hypersecretion of TSH, GH, and FSH with low alpha-subunit. Such a combination may represent a new subentity of TSHomas.

High clinical manifestation rate in an imported outbreak of hepatitis E genotype 1 infection in a German group of travellers returning from India.

PubMed

Pischke, Sven; Schulze-Zur-Wiesch, Julian; Lütgehetmann, Marc; Kreuels, Benno; Lueth, Stefan; Kapaun, Petra; Benten, Daniel; Schmiedel, Stefan; Sterneck, Martina; Lohse, Ansgar W; Polywka, Susanne

2017-01-01

Background. There are only few reports about travel-associated, imported tropical hepatitis E virus (HEV) genotype 1 infections within Western travellers. We describe the clinical course of a single outbreak of hepatitis E in a German travellers group returning from India and compare the results of two commercial HEV-seroassays. After identifying hepatitis E in an index patient returning from a journey to India all 24 members of this journey were tested for anti-HEV-IgG and IgM using two commercial seroassays (Wantai and Mikrogen), for HEV-RNA by PCR and HEV-Ag by an antigen-assay (Wantai). 5/24 (21%) individuals were viraemic with viral loads between 580-4,800,000 IU/mL. Bilirubin and ALT levels in these patients ranged from 1.3-14.9 mg/dL (mean 7.3 mg/dL, SD 5.6 mg/dL) and 151-4,820 U/L (mean 1,832U/L, SD 1842U/L), respectively and showed significant correlations with viral loads (r = 0.863, p < 0.001; r = 0.890, p < 0.001). No risk factor for food-borne HEV-transmission was identified. All viraemic patients (5/5) tested positive for anti-HEV-IgG and IgM in the Wantai-assay but only 4/5 in the Mikrogen-assay. Wantai-HEV-antigen-assay was negative in all patients. Six months later all previously viraemic patients tested positive for anti-HEV-IgG and negative for IgM in both assays. However, two non-viremic individuals who initially tested Wantai-IgM-positive stayed positive indicating false positive results. Despite the exact number of exposed individuals could not be determined HEV genotype 1 infections have a high manifestation rate of more than 20%.The Wantai-antigen-test failed, the Wantai-IgMrapid- test and the Mikrogen-IgM-recomblot showed a better performance but still they cannot replace real-time PCR for diagnosing ongoing HEV-infections.

41 CFR 302-3.508 - What relocation expenses are not authorized for new appointees or student trainees?

Code of Federal Regulations, 2012 CFR

2012-07-01

... 41 Public Contracts and Property Management 4 2012-07-01 2012-07-01 false What relocation expenses... Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities New Appointees § 302-3.508 What...

41 CFR 302-3.508 - What relocation expenses are not authorized for new appointees or student trainees?

Code of Federal Regulations, 2014 CFR

2014-07-01

... 41 Public Contracts and Property Management 4 2014-07-01 2014-07-01 false What relocation expenses... Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities New Appointees § 302-3.508 What...

41 CFR 302-3.508 - What relocation expenses are not authorized for new appointees or student trainees?

Code of Federal Regulations, 2011 CFR

2011-07-01

... 41 Public Contracts and Property Management 4 2011-07-01 2011-07-01 false What relocation expenses... Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities New Appointees § 302-3.508 What...

41 CFR 302-3.508 - What relocation expenses are not authorized for new appointees or student trainees?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 41 Public Contracts and Property Management 4 2013-07-01 2012-07-01 true What relocation expenses... Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities New Appointees § 302-3.508 What...

41 CFR 302-3.207 - Am I eligible to receive relocation allowances for overseas assignment and return travel?

Code of Federal Regulations, 2011 CFR

2011-07-01

... relocation allowances for overseas assignment and return travel? 302-3.207 Section 302-3.207 Public Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3... eligible to receive relocation allowances for overseas assignment and return travel? You may be eligible to...

41 CFR 302-3.207 - Am I eligible to receive relocation allowances for overseas assignment and return travel?

Code of Federal Regulations, 2013 CFR

2013-07-01

... relocation allowances for overseas assignment and return travel? 302-3.207 Section 302-3.207 Public Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3... eligible to receive relocation allowances for overseas assignment and return travel? You may be eligible to...

41 CFR 302-3.207 - Am I eligible to receive relocation allowances for overseas assignment and return travel?

Code of Federal Regulations, 2012 CFR

2012-07-01

... relocation allowances for overseas assignment and return travel? 302-3.207 Section 302-3.207 Public Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3... eligible to receive relocation allowances for overseas assignment and return travel? You may be eligible to...

41 CFR 302-3.207 - Am I eligible to receive relocation allowances for overseas assignment and return travel?

Code of Federal Regulations, 2010 CFR

2010-07-01

... relocation allowances for overseas assignment and return travel? 302-3.207 Section 302-3.207 Public Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3... eligible to receive relocation allowances for overseas assignment and return travel? You may be eligible to...

47 CFR 27.1111 - Relocation of fixed microwave service licensees in the 2110-2150 MHz band.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 2 2010-10-01 2010-10-01 false Relocation of fixed microwave service licensees..., 2110-2155 MHz, 2160-2180 MHz Bands Relocation of Incumbents § 27.1111 Relocation of fixed microwave... contain provisions governing the relocation of incumbent fixed microwave service licensees in the 2110...

47 CFR 27.1111 - Relocation of fixed microwave service licensees in the 2110-2150 MHz band.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 2 2011-10-01 2011-10-01 false Relocation of fixed microwave service licensees..., 2110-2155 MHz, 2160-2180 MHz Bands Relocation of Incumbents § 27.1111 Relocation of fixed microwave... contain provisions governing the relocation of incumbent fixed microwave service licensees in the 2110...

47 CFR 27.1111 - Relocation of fixed microwave service licensees in the 2110-2150 MHz band.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 2 2012-10-01 2012-10-01 false Relocation of fixed microwave service licensees..., 2110-2155 MHz, 2160-2180 MHz Bands Relocation of Incumbents § 27.1111 Relocation of fixed microwave... contain provisions governing the relocation of incumbent fixed microwave service licensees in the 2110...

41 CFR 302-12.100 - What are “relocation services”?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 41 Public Contracts and Property Management 4 2013-07-01 2012-07-01 true What are ârelocation servicesâ? 302-12.100 Section 302-12.100 Public Contracts and Property Management Federal Travel Regulation... Agency's Use of a Relocation Services Company § 302-12.100 What are “relocation services”? “Relocation...

SUMOylation Regulates the Homologous to E6-AP Carboxyl Terminus (HECT) Ubiquitin Ligase Rsp5p*

PubMed Central

Novoselova, Tatiana Vladislavovna; Rose, Ruth-Sarah; Marks, Helen Margaret; Sullivan, James Andrew

2013-01-01

The post-translational modifiers ubiquitin and small ubiquitin-related modifier (SUMO) regulate numerous critical signaling pathways and are key to controlling the cellular fate of proteins in eukaryotes. The attachment of ubiquitin and SUMO involves distinct, but related, machinery. However, it is now apparent that many substrates can be modified by both ubiquitin and SUMO and that some regulatory interaction takes place between the respective attachment machinery. Here, we demonstrate that the Saccharomyces cerevisiae ubiquitin ligase Rsp5p, a member of the highly conserved Nedd4 family of ubiquitin ligases, is SUMOylated in vivo. We further show that Rsp5p SUMOylation is mediated by the SUMO ligases Siz1p and Siz2p, members of the conserved family of PIAS SUMO ligases that are, in turn, substrates for Rsp5p-mediated ubiquitylation. Our experiments show that SUMOylated Rsp5p has reduced ubiquitin ligase activity, and similarly, ubiquitylated Siz1p demonstrates reduced SUMO ligase activity leading to respective changes in both ubiquitin-mediated sorting of the manganese transporter Smf1p and polySUMO chain formation. This reciprocal regulation of these highly conserved ligases represents an exciting and previously unidentified system of cross talk between the ubiquitin and SUMO systems. PMID:23443663

An essential role of ubiquitination in Cbl-mediated negative regulation of the Src-family kinase Fyn

PubMed Central

Rao, Navin; Ghosh, Amiya K.; Douillard, Patrice; Andoniou, Christopher E.; Zhou, Pengcheng; Band, Hamid

2009-01-01

SUMMARY The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using primary embryonic fibroblasts from Cbl+/+ and Cbl−/− mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases. PMID:19966925

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsumoto, Hotaru; Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp; Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto

2016-07-29

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of themore » SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. -- Highlights: •Puromycin exhibits the O-propargyl-puromycin effect. •Puromycin induces SUMO redistribution upon proteasome inhibition. •Ubiquitin and RNF4 accumulate at PML-nuclear bodies with SUMO-2/3. •The ubiquitin family may function in nuclear sequestration of immature proteins.« less

A tailored relocation stress intervention programme for family caregivers of patients transferred from a surgical intensive care unit to a general ward.

PubMed

Lee, Seul; Oh, HyunSoo; Suh, YeonOk; Seo, WhaSook

2017-03-01

To develop and examine a relocation stress intervention programme tailored for the family caregivers of patients scheduled for transfer from a surgical intensive care unit to a general ward. Family relocation stress syndrome has been reported to be similar to that exhibited by patients, and investigators have emphasised that nurses should make special efforts to relieve family relocation stress to maximise positive contributions to the well-being of patients by family caregivers. A nonequivalent control group, nonsynchronised pretest-post-test design was adopted. The study subjects were 60 family caregivers of patients with neurosurgical or general surgical conditions in the surgical intensive care unit of a university hospital located in Incheon, South Korea. Relocation stress and family burden were evaluated at three times, that is before intervention, immediately after transfer and four to five days after transfer. This relocation stress intervention programme was developed for the family caregivers based on disease characteristics and relocation-related needs. In the experimental group, relocation stress levels significantly and continuously decreased after intervention, whereas in the control group, a slight nonsignificant trend was observed. Family burden levels in the control group increased significantly after transfer, whereas burden levels in the experimental group increased only marginally and nonsignificantly. No significant between-group differences in relocation stress or family burden levels were observed after intervention. Relocation stress levels of family caregivers were significantly decreased after intervention in the experimental group, which indicates that the devised family relocation stress intervention programme effectively alleviated family relocation stress. The devised intervention programme, which was tailored to disease characteristics and relocation-related needs, may enhance the practicality and efficacy of relocation stress management and make meaningful contribution to the relief of family relocation stress, promote patient recovery and enhance the well-being of patients and family caregivers. © 2016 John Wiley & Sons Ltd.

