The Greek version of the Gagging Assessment Scale in children and adolescents: psychometric properties, prevalence of gagging, and the association between gagging and dental fear.

PubMed

Katsouda, Maria; Provatenou, Efthymia; Arapostathis, Konstantinos; Coolidge, Trilby; Kotsanos, Nikolaos

2017-03-01

No studies assessing the association between gagging and dental fear are available in pediatric samples. To assess the psychometric properties of the Greek version of the Gagging Assessment Scale (GAS), to explore the prevalence of gagging, and to evaluate the relationship between gagging and dental fear in a pediatric sample. A total of 849 8- and 14-year-old children filled out a questionnaire consisting of demographic items, the Greek version of the GAS, and the Greek Children's Fear Survey Schedule Dental Subscale (CFSS-DS); the older children also completed the Greek version of the Modified Dental Anxiety Scale (MDAS). The short form of dentist part of the Gagging Problem Assessment (GPA-de-c/SF) was used to objectively assess gagging. A total of 51 children (6.0%) demonstrated gagging on the GPA-de-c/SF. Children rated as gaggers on the GPA-de-c/SF had significantly higher GAS scores. There were no relationships between GPA-de-c/SF and the CFSS-DS or MDAS. The GAS ratings were significantly correlated with the CFSS-DS (rho = 0.420, P < 0.001) and MDAS (rho = 0.429, P < 0.001). The internal consistency was good (Cronbach's alpha = 0.697). The GAS demonstrated good psychometric properties. Dental fear was correlated with the self-report gagging assessment, but not with the objective gagging assessment. © 2016 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multiple lentigines, myxoid tumours and endocrine overactivity; four cases of Carney's complex.

PubMed

Handley, J; Carson, D; Sloan, J; Walsh, M; Thornton, C; Hadden, D; Bingham, E A

1992-04-01

Four patients with Carney's complex, one sporadic and three familial, are described. The sporadic case was a young male with centrofacial lentigines, who developed cyclical Cushing's syndrome secondary to bilateral pigmented nodular adrenocortical disease, two separate left atrial myxomas, and buccal mucosal myxomas. The three familial cases, who all had varying degrees of centrofacial/mucosal lentigines and cutaneous myxoid tumours, were a woman with myxoid mammary fibroadenomatosis and a left atrial myxoma, her daughter who developed a prolactin-secreting pituitary adenoma, and her son who had bilateral large-cell calcified Sertoli cell testicular tumours, and an axillary psammomatous melanotic schwannoma.

HIV-1 maturation inhibitor bevirimat stabilizes the immature Gag lattice.

PubMed

Keller, Paul W; Adamson, Catherine S; Heymann, J Bernard; Freed, Eric O; Steven, Alasdair C

2011-02-01

Maturation of nascent virions, a key step in retroviral replication, involves cleavage of the Gag polyprotein by the viral protease into its matrix (MA), capsid (CA), and nucleocapsid (NC) components and their subsequent reorganization. Bevirimat (BVM) defines a new class of antiviral drugs termed maturation inhibitors. BVM acts by blocking the final cleavage event in Gag processing, the separation of CA from its C-terminal spacer peptide 1 (SP1). Prior evidence suggests that BVM binds to Gag assembled in immature virions, preventing the protease from accessing the CA-SP1 cleavage site. To investigate this hypothesis, we used cryo-electron tomography to examine the structures of (noninfectious) HIV-1 viral particles isolated from BVM-treated cells. We find that these particles contain an incomplete shell of density underlying the viral envelope, with a hexagonal honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost, NC-related, layer. We conclude that the shell represents a remnant of the immature Gag lattice that has been processed, except at the CA-SP1 sites, but has remained largely intact. We also compared BVM-treated particles with virions formed by the mutant CA5, in which cleavage between CA and SP1 is also blocked. Here, we find a thinner CA-related shell with no visible evidence of honeycomb organization, indicative of an altered conformation and further suggesting that binding of BVM stabilizes the immature lattice. In both cases, the observed failure to assemble mature capsids correlates with the loss of infectivity.

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

PubMed Central

Laprevotte, I; Hampe, A; Sherr, C J; Galibert, F

1984-01-01

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing. PMID:6328019

Critical Role of the HTLV-1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly.