Enzymatic production of mono-ubiquitinated proteins for structural studies: The example of the Josephin domain of ataxin-3☆

PubMed Central

Faggiano, Serena; Menon, Rajesh P.; Kelly, Geoff P.; McCormick, John; Todi, Sokol V.; Scaglione, K. Matthew; Paulson, Henry L.; Pastore, Annalisa

2013-01-01

Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration. However, despite the undoubted biological importance, understanding the molecular basis of how ubiquitination regulates different pathways has up to now been strongly limited by the difficulty of producing the amounts of highly homogeneous samples that are needed for a structural characterization by X-ray crystallography and/or NMR. Here, we report on the production of milligrams of highly pure Josephin mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. We demonstrate that the samples produced with the described method are correctly folded and suitable for structural studies. The protocol allows facile selective labelling of the components. Our results provide an important proof-of-concept that may pave the way to new approaches to the in vitro study of ubiquitinated proteins. PMID:24251111

Distinct expression profile of HCMV encoded miRNAs in plasma from oral lichen planus patients.

PubMed

Ding, Meng; Wang, Xiang; Wang, Cheng; Liu, Xiaoshuang; Zen, Ke; Wang, Wenmei; Zhang, Chen-Yu; Zhang, Chunni

2017-06-07

Oral lichen planus (OLP) is a T cell-mediated autoimmune disease. The aetiology and molecular mechanisms of OLP remain unclear. Human cytomegalovirus (HCMV) infection is a causal factor in the development of various diseases, but the clinical relevance of HCMV in OLP has not been thoroughly investigated. In the present study, we firstly examined twenty-three HCMV-encoded microRNA (miRNA) expression profiles in plasma from training set that including 21 OLP patients and 18 healthy controls using RT-qPCR technology. Dysregulated miRNAs were subsequently confirmed in another larger cohort refereed as validation set consisting of 40 OLP patients and 33 healthy controls. HCMV DNA in peripheral blood leukocytes (PBLs) was also measured in an additional cohort of 13 OLP patients and 12 control subjects. Furthermore, bioinformatics analyses, luciferase reporter assay and western blotting were also performed to predict and verify the direct potential targets of HCMV-encoded miRNAs. The RT-qPCR results showed that the plasma levels of five HCMV-encoded miRNAs including hcmv-miR-UL112-3p, hcmv-miR-UL22a-5p, hcmv-miR-UL148d, hcmv-miR-UL36-5p and hcmv-miR-UL59 were significantly increased in OLP patients in both training and validation sets. HCMV DNA in PBLs was also significantly higher in OLP patients than in control subjects. Additionally, by using a combination of luciferase reporter assay and western blotting, we demonstrated that cytomegalovirus UL16-binding protein 1, a molecule that mediates the killing of virus-infected cells by natural killer cells, is a direct target of hcmv-miR-UL59. Our results demonstrate a distinct expression pattern of HCMV-encoded miRNAs in OLP patients, which may provide insight into the relationship between HCMV infection and OLP, and warrants additional study in the diagnosis and aetiology of OLP.

Antibodies against human cytomegalovirus late protein UL94 in the pathogenesis of scleroderma-like skin lesions in chronic graft-versus-host disease.

PubMed

Pastano, Rocco; Dell'Agnola, Chiara; Bason, Caterina; Gigli, Federica; Rabascio, Cristina; Puccetti, Antonio; Tinazzi, Elisa; Cetto, Gianluigi; Peccatori, Fedro; Martinelli, Giovanni; Lunardi, Claudio

2012-09-01

Human cytomegalovirus (hCMV) infection and its reactivation correlate both with the increased risk and with the worsening of graft-versus-host disease (GVHD). Because scleroderma-like skin lesions can occur in chronic GVHD (cGVHD) in allogeneic stem-cell transplant (HCT) patients and hCMV is relevant in the pathogenesis of systemic sclerosis (SSc), we evaluated the possible pathogenetic link between hCMV and skin cGVHD. Plasma from 18 HCT patients was tested for anti-UL94 and/or anti-NAG-2 antibodies, identified in SSc patients, by direct ELISA assays. Both donors and recipients were anti-hCMV IgG positive, without autoimmune diseases. Patients' purified anti-UL94 and anti-NAG-2 IgG binding to human umbilical endothelial cells (HUVECs) and fibroblasts was performed by FACS analysis and ELISA test. HUVECs apoptosis and fibroblasts proliferation induced by patients' anti-NAG-2 antibodies were measured by DNA fragmentation and cell viability, respectively. About 11/18 patients developed cGVHD and all of them showed skin involvement, ranging from diffuse SSc-like lesions to limited erythema. Eight of eleven cGVHD patients were positive for anti-UL94 and/or anti-NAG-2 antibodies. Remarkably, 4/5 patients who developed diffuse or limited SSc-like lesions had antibodies directed against both UL94 and NAG-2; their anti-NAG-2 IgG-bound HUVECs and fibroblasts induce both endothelial cell apoptosis and fibroblasts proliferation, similar to that induced by purified anti-UL94 and anti-NAG-2 antibodies obtained from SSc patients. In conclusion, our data suggest a pathogenetic link between hCMV infection and scleroderma-like skin cGVHD in HCT patients through a mechanism of molecular mimicry between UL94 viral protein and NAG-2 molecule, as observed in patients with SSc.

Load Dependency of Postural Control--Kinematic and Neuromuscular Changes in Response to over and under Load Conditions.

PubMed

Ritzmann, Ramona; Freyler, Kathrin; Weltin, Elmar; Krause, Anne; Gollhofer, Albert

2015-01-01

Load variation is associated with changes in joint torque and compensatory reflex activation and thus, has a considerable impact on balance control. Previous studies dealing with over (OL) and under loading (UL) used water buoyancy or additional weight with the side effects of increased friction and inertia, resulting in substantially modified test paradigms. The purpose of this study was to identify gravity-induced load dependency of postural control in comparable experimental conditions and to determine the underlying neuromuscular mechanisms. Balance performance was recorded under normal loading (NL, 1 g), UL (0.16 g 0.38 g) and OL (1.8 g) in monopedal stance. Center of pressure (COP) displacement and frequency distribution (low 0.15-0.5 Hz (LF), medium 0.5-2 Hz (MF), high 2-6 Hz (HF)) as well as ankle, knee and hip joint kinematics were assessed. Soleus spinal excitability was determined by H/M-recruitment curves (H/M-ratios). Compared to NL, OL caused an increase in ankle joint excursion, COP HF domain and H/M-ratio. Concomitantly, hip joint excursion and COP LF decreased. Compared to NL, UL caused modulations in the opposite direction: UL decreased ankle joint excursions, COP HF and H/M-ratio. Collaterally, hip joint excursion and COP LF increased. COP was augmented both in UL and in OL compared to NL. Subjects achieved postural stability in OL and UL with greater difficulty compared to NL. Reduced postural control was accompanied by modified balance strategies and compensatory reflex activation. With increasing load, a shift from hip to ankle strategy was observed. Accompanying, COP frequency distribution shifted from LF to HF and spinal excitability was enhanced. It is suggested that in OL, augmented ankle joint torques are compensated by quick reflex-induced postural reactions in distal muscles. Contrarily, UL is associated with diminished joint torques and thus, postural equilibrium may be controlled by the proximal segments to adjust the center of gravity above the base of support.

Human Cytomegalovirus Clinical Strain-Specific microRNA miR-UL148D Targets the Human Chemokine RANTES during Infection

PubMed Central

Kim, Sungchul; Kim, Donghyun; Ahn, Jin-Hyun; Ahn, Kwangseog

2012-01-01

The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b' sequences of the Toledo genome, targeted the 3′-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV. PMID:22412377

Factors associated with elevated serum alanine aminotransferase in patients with type 1 diabetes mellitus.

PubMed

Hatanaka, S A; Silva, N O; Colombo, B S; Correa, C G; Alcaire, B P; Coral, M H; Schiavon, L L; Narciso-Schiavon, J L

2015-09-01

Metabolic syndrome and type 2 diabetes are associated with insulin resistance and hepatic steatosis, which are common causes of alanine aminotransferase (ALT) elevation. This study aims to identify variables associated with altered ALT in type 1 diabetic (DM1) subjects. A cross-sectional study conducted in the outpatient endocrinology clinic of a university hospital. Patients with DM1 were seen between December 2012 and September 2013; clinical variables were collected from medical records. Fifty-six patients were included aged 27 ± 10.1 years; 60.7% were men. The study subjects exhibited an average ALT of 36.7 ± 10.3 U/L (median = 35 U/L) and their average Body Mass Index (BMI) was 23.8 ± 3.8 kg/m2. When comparing individuals with elevated ALT > 35 U/L (N. = 27) with those ALT ≤ 35 U/L (N. = 29), we found that individuals with ALT values > 35 U/L showed a higher proportion of men (77.8% vs. 44.8%, P = 0.012) and a higher mean age (30.2 ± 12.3 vs. 24.6 ± 6.9 years, P = 0.046). When new ALT reference values were applied (19 U/L for women and 30 U/L for men), five individuals had normal ALT values. Individuals with elevated ALT had higher BMI (24.3 vs. 20.9; P = 0.036), fasting glucose (194.8 ± 101.2 vs. 123.6 ± 42.0 mg/dL; P = 0.013) and higher HbA1c (9.9 ± 2.8 vs. 7.8 ± 0.7%; P < 0.001) levels. In Pearson correlation analysis, ALT values ​correlated with HbA1c (r = 0.285; P = 0.033). In patients with DM1, elevated ALT values ​​are associated with BMI, fasting glucose and HbA1c.

[Dynamic change study of dermatitis medicamentosa-like of trichloroethylene patients with liver damage].

PubMed

Liu, Wei; Zhang, Yan-fang; Zhang, Zhi-min; Li, Pei-mao; Jiang, Xiao-dong; Zhou, Gui-feng; Liu, Jian-jun

2011-10-01

Observing the dynamic change characteristics of serum liver function indexes in occupational dermatitis medicamentosa-like of trichloroethylene patients with liver damage, we can underlie for guiding therapy, prognosis and mechanism of dermatitis medicamentosa-like of trichloroethylene patients with liver damage. We collected serum of 10 cases of occupational dermatitis medicamentosa-like of trichloro-ethylene patients with liver damage from different time points since they were hospitalized, using automatic biochemistry analyzer to detect total protein (TP), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), albumin/globulin ratio etc 11 liver function biochemical indicators. We used Excel to establish database, professional drawing software gnuplot to draw dynamic variation diagram of each index. The variation range of 11 liver function indexes of 10 cases was TP 43.2-74.2 g/L, ALB 24.6-44.6 g/L, A/G 0.77-2.10, TBIL 3.7-268.2 umol/L, DBIL 1.0-166.0 umol/L, IBIL 2.4 -167.5 umol/L, ALT 11-5985 U/L, AST 14-5586 U/L, GGT 15-1500 U/L, ALP 35-309 U/L, S/L 0.07-1.94, respectively. TBIL, DBIL, ALT, AST, GGT, ALP concentration significantly increased, especially ALT, AST, GGT, ALT topped 5985 U/L, AST topped 5586 U/L, GGT topped 1500 U/L. But TP, ALB and S/L significantly decreased, TP lowest to 43.2 g/L, S/L lowest to 0.07. A/G basically remained unchanged, but IBIL didn't change regularly. The early liver damage in dermatitis medicamentosa-like of trichloroethylene patients was serious, and repeatedly attacked, so we should lead to enough attention to the clinical work and prevention. This also provided the basis for studying the mechanism of trichloroethylene poisoning.