PubMed

Martin, Jessica L; Mendonça, Luiza; Marusinec, Rachel; Zuczek, Jennifer; Angert, Isaac; Blower, Ruth J; Mueller, Joachim D; Perilla, Juan R; Zhang, Wei; Mansky, Louis M

2018-04-25

The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a β-hairpin turn and a centralized coiled-coil-like structure of six α helices in the CA amino-terminal domain (NTD) as well as four α-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regions and amino acids at the beginning and ends of α-helices due to their structural dissimilarity from the HIV-1 CA NTD structure. We analyzed both Gag subcellular distribution and efficiency of particle production for these mutants. We discovered several important residues (i.e., M17, Q47/F48, and Y61). Modeling implicated that these residues reside at the dimer interface (i.e., M17 and Y61) or at the trimer interface (i.e., Q47/F48). Taken together, these observations highlight the critical role of the HTLV-1 CA NTD in Gag-Gag interactions and particle assembly, which is, to the best of our knowledge, in contrast to HIV-1 and other retroviruses. Importance Retrovirus particle assembly and release from infected cells is driven by the Gag structural protein. Gag-Gag interactions, which form an oligomeric lattice structure at a particle budding site, are essential to the biogenesis of an infectious virus particle. The capsid (CA) domain of Gag is generally thought to possess the key

NC-Mediated Nucleolar Localization of Retroviral Gag Proteins

PubMed Central

Lochmann, Timothy L.; Bann, Darrin V.; Ryan, Eileen P.; Beyer, Andrea R.; Mao, Annie; Cochrane, Alan

2012-01-01

The assembly and release of retrovirus particles from the cell membrane is directed by the Gag polyprotein. The Gag protein of Rous sarcoma virus (RSV) traffics through the nucleus prior to plasma membrane localization. We previously reported that nuclear localization of RSV Gag is linked to efficient packaging of viral genomic RNA, however the intranuclear activities of RSV Gag are not well understood. To gain insight into the properties of the RSV Gag protein within the nucleus, we examined the subnuclear localization and dynamic trafficking of RSV Gag. Restriction of RSV Gag to the nucleus by mutating its nuclear export signal (NES) in the p10 domain or interfering with CRM1-mediated nuclear export of Gag by leptomycin B (LMB) treatment led to the accumulation of Gag in nucleoli and discrete nucleoplasmic foci. Retention of RSV Gag in nucleoli was reduced with cis-expression of the 5′ untranslated RU5 region of the viral RNA genome, suggesting the psi (ψ packaging signal may alter the subnuclear localization of Gag. Fluorescence recovery after photobleaching (FRAP) demonstrated that the nucleolar fraction of Gag was highly mobile, indicating that the there was rapid exchange with Gag proteins in the nucleoplasm. RSV Gag is targeted to nucleoli by a nucleolar localization signal (NoLS) in the NC domain, and similarly, the human immunodeficiency virus type 1 (HIV-1) NC protein also contains an NoLS consisting of basic residues. Interestingly, co-expression of HIV-1 NC or Rev with HIV-1 Gag resulted in accumulation of Gag in nucleoli. Moreover, a subpopulation of HIV-1 Gag was detected in the nucleoli of HeLa cells stably expressing the entire HIV-1 genome in a Rev-dependent fashion. These findings suggest that the RSV and HIV-1 Gag proteins undergo nucleolar trafficking in the setting of viral infection. PMID:23036987

Diagnosis of extraskeletal myxoid chondrosarcoma in the thigh using EWSR1-NR4A3 gene fusion: a case report.

PubMed

Kobayashi, Hiroki; Kikuta, Kazutaka; Sekita, Tetsuya; Susa, Michiro; Nishimoto, Kazumasa; Sasaki, Aya; Kameyama, Kaori; Sugita, Shintaro; Hasegawa, Tadashi; Nakamura, Masaya; Matsumoto, Morio; Morioka, Hideo