Facile green synthesis of zinc oxide nanoparticles using Ulva lactuca seaweed extract and evaluation of their photocatalytic, antibiofilm and insecticidal activity.

PubMed

Ishwarya, Ramachandran; Vaseeharan, Baskaralingam; Kalyani, Subramanian; Banumathi, Balan; Govindarajan, Marimuthu; Alharbi, Naiyf S; Kadaikunnan, Shine; Al-Anbr, Mohammed N; Khaled, Jamal M; Benelli, Giovanni

2018-01-01

The bioactivity of semiconductor nanocomplexes has been poorly studied in the field of pesticide science. In this research, the synthesis of zinc nanoparticles was accomplished through new effortless green chemistry process, using the Ulva lactuca seaweed extract as a reducing and capping agent. The production of U. lactuca-fabricated ZnO nanoparticles (Ul-ZnO Nps) was characterized by powder X-ray diffraction (XRD), UV-visible, Fourier transform infrared (FTIR) spectroscopy, selected area electron diffraction (SAED) analysis and transmission electron microscopy (TEM). The U. lactuca-fabricated ZnO NPs were tested for their photodegradative action against organic dyes, as well as for antibiofilm and larvicidal activities. The UV visible absorbance spectrum of Ul-ZnO NPs exhibited the absorbance band at 325nm and TEM highlighted average crystallite sizes of nanoparticles of 10-50nm. Methylene blue (MB) dye was efficiently corrupted under sunlight in presence of Ul-ZnO NPs. Excellent bactericidal activity was shown by the Ul-ZnO Nps on Gram positive (Bacillus licheniformis and Bacillus pumilis) and Gram negative (Escherichia coliand Proteus vulgaris) bacteria. High antibiofilm potential was noted under both dark and sunlight conditions. The impact of a single treatment with Ul-ZnO NPs on biofilm architecture was also analyzed by confocal laser scanning microscopy (CLSM) on both Gram positive and Gram negative bacteria. Moreover, Ul-ZnO NPs led to 100% mortality of Aedes aegypti fourth instar larvae at the concentration of 50μg/ml within 24h. The effects of ZnO nanoparticle-based treatment on mosquito larval morphology and histology were monitored. Overall, based on our results, we believe that the synthesis of multifunctional Ul-ZnO Nps using widely available seaweed products can be promoted as a potential eco-friendly option to chemical methods currently used for nanosynthesis of antimicrobials and insecticides. Copyright © 2017 Elsevier B.V. All rights reserved.

Ubiquitin acetylation inhibits polyubiquitin chain elongation

PubMed Central

Ohtake, Fumiaki; Saeki, Yasushi; Sakamoto, Kensaku; Ohtake, Kazumasa; Nishikawa, Hiroyuki; Tsuchiya, Hikaru; Ohta, Tomohiko; Tanaka, Keiji; Kanno, Jun

2015-01-01

Ubiquitylation is a versatile post-translational modification (PTM). The diversity of ubiquitylation topologies, which encompasses different chain lengths and linkages, underlies its widespread cellular roles. Here, we show that endogenous ubiquitin is acetylated at lysine (K)-6 (AcK6) or K48. Acetylated ubiquitin does not affect substrate monoubiquitylation, but inhibits K11-, K48-, and K63-linked polyubiquitin chain elongation by several E2 enzymes in vitro. In cells, AcK6-mimetic ubiquitin stabilizes the monoubiquitylation of histone H2B—which we identify as an endogenous substrate of acetylated ubiquitin—and of artificial ubiquitin fusion degradation substrates. These results characterize a mechanism whereby ubiquitin, itself a PTM, is subject to another PTM to modulate mono- and polyubiquitylation, thus adding a new regulatory layer to ubiquitin biology. PMID:25527407

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction.

PubMed

Saini, Vikas; Marchese, Adriano; Tang, Wei-Jen; Majetschak, Matthias

2011-12-23

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.

Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

PubMed Central

Solomon, Vered; Baracos, Vickie; Sarraf, Pasha; Goldberg, Alfred L.

1998-01-01

The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin–proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3α, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3α-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway. PMID:9770532

Differential ubiquitination in NETs regulates macrophage responses in systemic lupus erythematosus.

PubMed

Barrera-Vargas, Ana; Gómez-Martín, Diana; Carmona-Rivera, Carmelo; Merayo-Chalico, Javier; Torres-Ruiz, Jiram; Manna, Zerai; Hasni, Sarfaraz; Alcocer-Varela, Jorge; Kaplan, Mariana J

2018-06-01

To assess if ubiquitinated proteins potentially present in neutrophil extracellular traps (NETs) can modify cellular responses and induce inflammatory mechanisms in patients with systemic lupus erythematosus (SLE) and healthy subjects. We studied 74 subjects with SLE and 77 healthy controls. Neutrophils and low-density granulocytes were isolated, and NETs were induced. Ubiquitin content was quantified in NETs by western blot analysis, ELISA and immunofluorescence microscopy, while ubiquitination of NET proteins was assessed by immunoprecipitation. Monocyte-derived macrophages from SLE and controls were isolated and stimulated with NETs or ubiquitin. Calcium flux and cytokine synthesis were measured following these stimuli. NETs contain ubiquitinated proteins, with a lower expression of polyubiquitinated proteins in subjects with SLE than in controls. Myeloperoxidase (MPO) is present in ubiquitinated form in NETs. Patients with SLE develop antiubiquitinated MPO antibodies, and titres positively correlate with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (P<0.01), and negatively correlate with complement components (P<0.01). Stimulation of monocyte-derived macrophages with NETs or with ubiquitin led to enhanced calcium flux. In addition, stimulation with NETs led to enhanced cytokine (tumour necrosis factor-α and interleukin-10) production in macrophages from patients with SLE when compared with controls, which was hampered by inhibition of NET internalisation by macrophages. This is the first study to find ubiquitinated proteins in NETs, and evidence for adaptive immune responses directed towards ubiquitinated NET proteins in SLE. The distinct differences in ubiquitin species profile in NETs compared with healthy controls may contribute to dampened anti-inflammatory responses observed in SLE. These results also support a role for extracellular ubiquitin in inflammation in SLE. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

41 CFR 302-3.208 - What relocation expenses will my agency pay for my overseas assignment and return?

Code of Federal Regulations, 2011 CFR

2011-07-01

... 41 Public Contracts and Property Management 4 2011-07-01 2011-07-01 false What relocation expenses... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Types of Transfers Overseas Assignment and Return § 302-3.208 What...

41 CFR 302-3.208 - What relocation expenses will my agency pay for my overseas assignment and return?

Code of Federal Regulations, 2014 CFR

2014-07-01

... 41 Public Contracts and Property Management 4 2014-07-01 2014-07-01 false What relocation expenses... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Types of Transfers Overseas Assignment and Return § 302-3.208 What...

41 CFR 302-3.208 - What relocation expenses will my agency pay for my overseas assignment and return?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 41 Public Contracts and Property Management 4 2013-07-01 2012-07-01 true What relocation expenses... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Types of Transfers Overseas Assignment and Return § 302-3.208 What...

41 CFR 302-3.208 - What relocation expenses will my agency pay for my overseas assignment and return?

Code of Federal Regulations, 2012 CFR

2012-07-01

... 41 Public Contracts and Property Management 4 2012-07-01 2012-07-01 false What relocation expenses... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Types of Transfers Overseas Assignment and Return § 302-3.208 What...

41 CFR 302-3.506 - May we pay relocation expenses if the employee violates his/her service agreement?

Code of Federal Regulations, 2011 CFR

2011-07-01

... 41 Public Contracts and Property Management 4 2011-07-01 2011-07-01 false May we pay relocation... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities Service Agreements § 302-3.506 May we pay...

41 CFR 302-3.506 - May we pay relocation expenses if the employee violates his/her service agreement?

Code of Federal Regulations, 2012 CFR

2012-07-01

... 41 Public Contracts and Property Management 4 2012-07-01 2012-07-01 false May we pay relocation... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities Service Agreements § 302-3.506 May we pay...

41 CFR 302-3.506 - May we pay relocation expenses if the employee violates his/her service agreement?

Code of Federal Regulations, 2014 CFR

2014-07-01

... 41 Public Contracts and Property Management 4 2014-07-01 2014-07-01 false May we pay relocation... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities Service Agreements § 302-3.506 May we pay...

41 CFR 302-3.506 - May we pay relocation expenses if the employee violates his/her service agreement?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 41 Public Contracts and Property Management 4 2013-07-01 2012-07-01 true May we pay relocation... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities Service Agreements § 302-3.506 May we pay...

41 CFR 302-3.506 - May we pay relocation expenses if the employee violates his/her service agreement?

Code of Federal Regulations, 2010 CFR

2010-07-01

... 41 Public Contracts and Property Management 4 2010-07-01 2010-07-01 false May we pay relocation... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Agency Responsibilities Service Agreements § 302-3.506 May we pay...

41 CFR 302-3.208 - What relocation expenses will my agency pay for my overseas assignment and return?

Code of Federal Regulations, 2010 CFR

2010-07-01

... 41 Public Contracts and Property Management 4 2010-07-01 2010-07-01 false What relocation expenses... and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES RELOCATION ALLOWANCES 3-RELOCATION ALLOWANCE BY SPECIFIC TYPE Types of Transfers Overseas Assignment and Return § 302-3.208 What...

41 CFR 302-12.103 - May we separately contract for each type of relocation service?

Code of Federal Regulations, 2010 CFR

2010-07-01

... contract for each type of relocation service? 302-12.103 Section 302-12.103 Public Contracts and Property... A RELOCATION SERVICES COMPANY Agency's Use of a Relocation Services Company § 302-12.103 May we separately contract for each type of relocation service? Yes, you may separately contract for each type of...

46 CFR 120.410 - Lighting fixtures.

Code of Federal Regulations, 2013 CFR

2013-10-01

... or be made of high strength material, except in an accommodation space, radio room, galley, or...,” UL 1573, “Stage and Studio Lighting Units,” or UL 1574, “Track Lighting Systems,” as long as the...

46 CFR 120.410 - Lighting fixtures.

Code of Federal Regulations, 2011 CFR

2011-10-01

... or be made of high strength material, except in an accommodation space, radio room, galley, or...,” UL 1573, “Stage and Studio Lighting Units,” or UL 1574, “Track Lighting Systems,” as long as the...

46 CFR 120.410 - Lighting fixtures.

Code of Federal Regulations, 2012 CFR

2012-10-01

... or be made of high strength material, except in an accommodation space, radio room, galley, or...,” UL 1573, “Stage and Studio Lighting Units,” or UL 1574, “Track Lighting Systems,” as long as the...

46 CFR 120.410 - Lighting fixtures.

Code of Federal Regulations, 2014 CFR

2014-10-01

... or be made of high strength material, except in an accommodation space, radio room, galley, or...,” UL 1573, “Stage and Studio Lighting Units,” or UL 1574, “Track Lighting Systems,” as long as the...

Identification of a small molecule that inhibits herpes simplex virus DNA Polymerase subunit interactions and viral replication.

PubMed

Pilger, Beatrice D; Cui, Can; Coen, Donald M

2004-05-01

The interaction between the catalytic subunit Pol and the processivity subunit UL42 of herpes simplex virus DNA polymerase has been characterized structurally and mutationally and is a potential target for novel antiviral drugs. We developed and validated an assay for small molecules that could disrupt the interaction of UL42 and a Pol-derived peptide and used it to screen approximately 16,000 compounds. Of 37 "hits" identified, four inhibited UL42-stimulated long-chain DNA synthesis by Pol in vitro, of which two exhibited little inhibition of polymerase activity by Pol alone. One of these specifically inhibited the physical interaction of Pol and UL42 and also inhibited viral replication at concentrations below those that caused cytotoxic effects. Thus, a small molecule can inhibit this protein-protein interaction, which provides a starting point for the discovery of new antiviral drugs.

Regulation of Proteolysis by Human Deubiquitinating Enzymes

PubMed Central

Eletr, Ziad M.; Wilkinson, Keith D.

2013-01-01

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. PMID:23845989

Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

2015-01-01

In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

PubMed

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017 Elsevier Inc. All rights reserved.

Correlation between ubiquitination and defects of bull spermatozoa and removal of defective spermatozoa using anti-ubiquitin antibody-coated magnetized beads.

PubMed

Zhang, Jian; Su, Jie; Hu, Shuxiang; Zhang, Jindun; Ding, Rui; Guo, Jitong; Cao, Guifang; Li, Rongfeng; Sun, Qing-Yuan; Li, Xihe

2018-05-01

Ubiquitination is an important cellular process in spermatogenesis and involves the regulation of spermatid differentiation and spermiogenesis. In the current study, the correlation between bull sperm ubiquitination and sperm defects was analyzed, and the feasibility using anti-ubiquitin specific antibody immobilized magnetic beads to remove the spermatozoa with defects was assessed. A total of nine bulls were examined, and the amount of sperm ubiquitination ranged from 55 to 151. Correspondingly, the percentage of sperm deformity ranged from 9.3% to 28.1%. The coefficient of correlation was r = 0.92, indicating a significant correlation between the percentage of sperm deformity and the amount of ubiquitination (P < 0.05). The results from use of fluorescence staining and single-channel flow cytometry indicated there was a significant correlation between the sperm deformity and amount of ubiquitination (r = 0.86, P < 0.05). Results gained by use of the TUNEL and ubiquitination assays by double-channel flow cytometry indicated that the proportion of genetically defective spermatozoa with ubiquitination in Q3 and Q2 quartiles was markedly greater than that of spermatozoa with ubiquitination in Q1 and Q4 quartiles (82.1% compared with 17.9%). All these results confirmed that sperm ubiquitination is associated with genetic DNA defects (P < 0.01). Furthermore, nine semen samples with sperm motility of less than 50% (minimal motility), 50% to 70% (moderate motility) and greater than 70% (greatest motility) were selected for sorting defective spermatozoa using anti-ubiquitin specific antibody-coated magnetic beads. Strikingly, the percentage of sperm deformity significantly decreased from 18.8%, 19.0% and 17.1% to 11.7%, 11.0% and 11.0%, respectively (P < 0.05), suggesting that this method might be a feasible technology to improve the productivity via removal of the defective spermatozoa from bull semen. Copyright © 2018 Elsevier B.V. All rights reserved.

p62/SQSTM1 promotes rapid ubiquitin conjugation to target proteins after endosome rupture during xenophagy.

PubMed

Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Mori, Chie; Osakada, Hiroko; Kobayashi, Shouhei; Hiraoka, Yasushi; Haraguchi, Tokuko

2018-03-01

Autophagy is a bulk degradation pathway, and selective autophagy to remove foreign entities is called xenophagy. The conjugation of ubiquitin to target pathogens is an important process in xenophagy but when and where this ubiquitination occurs remains unclear. Here, we analyzed the temporal sequence and subcellular location of ubiquitination during xenophagy using time-lapse observations, with polystyrene beads mimicking invading pathogens. Results revealed accumulation of a ubiquitination marker around the beads within 3 min after endosome rupture. Recruitment of ubiquitin to the beads was significantly delayed in p62-knockout murine embryonic fibroblast cells, and this delay was rescued by ectopic p62 expression. Ectopic expression of a phosphorylation-mimicking p62 mutated at serine residue 405 (equivalent to human serine residue 403) rescued this delay, but its unphosphorylated form did not. These results indicate that ubiquitination mainly occurs after endosome rupture and suggest that p62, specifically the phosphorylated form, promotes ubiquitin conjugation to target proteins in xenophagy.

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination.

PubMed

Ahmed, M Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V; Gurevich, Eugenia V

2011-05-10

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmunoprecipitation of endogenous proteins from brain tissue and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both nonvisual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology.

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-11-14

TGF-{beta} activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-{beta} enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-{beta} type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smadmore » complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes.« less

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes.

PubMed

Wang, Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-11-14

TGF-beta activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-beta enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-beta type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smad complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes.

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

Ubiquitin-Modifying Enzymes and Regulation of the Inflammasome.

PubMed

Kattah, Michael G; Malynn, Barbara A; Ma, Averil

2017-11-10

Ubiquitin and ubiquitin-modifying enzymes play critical roles in a wide variety of intracellular signaling pathways. Inflammatory signaling cascades downstream of TNF, TLR agonists, antigen receptor cross-linking, and cytokine receptors, all rely on ubiquitination events to direct subsequent immune responses. In the past several years, inflammasome activation and subsequent signal transduction have emerged as an excellent example of how ubiquitin signals control inflammatory responses. Inflammasomes are multiprotein signaling complexes that ultimately lead to caspase activation and release of the interleukin-1 (IL-1) family members, IL-1β and IL-18. Inflammasome activation is critical for the host's defense against pathogens, but dysregulation of inflammasomes may contribute to the pathogenesis of multiple diseases. Ultimately, understanding how various ubiquitin interacting proteins control inflammatory signaling cascades could provide new pathways for therapeutic intervention. Here we review specific ubiquitin-modifying enzymes and ubiquitination events that orchestrate inflammatory responses, with an emphasis on the NLRP3 inflammasome. Copyright © 2017 Elsevier Ltd. All rights reserved.

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition.

PubMed

Matsumoto, Hotaru; Saitoh, Hisato

2016-07-29

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of covalent modification by 4-hydroxy-2-nonenal on the noncovalent oligomerization of ubiquitin.

PubMed

Grasso, Giuseppe; Axelsen, Paul H

2017-01-01

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius

NASA Astrophysics Data System (ADS)

Anjum, Rana S.; Bray, Sian M.; Blackwood, John K.; Kilkenny, Mairi L.; Coelho, Matthew A.; Foster, Benjamin M.; Li, Shurong; Howard, Julie A.; Pellegrini, Luca; Albers, Sonja-Verena; Deery, Michael J.; Robinson, Nicholas P.

2015-09-01

In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

Ubiquitin Utilizes an Acidic Surface Patch to Alter Chromatin Structure

PubMed Central

Debelouchina, Galia T.; Gerecht, Karola; Muir, Tom W.

2016-01-01

Ubiquitylation of histone H2B, associated with gene activation, leads to chromatin decompaction through an unknown mechanism. We used a hydrogen-deuterium exchange strategy coupled with nuclear magnetic resonance spectroscopy to map the ubiquitin surface responsible for its structural effects on chromatin. Our studies revealed that a previously uncharacterized acidic patch on ubiquitin comprising residues Glu16 and Glu18 is essential for decompaction. These residues mediate promiscuous electrostatic interactions with the basic histone proteins, potentially positioning the ubiquitin moiety as a dynamic “wedge” that prevents the intimate association of neighboring nucleosomes. Using two independent cross-linking strategies and an oligomerization assay, we also showed that ubiquitin-ubiquitin contacts occur in the chromatin environment and are important for the solubilization of the chromatin polymers. Our work highlights a novel, chromatin-related aspect of the “ubiquitin code”, and sheds light on how the information rich ubiquitin modification can orchestrate different biochemical outcomes using different surface features. PMID:27870837

Activation of the Slx5–Slx8 Ubiquitin Ligase by Poly-small Ubiquitin-like Modifier Conjugates*S⃞

PubMed Central

Mullen, Janet R.; Brill, Steven J.

2008-01-01

Protein sumoylation is a regulated process that is important for the health of human and yeast cells. In budding yeast, a subset of sumoylated proteins is targeted for ubiquitination by a conserved heterodimeric ubiquitin (Ub) ligase, Slx5–Slx8, which is needed to suppress the accumulation of high molecular weight small ubiquitin-like modifier (SUMO) conjugates. Structure-function analysis indicates that the Slx5–Slx8 complex contains multiple SUMO-binding domains that are collectively required for in vivo function. To determine the specificity of Slx5–Slx8, we assayed its Ub ligase activity using sumoylated Siz2 as an in vitro substrate. In contrast to unsumoylated or multisumoylated Siz2, substrates containing poly-SUMO conjugates were efficiently ubiquitinated by Slx5–Slx8. Although Siz2 itself was ubiquitinated, the bulk of the Ub was conjugated to SUMO residues. Slx5–Slx8 primarily mono-ubiquitinated the N-terminal SUMO moiety of the chain. These data indicate that the Slx5–Slx8 Ub ligase is stimulated by poly-SUMO conjugates and that it can ubiquitinate a poly-SUMO chain. PMID:18499666

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

Responses of non-eye movement central vestibular neurons to sinusoidal horizontal translation in compensated macaques after unilateral labyrinthectomy

PubMed Central

Lin, Nan; Wei, Min

2014-01-01

After vestibular labyrinth injury, behavioral deficits partially recover through the process of vestibular compensation. The present study was performed to improve our understanding of the physiology of the macaque vestibular system in the compensated state (>7 wk) after unilateral labyrinthectomy (UL). Three groups of vestibular nucleus neurons were included: pre-UL control neurons, neurons ipsilateral to the lesion, and neurons contralateral to the lesion. The firing responses of neurons sensitive to linear acceleration in the horizontal plane were recorded during sinusoidal horizontal translation directed along six different orientations (30° apart) at 0.5 Hz and 0.2 g peak acceleration (196 cm/s2). This data defined the vector of best response for each neuron in the horizontal plane, along which sensitivity, symmetry, detection threshold, and variability of firing were determined. Additionally, the responses of the same cells to translation over a series of frequencies (0.25–5.0 Hz) either in the interaural or naso-occipital orientation were obtained to define the frequency response characteristics in each group. We found a decrease in sensitivity, increase in threshold, and alteration in orientation of best responses in the vestibular nuclei after UL. Additionally, the phase relationship of the best neural response to translational stimulation changed with UL. The symmetry of individual neuron responses in the excitatory and inhibitory directions was unchanged by UL. Bilateral central utricular neurons still demonstrated two-dimension tuning after UL, consistent with spatio-temporal convergence from a single vestibular end-organ. These neuronal data correlate with known behavioral deficits after unilateral vestibular compromise. PMID:24717349

Novel mode of phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus.

PubMed

Milbradt, Jens; Webel, Rike; Auerochs, Sabrina; Sticht, Heinrich; Marschall, Manfred

2010-04-30

The nucleocytoplasmic egress of viral capsids is a rate-limiting step in the replication of the human cytomegalovirus (HCMV). As reported recently, an HCMV-specific nuclear egress complex is composed of viral and cellular proteins, in particular protein kinases with the capacity to induce destabilization of the nuclear lamina. Viral protein kinase pUL97 and cellular protein kinase C (PKC) play important roles by phosphorylating several types of nuclear lamins. Using pUL97 mutants, we show that the lamin-phosphorylating activity of pUL97 is associated with a reorganization of nuclear lamin A/C. Either pUL97 or PKC has the potential to induce distinct punctate lamina-depleted areas at the periphery of the nuclear envelope, which were detectable in transiently transfected and HCMV-infected cells. Using recombinant HCMV, which produces green fluorescent protein-labeled viral capsids, the direct transition of viral capsids through these areas could be visualized. This process was sensitive to an inhibitor of pUL97/PKC activity. The pUL97-mediated phosphorylation of lamin A/C at Ser(22) generated a novel binding motif for the peptidyl-prolyl cis/trans-isomerase Pin1. In HCMV-infected fibroblasts, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to viral replication centers and to the nuclear lamina. The local increase in Pin1 peptidyl-prolyl cis/trans-isomerase activity may promote conformational modulation of lamins. Thus, we postulate a novel phosphorylation-triggered mechanism for the reorganization of the nuclear lamina in HCMV-infected cells.

RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions.

PubMed

Silva, Amanda Perse da; Lopes, Juliana Freitas; Paula, Vanessa Salete de

2014-01-01

The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions' samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative. Copyright © 2014. Published by Elsevier Editora Ltda.

Proteomic Analysis of the Multimeric Nuclear Egress Complex of Human Cytomegalovirus*

PubMed Central

Milbradt, Jens; Kraut, Alexandra; Hutterer, Corina; Sonntag, Eric; Schmeiser, Cathrin; Ferro, Myriam; Wagner, Sabrina; Lenac, Tihana; Claus, Claudia; Pinkert, Sandra; Hamilton, Stuart T.; Rawlinson, William D.; Sticht, Heinrich; Couté, Yohann; Marschall, Manfred

2014-01-01

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC. PMID:24969177

Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus*

PubMed Central

Milbradt, Jens; Webel, Rike; Auerochs, Sabrina; Sticht, Heinrich; Marschall, Manfred

2010-01-01

The nucleocytoplasmic egress of viral capsids is a rate-limiting step in the replication of the human cytomegalovirus (HCMV). As reported recently, an HCMV-specific nuclear egress complex is composed of viral and cellular proteins, in particular protein kinases with the capacity to induce destabilization of the nuclear lamina. Viral protein kinase pUL97 and cellular protein kinase C (PKC) play important roles by phosphorylating several types of nuclear lamins. Using pUL97 mutants, we show that the lamin-phosphorylating activity of pUL97 is associated with a reorganization of nuclear lamin A/C. Either pUL97 or PKC has the potential to induce distinct punctate lamina-depleted areas at the periphery of the nuclear envelope, which were detectable in transiently transfected and HCMV-infected cells. Using recombinant HCMV, which produces green fluorescent protein-labeled viral capsids, the direct transition of viral capsids through these areas could be visualized. This process was sensitive to an inhibitor of pUL97/PKC activity. The pUL97-mediated phosphorylation of lamin A/C at Ser22 generated a novel binding motif for the peptidyl-prolyl cis/trans-isomerase Pin1. In HCMV-infected fibroblasts, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to viral replication centers and to the nuclear lamina. The local increase in Pin1 peptidyl-prolyl cis/trans-isomerase activity may promote conformational modulation of lamins. Thus, we postulate a novel phosphorylation-triggered mechanism for the reorganization of the nuclear lamina in HCMV-infected cells. PMID:20202933

Ubiquitination as an efficient molecular strategy employed in salmonella infection

USDA-ARS?s Scientific Manuscript database

The ubiquitin modification has various functions in the host innate immune system in response to the bacterial infection. To counteract the host immunity, Salmonella can specifically target ubiquitin pathways by its effector proteins. In this review, we describe the multiple facets of ubiquitin func...

41 CFR 302-2.5 - May I relocate from a location other than the location specified in my relocation travel...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 41 Public Contracts and Property Management 4 2010-07-01 2010-07-01 false May I relocate from a... Public Contracts and Property Management Federal Travel Regulation System RELOCATION ALLOWANCES INTRODUCTION 2-EMPLOYEES ELIGIBILITY REQUIREMENTS General Rules § 302-2.5 May I relocate from a location other...

Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

PubMed Central

Bowman, James; Rodgers, Mary A.; Shi, Mude; Amatya, Rina; Hostager, Bruce; Iwai, Kazuhiro; Gao, Shou-Jiang

2015-01-01

ABSTRACT Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. PMID:26578682

Ubiquitin conjugating enzyme E2-N and sequestosome-1 (p62) are components of the ubiquitination process mediated by the malin-laforin E3-ubiquitin ligase complex.

PubMed

Sánchez-Martín, Pablo; Romá-Mateo, Carlos; Viana, Rosa; Sanz, Pascual

2015-12-01

Lafora disease (LD, OMIM254780, ORPHA501) is a rare neurodegenerative form of epilepsy related to mutations in two proteins: laforin, a dual specificity phosphatase, and malin, an E3-ubiquitin ligase. Both proteins form a functional complex, where laforin recruits specific substrates to be ubiquitinated by malin. However, little is known about the mechanism driving malin-laforin mediated ubiquitination of its substrates. In this work we present evidence indicating that the malin-laforin complex interacts physically and functionally with the ubiquitin conjugating enzyme E2-N (UBE2N). This binding determines the topology of the chains that the complex is able to promote in the corresponding substrates (mainly K63-linked polyubiquitin chains). In addition, we demonstrate that the malin-laforin complex interacts with the selective autophagy adaptor sequestosome-1 (p62). Binding of p62 to the malin-laforin complex allows its recognition by LC3, a component of the autophagosomal membrane. In addition, p62 enhances the ubiquitinating activity of the malin-laforin E3-ubiquitin ligase complex. These data enrich our knowledge on the mechanism of action of the malin-laforin complex as an E3-ubiquitin ligase and reinforces the role of this complex in targeting substrates toward the autophagy pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of TRAF6 and ubiquitin mRNA in skeletal muscle of gastric cancer patients

PubMed Central

2012-01-01

Objective To investigate the prognostic significance of tumor necrosis factor receptor (TNFR),-associated factor 6 (TRAF6),-and ubiquitin in gastric cancer patients. Methods Biopsies of the rectus abdominis muscle were obtained intra operatively from 102 gastric cancer patients and 29 subjects undergoing surgery for benign abdominal diseases, and muscle TRAF6 and ubiquitin mRNA expression and proteasome proteolytic activities were assessed. Results TRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles. TRAF6 was upregulated in 67.65% (69/102) muscle of gastric cancer. Over expression of TRAF6 in muscles of gastric cancer were associated with TNM stage, level of serum albumin and percent of weight loss. Ubiquitin was significantly upregulated in muscle of gastric cancer compared with the control muscles. Ubiquitin was upregulated in 58.82% (60/102) muscles of gastric cancer. Over expression of ubiquitin in muscles of gastric cancer were associated with TNM (Tumor-Node-Metastasis) stage and weight loss. There was significant relation between TRAF6 and ubiquitin expression. Conclusions We found a positive correlation between TRAF6 and ubiquitin expression, suggesting that TRAF6 may up regulates ubiquitin activity in cancer cachexia. While more investigations are required to understand its mechanisms of TRAF6 and ubiquitin in skeletal muscle. Correct the catabolic-anabolic imbalance is essential for the effective treatment of cancer cachexia. PMID:23013936

Ubiquitin-dependent Regulation of Phospho-AKT Dynamics by the Ubiquitin E3 Ligase, NEDD4-1, in the Insulin-like Growth Factor-1 Response*

PubMed Central

Fan, Chuan-Dong; Lum, Michelle A.; Xu, Chao; Black, Jennifer D.; Wang, Xinjiang

2013-01-01

AKT is a critical effector kinase downstream of the PI3K pathway that regulates a plethora of cellular processes including cell growth, death, differentiation, and migration. Mechanisms underlying activated phospho-AKT (pAKT) translocation to its action sites remain unclear. Here we show that NEDD4-1 is a novel E3 ligase that specifically regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. NEDD4-1 physically interacts with AKT and promotes HECT domain-dependent ubiquitination of exogenous and endogenous AKT. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in vitro. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4-1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is released into the cytoplasm, imported into the nucleus, or coupled with proteasomal degradation. IGF-1 signaling specifically stimulates NEDD4-1-mediated ubiquitination of pAKT, without altering total AKT ubiquitination. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with wild type AKT. This study reveals a novel mechanism by which a specific E3 ligase is required for ubiquitin-dependent control of pAKT dynamics in a ligand-specific manner. PMID:23195959

Natural products inhibiting the ubiquitin-proteasome proteolytic pathway, a target for drug development.

PubMed

Tsukamoto, Sachiko; Yokosawa, Hideyoshi

2006-01-01

The ubiquitin-proteasome proteolytic pathway plays a major role in selective protein degradation and regulates various cellular events including cell cycle progression, transcription, DNA repair, signal transduction, and immune response. Ubiquitin, a highly conserved small protein in eukaryotes, attaches to a target protein prior to degradation. The polyubiquitin chain tagged to the target protein is recognized by the 26S proteasome, a high-molecular-mass protease subunit complex, and the protein portion is degraded by the 26S proteasome. The potential of specific proteasome inhibitors, which act as anti-cancer agents, is now under intensive investigation, and bortezomib (PS-341), a proteasome inhibitor, has been recently approved by FDA for multiple myeloma treatment. Since ubiquitination of proteins requires the sequential action of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3), and polyubiquitination is a prerequisite for proteasome-mediated protein degradation, inhibitors of E1, E2, and E3 are reasonably thought to be drug candidates for treatment of diseases related to ubiquitination. Recently, various compounds inhibiting the ubiquitin-proteasome pathway have been isolated from natural resources. We also succeeded in isolating inhibitors against the proteasome and E1 enzyme from marine natural resources. In this review, we summarize the structures and biological activities of natural products that inhibit the ubiquitin-proteasome proteolytic pathway.

A New Scheme to Characterize and Identify Protein Ubiquitination Sites.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Lai, K Robert; Lee, Tzong-Yi

2017-01-01

Protein ubiquitination, involving the conjugation of ubiquitin on lysine residue, serves as an important modulator of many cellular functions in eukaryotes. Recent advancements in proteomic technology have stimulated increasing interest in identifying ubiquitination sites. However, most computational tools for predicting ubiquitination sites are focused on small-scale data. With an increasing number of experimentally verified ubiquitination sites, we were motivated to design a predictive model for identifying lysine ubiquitination sites for large-scale proteome dataset. This work assessed not only single features, such as amino acid composition (AAC), amino acid pair composition (AAPC) and evolutionary information, but also the effectiveness of incorporating two or more features into a hybrid approach to model construction. The support vector machine (SVM) was applied to generate the prediction models for ubiquitination site identification. Evaluation by five-fold cross-validation showed that the SVM models learned from the combination of hybrid features delivered a better prediction performance. Additionally, a motif discovery tool, MDDLogo, was adopted to characterize the potential substrate motifs of ubiquitination sites. The SVM models integrating the MDDLogo-identified substrate motifs could yield an average accuracy of 68.70 percent. Furthermore, the independent testing result showed that the MDDLogo-clustered SVM models could provide a promising accuracy (78.50 percent) and perform better than other prediction tools. Two cases have demonstrated the effective prediction of ubiquitination sites with corresponding substrate motifs.

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

PubMed Central

Zhou, Yan; Dong, Na; Hu, Liyan; Shao, Feng

2013-01-01

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells. PMID:23469023

Clinical validity of a relocation stress scale for the families of patients transferred from intensive care units.

PubMed

Oh, HyunSoo; Lee, Seul; Kim, JiSun; Lee, EunJu; Min, HyoNam; Cho, OkJa; Seo, WhaSook

2015-07-01

This study was conducted to develop a family relocation stress scale by modifying the Son's Relocation Stress Syndrome Scale, to examine its clinical validity and reliability and to confirm its suitability for measuring family relocation stress. The transfer of ICU patients to general wards is a significant anxiety-producing event for family members. However, no relocation stress scale has been developed specifically for families. A nonexperimental, correlation design was adopted. The study subjects were 95 family members of 95 ICU patients at a university hospital located in Incheon, South Korea. Face and construct validities of the devised family relocation stress scale were examined. Construct validity was examined using factor analysis and by using a nomological validity test. Reliability was also examined. Face and content validity of the scale were verified by confirming that its items adequately measured family relocation stress. Factor analysis yielded four components, and the total variance explained by these four components was 63·0%, which is acceptable. Nomological validity was well supported by significant relationships between relocation stress and degree of preparation for relocation, patient self-care ability, family burden and satisfaction with the relocation process. The devised scale was also found to have good reliability. The family relocation stress scale devised in this study was found to have good validity and reliability, and thus, is believed to offer a means of assessing family relocation stress. The findings of this study provide a reliable and valid assessment tool when nurses prepare families for patient transfer from an ICU to a ward setting, and may also provide useful information to those developing an intervention programme for family relocation stress management. © 2015 John Wiley & Sons Ltd.

Combined Satellite - and ULS-Derived Sea-Ice Flux in the Weddell Sea

NASA Technical Reports Server (NTRS)

Drinkwater, M.; Liu, X.; Harms, S.

2000-01-01

Several years of daily microwave satellite ice-drift are combined with moored Upward Looking Sonar (ULS) ice-drafts into an ice volume flux record at points along a flux gate across the Weddell Sea, Antarctica.

46 CFR 125.180 - Incorporation by reference.

Code of Federal Regulations, 2014 CFR

2014-10-01

...) American Yacht and Boat Council, Inc. (AYBC): 3069 Solomon's Island Rd., Edgewater, MD 21037-1416, 410-990..., Research Triangle Park, NC 27709-3995, 919-549-1400, http://www.ul.com: (1) UL 19-1992—Lined Fire Hose and...

Understanding Cullin-RING E3 Biology through Proteomics-based Substrate Identification*

PubMed Central

Harper, J. Wade; Tan, Meng-Kwang Marcus

2012-01-01

Protein turnover through the ubiquitin-proteasome pathway controls numerous developmental decisions and biochemical processes in eukaryotes. Central to protein ubiquitylation are ubiquitin ligases, which provide specificity in targeted ubiquitylation. With more than 600 ubiquitin ligases encoded by the human genome, many of which remain to be studied, considerable effort is being placed on the development of methods for identifying substrates of specific ubiquitin ligases. In this review, we describe proteomic technologies for the identification of ubiquitin ligase targets, with a particular focus on members of the cullin-RING E3 class of ubiquitin ligases, which use F-box proteins as substrate specific adaptor proteins. Various proteomic methods are described and are compared with genetic approaches that are available. The continued development of such methods is likely to have a substantial impact on the ubiquitin-proteasome field. PMID:22962057

Understanding cullin-RING E3 biology through proteomics-based substrate identification.

PubMed

Harper, J Wade; Tan, Meng-Kwang Marcus

2012-12-01

Protein turnover through the ubiquitin-proteasome pathway controls numerous developmental decisions and biochemical processes in eukaryotes. Central to protein ubiquitylation are ubiquitin ligases, which provide specificity in targeted ubiquitylation. With more than 600 ubiquitin ligases encoded by the human genome, many of which remain to be studied, considerable effort is being placed on the development of methods for identifying substrates of specific ubiquitin ligases. In this review, we describe proteomic technologies for the identification of ubiquitin ligase targets, with a particular focus on members of the cullin-RING E3 class of ubiquitin ligases, which use F-box proteins as substrate specific adaptor proteins. Various proteomic methods are described and are compared with genetic approaches that are available. The continued development of such methods is likely to have a substantial impact on the ubiquitin-proteasome field.

RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

PubMed Central

Metzger, Meredith B.; Pruneda, Jonathan N.; Klevit, Rachel E.; Weissman, Allan M.

2013-01-01

RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. PMID:23747565

A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Jiazhang; Yu, Kaiwen; Fei, Xiaowen

Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attacked several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. Following ubiquitin activation by ADP- ribosylation via a mono-ADP-ribosylation motif, ADP-ribosylated ubiquitin is cleaved by a phosphodiesterasedomainwithinSdeA,whichisconcomitantwiththelinkof phosphoribosylated ubiquitin to serine residues in the substrate. Here we demonstrate that the activity of SidEs is regulated by SidJ, another effector encodedmore » by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ functions to remove ubiquitin from SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. Further, the deubiquitinase activity of SidJ is essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a deubiquitinase that functions to impose temporal regulation of the activity of the SidE effectors. The identification of SidJ may shed light on future study of signaling cascades mediated by this unique ubiquitination that also potentially regulates cellular processes in eukaryotic cells.« less

Dual Function of Phosphoubiquitin in E3 Activation of Parkin*

PubMed Central

Walinda, Erik; Morimoto, Daichi; Sugase, Kenji; Shirakawa, Masahiro

2016-01-01

Mutations in the gene encoding parkin, an auto-inhibited E3 ubiquitin ligase that functions in the clearance of damaged mitochondria, are the most common cause of autosomal recessive juvenile Parkinsonism. The mechanism regulating parkin activation remains poorly understood. Here we show, by using isothermal titration calorimetry, solution NMR, and fluorescence spectroscopy, that parkin can bind ubiquitin and phosphomimetic ubiquitin by recognizing the canonical hydrophobic patch and C terminus of ubiquitin. The affinity of parkin for both phosphomimetic and unmodified ubiquitin is markedly enhanced upon removal of the ubiquitin-like (UBL) domain of parkin. This suggests that the agonistic binding of ubiquitin to parkin in trans is counterbalanced by the antagonistic activity of the parkin UBL domain in cis. Intriguingly, UBL binding is enthalpy-driven, whereas ubiquitin binding is driven by an increase in the total entropy of the system. These thermodynamic differences are explained by different chemistry in the ubiquitin- and UBL-binding pockets of parkin and, as shown by molecular dynamics simulations, are not a consequence of changes in protein conformational entropy. Indeed, comparison of conformational fluctuations reveals that the RING1-IBR element becomes considerably more rigid upon complex formation. A model of parkin activation is proposed in which E2∼Ub binding triggers large scale diffusional motion of the RING2 domain toward the ubiquitin-stabilized RING1-IBR assembly to complete formation of the active parkin-E2∼Ub transfer complex. Thus, ubiquitin plays a dual role in parkin activation by competing with the inhibitory UBL domain and stabilizing the active form of parkin. PMID:27284007

Surgical relocation of the papillary muscles in functional ischemic mitral regurgitation: what are the forces of the relocation stitches acting on the myocardium?

PubMed

Jensen, Henrik; Jensen, Morten O; Vind-Kezunovic, Stefan; Vestergaard, Rikke; Ringgaard, Steffen; Smerup, Morten H; Hønge, Jesper L; Hasenkam, J Michael; Nielsen, Sten L

2013-07-01

In patients with chronic functional ischemic mitral regurgitation (FIMR), papillary muscle relocation has the potential to induce reverse left ventricular remodeling. However, in order to optimize function and durability, the forces imposed on the left ventricular myocardium by papillary muscle relocation should be assessed. Eight pigs with FIMR were subjected to down-sized ring annuloplasty in combination with relocation of the anterior (5 mm) and posterior (15 mm) papillary muscles towards the respective trigone. Papillary muscle relocation was obtained by a 2-0 expanded polytetrafluoroethylene stitch fixed to the trigone, exteriorized through the myocardium overlying the papillary muscle, and fixed to an epicardial disc. Tension in these stitches was measured at a systolic blood pressure > 80 mmHg using a custom-made sliding caliper with a strain gauge mounted in line. This allowed assessment of the cyclic change from minimal diastolic to maximum systolic papillary muscle relocation stitch tension. Maximum cyclic change in the posterior papillary muscle (PPM) stitch tension was 1.1 N at 15 mm relocation. In comparison, the anterior papillary muscle (APM) tension was increased to a maximum of 1.4 N with only 5 mm relocation. Surprisingly, during each step of isolated PPM relocation, the APM stitch tension increased concomitantly, but in contrast APM relocation did not influence the magnitude of PPM stitch tension. There was no statistically significant difference between cyclic changes in APM and PPM stitch tension at any step of relocation. Papillary muscle relocation using stitches attached between epicardial discs and respective trigones induced a cyclic change in papillary muscle relocation stitch tension of 1.1-1.4 N. These values were in the range of normal tension in the mitral valve apparatus, and equivalent to only 19-24% of the total papillary muscle forces. Therefore, this technique does not appear to induce a non-physiologically high cyclic load on the mitral valve complex.

Transjugular intrahepatic portosystemic shunt for severe jaundice in patients with acute Budd-Chiari syndrome.

PubMed

He, Fu-Liang; Wang, Lei; Zhao, Hong-Wei; Fan, Zhen-Hua; Zhao, Meng-Fei; Dai, Shan; Yue, Zhen-Dong; Liu, Fu-Quan

2015-02-28

To evaluate the feasibility of transjugular intrahepatic portosystemic shunt (TIPS) for severe jaundice secondary to acute Budd-Chiari syndrome (BCS). From February 2009 to March 2013, 37 patients with severe jaundice secondary to acute BCS were treated. Sixteen patients without hepatic venule, hepatic veins (HV) obstruction underwent percutaneous angioplasty of the inferior vena cava (IVC) and/or HVs. Twenty-one patients with HV occlusion underwent TIPS. Serum bilirubin, liver function, demographic data and operative data of the two groups of patients were analyzed. Twenty-one patients underwent TIPS and the technical success rate was 100%, with no technical complications. Sixteen patients underwent recanalization of the IVC and/or HVs and the technical success rate was 100%. The mean procedure time for TIPS was 84.0±12.11 min and angioplasty was 44.11±5.12 min (P<0.01). The mean portosystemic pressure in the TIPS group decreased significantly from 40.50±4.32 to 16.05±3.50 mmHg (P<0.01). The mean portosystemic pressure gradient decreased significantly from 33.60±2.62 to 7.30±2.21 mmHg (P<0.01). At 8 wk after the procedures, in the TIPS group, total bilirubin (TBIL) decreased significantly from 266.24±122.03 before surgery to 40.11±3.52 μmol/L (P<0.01) and direct bilirubin (DBIL) decreased significantly from 194.22±69.82 μmol/L to 29.82±3.10 μmol/L (P<0.01). In the angioplasty group, bilirubin returned to the normal range, with TBIL decreased significantly from 258.22±72.71 μmol/L to 13.33±3.54 μmol/L (P<0.01) and DBIL from 175.08±39.27 to 4.03±1.74 μmol/L (P<0.01). Liver function improved faster than TBIL. After 2 wk, in the TIPS group, alanine aminotransferase (ALT) decreased significantly from 50.33±40.61 U/L to 28.67±7.02 U/L (P<0.01) and aspartate aminotransferase (AST) from 49.46±34.33 U/L to 26.89±8.68 U/L (P<0.01). In the angioplasty group, ALT decreased significantly from 51.56±27.90 to 14.22±2.59 μmol/L (P<0.01) and AST from 60.66±39.89 μmol/L to 8.18±1.89 μmol/L (P<0.01). After mean follow-up of 12.6 mo, there was no recurrence of jaundice in either group. Severe jaundice is not a contraindication for TIPS in patients with acute BCS and TIPS is appropriate for severe jaundice due to BCS.

Phosphate Salts

MedlinePlus

... UNSAFE when taken in doses higher than 4 grams per day for adults younger than 70 years of age and 3 grams per day for people who are older. Regular ... upper intake level (UL). The ULs are 3 grams per day for children 1-8 years; and ...

Epidemiology of Uterine Fibroids – From Menarche to Menopause

PubMed Central

Wise, Lauren A.; Laughlin-Tommaso, Shannon K.

2015-01-01

Uterine leiomyomata (UL) have a substantial impact on women's health, but relatively few studies have identified opportunities for primary prevention of these neoplasms. Most established risk factors are not modifiable, including premenopausal age, African ancestry, age at menarche, and childbearing history. The main challenge in studying UL is that a large proportion of tumors are asymptomatic. Herein, we review the epidemiology of UL from published studies to date. We highlight the advantages of ultrasound screening studies and the ways in which their innovative methods have helped clarify the etiology of disease. We conclude with a discussion of promising new hypotheses. PMID:26744813

Sodium Content of Lunches and Snacks Provided in Australian Long Day Care Centres: A Cross-Sectional Study

PubMed Central

Campbell, Karen J.

2018-01-01

We determined the average amount of sodium provided in lunches and snacks and the average amount of sodium consumed at lunch in a convenience sample of Australian preschool children attending Long Day Care (LDC). Sodium content of lunches and snacks was determined from standardised recipes. Individual children’s sodium intake was estimated by a validated visual plate waste scale method. Five recipes (lunch n = 35, snacks n = 70) collected from 7 LDC centres; 95 children (50 boys) mean age 3.5 (SD) (0.2) years lunch intakes were assessed. Average total amount of sodium provided from two snacks and one lunch: 590 (146) mg, representing ~59% of the Australian Upper Level (UL) of intake (1000 mg/day sodium). Average total amount of sodium consumed: 541 (98) mg representing ~54% of the UL. Across all centres, the average sodium and energy consumed from lunch: 186 (108) mg (~19% of UL); 948 (437) kJ (38% of energy allowance); morning snacks: 63 (45) mg (6% of UL), 535 (183) kJ (21% of energy allowance); afternoon snacks: 291 (97) mg (29% of UL), 464 (171) kJ energy (46% of energy allowance). Australian LDC centres providing lunches cooked on site resulted in relatively low-sodium lunches. PMID:29495628

47 CFR 24.245 - Reimbursement under the Cost-Sharing Plan.

Code of Federal Regulations, 2013 CFR

2013-10-01

... SERVICES PERSONAL COMMUNICATIONS SERVICES Broadband PCS Policies Governing Microwave Relocation from the... incumbent. (2) To obtain reimbursement, a voluntarily relocating microwave incumbent must submit... PCS relocator or the voluntarily relocating microwave incumbent, must submit documentation itemizing...

47 CFR 24.245 - Reimbursement under the Cost-Sharing Plan.

Code of Federal Regulations, 2011 CFR

2011-10-01

... SERVICES PERSONAL COMMUNICATIONS SERVICES Broadband PCS Policies Governing Microwave Relocation from the... incumbent. (2) To obtain reimbursement, a voluntarily relocating microwave incumbent must submit... PCS relocator or the voluntarily relocating microwave incumbent, must submit documentation itemizing...

47 CFR 24.245 - Reimbursement under the Cost-Sharing Plan.

Code of Federal Regulations, 2014 CFR

2014-10-01

... SERVICES PERSONAL COMMUNICATIONS SERVICES Broadband PCS Policies Governing Microwave Relocation from the... incumbent. (2) To obtain reimbursement, a voluntarily relocating microwave incumbent must submit... PCS relocator or the voluntarily relocating microwave incumbent, must submit documentation itemizing...

47 CFR 24.245 - Reimbursement under the Cost-Sharing Plan.

Code of Federal Regulations, 2010 CFR

2010-10-01

... SERVICES PERSONAL COMMUNICATIONS SERVICES Broadband PCS Policies Governing Microwave Relocation from the... incumbent. (2) To obtain reimbursement, a voluntarily relocating microwave incumbent must submit... PCS relocator or the voluntarily relocating microwave incumbent, must submit documentation itemizing...

Targeting ubiquitination for cancer therapies.

PubMed

Morrow, John Kenneth; Lin, Hui-Kuan; Sun, Shao-Cong; Zhang, Shuxing

2015-01-01

Ubiquitination, the structured degradation and turnover of cellular proteins, is regulated by the ubiquitin-proteasome system (UPS). Most proteins that are critical for cellular regulations and functions are targets of the process. Ubiquitination is comprised of a sequence of three enzymatic steps, and aberrations in the pathway can lead to tumor development and progression as observed in many cancer types. Recent evidence indicates that targeting the UPS is effective for certain cancer treatment, but many more potential targets might have been previously overlooked. In this review, we will discuss the current state of small molecules that target various elements of ubiquitination. Special attention will be given to novel inhibitors of E3 ubiquitin ligases, especially those in the SCF family.

Specificity and disease in the ubiquitin system

PubMed Central

Chaugule, Viduth K.; Walden, Helen

2016-01-01

Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation. PMID:26862208

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin.

PubMed

Wang, Chien-Hsiang; Huang, Yi-Chun; Chen, Pei-Yi; Cheng, Ying-Ju; Kao, Hsiu-Hua; Pi, Haiwei; Chien, Cheng-Ting

2017-05-10

Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth.

Hormone signaling through protein destruction: a lesson from plants.

PubMed

Tan, Xu; Zheng, Ning

2009-02-01

Ubiquitin-dependent protein degradation has emerged as a major pathway regulating eukaryotic biology. By employing a variety of ubiquitin ligases to target specific cellular proteins, the ubiquitin-proteasome system controls physiological processes in a highly regulated fashion. Recent studies on a plant hormone auxin have unveiled a novel paradigm of signal transduction in which ubiquitin ligases function as hormone receptors. Perceived by the F-box protein subunit of the SCF(TIR1) ubiquitin ligase, auxin directly promotes the recruitment of a family of transcriptional repressors for ubiquitination, thereby activating extensive transcriptional programs. Structural studies have revealed that auxin functions through a "molecular glue" mechanism to enhance protein-protein interactions with the assistance of another small molecule cofactor, inositol hexakisphosphate. Given the extensive repertoire of similar ubiquitin ligases in eukaryotic cells, this novel and widely adopted hormone-signaling mechanism in plants may also exist in other organisms.

Molecular characterization and functional analysis of ubiquitin extension genes from the potato cyst nematode Globodera rostochiensis

USDA-ARS?s Scientific Manuscript database

Ubiquitin is a highly conserved 76-amino acid protein found in every eukaryotic cell. It has been proposed that ubiquitin has many cellular functions including DNA repair, transcription regulation, regulation of cell cycle and apoptosis. We identified two ubiquitin extension genes (Gr-Ubi1 and Gr-Ub...

The ubiquitin–proteasome system regulates membrane fusion of yeast vacuoles

PubMed Central

Kleijnen, Maurits F; Kirkpatrick, Donald S; Gygi, Steven P

2007-01-01

Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin–proteasome regulation may be involved in membrane fusion elsewhere. PMID:17183369

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

PubMed Central

Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

2011-01-01

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

Lysine ubiquitination and acetylation of human cardiac 20S proteasomes.

PubMed

Zong, Nobel; Ping, Peipei; Lau, Edward; Choi, Howard Jh; Ng, Dominic Cm; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie Py

2014-08-01

Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets polyubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. This is the most comprehensive characterization of cardiac proteasome ubiquitination to date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2

PubMed Central

Xie, Xingqiao; Li, Faxiang; Wang, Yuanyuan; Wang, Yingli; Lin, Zhijie; Cheng, Xiaofang; Liu, Jianping; Chen, Changbin; Pan, Lifeng

2015-01-01

The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C2H2-type zinc-finger, and only the C2H2-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C2H2-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations. PMID:26506893

Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

PubMed Central

Yoshida, Yukiko; Yasuda, Sayaka; Fujita, Toshiharu; Hamasaki, Maho; Murakami, Arisa; Kawawaki, Junko; Iwai, Kazuhiro; Saeki, Yasushi; Yoshimori, Tamotsu; Matsuda, Noriyuki; Tanaka, Keiji

2017-01-01

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy. PMID:28743755

The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

PubMed

Miao, Min; Niu, Xiangli; Kud, Joanna; Du, Xinran; Avila, Julian; Devarenne, Timothy P; Kuhl, Joseph C; Liu, Yongsheng; Xiao, Fangming

2016-07-01

We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways

PubMed Central

Seigneurin-Berny, Daphné; Verdel, André; Curtet, Sandrine; Lemercier, Claudie; Garin, Jérôme; Rousseaux, Sophie; Khochbin, Saadi

2001-01-01

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination. PMID:11689694

Crystal Structures of Lys-63-linked tri- and di-ubiquitin Reveal a Highly Extended Chain Architecture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weeks, S.; Grasty, K; Hernandez-Cuebas, L

2009-01-01

The covalent attachment of different types of poly-ubiquitin chains signal different outcomes for the proteins so targeted. For example, a protein modified with Lys-48-linked poly-ubiquitin chains is targeted for proteasomal degradation, whereas Lys-63-linked chains encode nondegradative signals. The structural features that enable these different types of chains to encode different signals have not yet been fully elucidated. We report here the X-ray crystal structures of Lys-63-linked tri- and di-ubiquitin at resolutions of 2.3 and 1.9 {angstrom}, respectively. The tri- and di-ubiquitin species adopt essentially identical structures. In both instances, the ubiquitin chain assumes a highly extended conformation with a left-handedmore » helical twist; the helical chain contains four ubiquitin monomers per turn and has a repeat length of {approx}110 {angstrom}. Interestingly, Lys-48 ubiquitin chains also adopt a left-handed helical structure with a similar repeat length. However, the Lys-63 architecture is much more open than that of Lys-48 chains and exposes much more of the ubiquitin surface for potential recognition events. These new crystal structures are consistent with the results of solution studies of Lys-63 chain conformation, and reveal the structural basis for differential recognition of Lys-63 versus Lys-48 chains.« less

Linear ubiquitin chains: enzymes, mechanisms and biology

PubMed Central

2017-01-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. PMID:28446710

Linear ubiquitin chains: enzymes, mechanisms and biology.

PubMed

Rittinger, Katrin; Ikeda, Fumiyo

2017-04-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. © 2017 The Authors.

Resourcefulness, positive cognitions, relocation controllability and relocation adjustment among older people: a cross-sectional study of cultural differences.

PubMed

Bekhet, Abir K; Zauszniewski, Jaclene A

2013-09-01

The population of older people in both the United States and Egypt is expected to double by the year 2030. With ageing, chronic illnesses increase and many older people need to relocate to retirement communities. Research has shown that positive cognitions and resourcefulness are positively correlated with adaptive functioning and better adjustment. The purpose of this study was to examine and compare relocation controllability, positive cognitions, resourcefulness and relocation adjustment between American and Egyptian older people living in retirement communities. The purpose of this cultural comparison is to gain insight into influencing factors in each culture that might lead to interventions to help relocated older adults in both cultures adjust to their new surroundings. A cross-sectional, descriptive design was used to compare relocation controllability, positive cognitions, resourcefulness and relocation adjustment of a convenience sample of American older people (n = 104) and a convenience sample of Egyptian older people (n = 94). The study was a secondary analysis of two studies of older people residing in six retirement communities in Northeast Ohio and in five retirement communities in Alexandria, Egypt. Examination of mean scores and standard deviations on the measure of positive cognitions using independent sample t-tests indicated that on average, the American older people reported more positive cognitions (t (131.16) = 11.29, P < 0.001), more relocation controllability (t (196) = -6.78, P < 0.001) and more relocation adjustment (t (196) = 9.42, P < 0.001) than the Egyptian older people. However, there was no significant difference between Egyptians and Americans in resourcefulness (t (174.16) = -0.97, P > 0.05). The results provide direction for the development of positive cognition interventions and engaging older people in the decision-making process to help them to adjust to relocation. Implications for practice.  Positive thinking and resourcefulness training interventions can be used by nurses to help relocated older people to adjust to the stress of relocation to retirement communities. These interventions can be used on primary, secondary, and tertiary levels. Primary interventions can help to prevent the stress of relocation before happening by helping older people to use their positive thinking and their resources and work with them before relocating to retirement communities. Secondary prevention can be used by nurses to help older people who have already relocated to retirement communities and have already experienced stress of relocation to help them out by decreasing the stress that they are suffering. Tertiary prevention can be used to prevent further stress and deterioration for those who have suffered physical and psychological symptoms as a result of relocation. © 2012 Blackwell Publishing Ltd.

Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma.

PubMed

Chen, Xiangyun; Wu, Jingjing; Chen, Yitian; Ye, Dongxia; Lei, Hu; Xu, Hanzhang; Yang, Li; Wu, Yingli; Gu, Wenli

2016-10-01

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage*

PubMed Central

Baiady, Nardeen; Padala, Prasanth; Mashahreh, Bayan; Cohen-Kfir, Einav; Todd, Emily A.; Du Pont, Kelly E.; Berndsen, Christopher E.; Wiener, Reuven

2016-01-01

The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the kcat of Lys63-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the Km value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys63-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys63-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage. PMID:26601948

PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

PubMed Central

Barrera, Susana P.; Castrejon-Tellez, Vicente; Trinidad, Margarita; Robles-Escajeda, Elisa; Vargas-Medrano, Javier; Varela-Ramirez, Armando; Miranda, Manuel

2015-01-01

Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications. PMID:26418248

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism

DOE PAGES

VanderLinden, Ryan T.; Hemmis, Casey W.; Yao, Tingting; ...

2017-04-25

This work presents that the 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to themore » C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. In conclusion, these findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics.« less

Dual Function of Phosphoubiquitin in E3 Activation of Parkin.

PubMed

Walinda, Erik; Morimoto, Daichi; Sugase, Kenji; Shirakawa, Masahiro

2016-08-05

Mutations in the gene encoding parkin, an auto-inhibited E3 ubiquitin ligase that functions in the clearance of damaged mitochondria, are the most common cause of autosomal recessive juvenile Parkinsonism. The mechanism regulating parkin activation remains poorly understood. Here we show, by using isothermal titration calorimetry, solution NMR, and fluorescence spectroscopy, that parkin can bind ubiquitin and phosphomimetic ubiquitin by recognizing the canonical hydrophobic patch and C terminus of ubiquitin. The affinity of parkin for both phosphomimetic and unmodified ubiquitin is markedly enhanced upon removal of the ubiquitin-like (UBL) domain of parkin. This suggests that the agonistic binding of ubiquitin to parkin in trans is counterbalanced by the antagonistic activity of the parkin UBL domain in cis Intriguingly, UBL binding is enthalpy-driven, whereas ubiquitin binding is driven by an increase in the total entropy of the system. These thermodynamic differences are explained by different chemistry in the ubiquitin- and UBL-binding pockets of parkin and, as shown by molecular dynamics simulations, are not a consequence of changes in protein conformational entropy. Indeed, comparison of conformational fluctuations reveals that the RING1-IBR element becomes considerably more rigid upon complex formation. A model of parkin activation is proposed in which E2∼Ub binding triggers large scale diffusional motion of the RING2 domain toward the ubiquitin-stabilized RING1-IBR assembly to complete formation of the active parkin-E2∼Ub transfer complex. Thus, ubiquitin plays a dual role in parkin activation by competing with the inhibitory UBL domain and stabilizing the active form of parkin. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism.

PubMed

VanderLinden, Ryan T; Hemmis, Casey W; Yao, Tingting; Robinson, Howard; Hill, Christopher P

2017-06-09

The 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to the C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. These findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration.

PubMed

Del Prete, Dolores; Rice, Richard C; Rajadhyaksha, Anjali M; D'Adamio, Luciano

2016-08-12

The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of exogenous ubiquitin in a polytrauma model with blunt chest trauma

PubMed Central

Baker, Todd A.; Romero, Jacqueline; Bach, Harold H.; Strom, Joel A.; Gamelli, Richard L.; Majetschak, Matthias

2013-01-01

Objective To determine whether treatment with the CXC chemokine receptor (CXCR) 4 agonist ubiquitin results in beneficial effects in a polytrauma model consisting of bilateral femur fractures plus blunt chest trauma (Injury Severity Score 18-25). Design Treatment study. Setting Research Laboratory. Subjects Seventeen Yorkshire pigs. Interventions Intravenous (i.v.) injection of 1.5 mg/kg ubiquitin or albumin (=control) at 60 min after polytrauma. Measurements and Main Results Anesthetized, mechanically ventilated pigs underwent polytrauma, followed by a simulated 60 min shock phase. At the end of the shock phase ubiquitin or albumin were administered and animals were resuscitated to a mean arterial blood pressure of 70 mmHg until t = 420 min. After i.v. ubiquitin, ubiquitin plasma concentrations increased sixteen-fold to 2870 ± 1015 ng/mL at t = 90 min and decreased with t1/2 = 60 min. Endogenous plasma ubiquitin increased two-fold in the albumin group with peak levels of 359 ± 210 ng/mL. Plasma levels of the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α were unchanged in both groups. Ubiquitin treatment reduced arterial lactate levels and prevented a continuous decrease in arterial oxygenation, which occurred in the albumin group during resuscitation. Wet weight to dry weight ratios of the lung contralateral from the injury, heart, spleen and jejunum were lower with ubiquitin. With ubiquitin treatment, tissue levels of IL-8, IL-10, TNFα and SDF-1α were reduced in the injured lung and of IL-8 in the contralateral lung, respectively. Conclusions Administration of exogenous ubiquitin modulates the local inflammatory response, improves resuscitation, reduces fluid shifts into tissues and preserves arterial oxygenation after blunt polytrauma with lung injury. This study further supports the notion that ubiquitin is a promising protein therapeutic and implies CXCR4 as a drug target after polytrauma. PMID:22622399