2016-11-10

Extraskeletal myxoid chondrosarcoma is a rare soft tissue sarcoma that has unusual ultrastructural and molecular features. However, unlike other soft tissue sarcomas, it does not have specific clinical symptoms or radiological features, which can make its diagnosis difficult. Nevertheless, extraskeletal myxoid chondrosarcoma has a rare gene fusion (EWSR1-NR4A3) that is useful for making a differential diagnosis. A 43-year-old Japanese man presented with a soft tissue mass in his right thigh. A physical examination and radiography revealed a large soft tissue mass. During magnetic resonance imaging, the mass exhibited isointensity on T1-weighted images and high intensity on T2-weighted images, as well as gadolinium enhancement at the side edge of the partition structure. Thus, we considered a possible diagnosis of a malignant myxoid soft tissue tumor, such as myxoid liposarcoma, myxofibrosarcoma, or metastatic carcinomas, including myoepithelial tumor and neuroendocrine tumor, and performed an incisional biopsy to make a definitive diagnosis. The pathological findings revealed a lobulated tumor with a myxoid structure and atypical spindle-shaped cells that created eosinophilic cord-like forms. Immunohistochemistry revealed that the tumor was positive for S-100 and negative for synaptophysin, chromogranin A, and pan keratin (AE1/AE3). The percentage of Ki-67 was 10 % in the hot spot area. Based on these clinicopathological findings, we initially considered the possibility of a myxoid liposarcoma, although we did not observe any lipoblasts. Therefore, we considered the possibility of an extraskeletal myxoid chondrosarcoma. As this tumor is very rare, we searched for the EWSR1-NR4A3 gene fusion using fluorescence in situ hybridization, which confirmed the diagnosis of extraskeletal myxoid chondrosarcoma. Positron emission tomography-computed tomography did not identify any obvious metastases, and we performed radical resection of our patient's vastus medialis and

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

PubMed Central

2012-01-01

Background Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1. Results A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named AnkGAG1D4 (16.5 kDa) was isolated. AnkGAG1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of Kd ~ 1 μM, and the AnkGAG1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing AnkGAG1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. AnkGAG1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The AnkGAG1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of AnkGAG1D4-CA with the Gag assembly and budding pathway. Conclusions The resistance of AnkGAG1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin AnkGAG1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules. PMID:22348230

Proteolytic Processing and Assembly of gag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family

PubMed Central

Hajek, Kathryn L.; Friesen, Paul D.

1998-01-01

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55gag) is cleaved to produce a single VLP structural protein, p37gag. Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55gag cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195gag-pol. The PR cleavage site within Pr55gag was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55gag truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55gag abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37gag provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging. PMID:9765414

Proteolytic processing and assembly of gag and gag-pol proteins of TED, a baculovirus-associated retrotransposon of the gypsy family.

PubMed

Hajek, K L; Friesen, P D

1998-11-01

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55(gag)) is cleaved to produce a single VLP structural protein, p37(gag). Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55(gag) cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195(gag-pol). The PR cleavage site within Pr55(gag) was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55(gag) truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55(gag) abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37(gag) provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.

Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production

PubMed Central

Wang, Margaret Q; Kim, Wankee; Gao, Guangxia; Torrey, Ted A; Morse, Herbert C; De Camilli, Pietro; Goff, Stephen P

2004-01-01

Background The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. Results A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. Conclusions This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production. PMID:14659004

Prognostic significance of MRI findings in patients with myxoid-round cell liposarcoma.

PubMed

Tateishi, Ukihide; Hasegawa, Tadashi; Beppu, Yasuo; Kawai, Akira; Satake, Mitsuo; Moriyama, Noriyuki

2004-03-01

The aims of this study were to determine the prognostic significance of MRI findings in patients with myxoid-round cell liposarcomas and to clarify which MRI features best indicate tumors with adverse clinical behavior. The initial MRI studies of 36 pathologically confirmed myxoid-round cell liposarcomas were retrospectively reviewed, and observations from this review were correlated with the histopathologic features. MR images were evaluated by two radiologists with agreement by consensus, and both univariate and multivariate analyses were conducted to evaluate survival with a median clinical follow-up of 33 months (range, 9-276 months). Statistically significant MRI findings that favored a diagnosis of intermediate- or high-grade tumor were large tumor size (> 10 cm), deeply situated tumor, tumor possessing irregular contours, absence of lobulation, absence of thin septa, presence of thick septa, absence of tumor capsule, high-intensity signal pattern, pronounced enhancement, and globular or nodular enhancement. Of these MRI findings, thin septa (p < 0.05), a tumor capsule (p < 0.01), and pronounced enhancement (p < 0.01) were associated significantly, according to univariate analysis, with overall survival. Multivariate analysis indicated that pronounced enhancement was associated significantly with overall survival (p < 0.05). Contrast-enhanced MRI findings can indicate a good or adverse prognosis in patients with myxoid-round cell liposarcomas.

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.

PubMed

Webb, Joseph A; Jones, Christopher P; Parent, Leslie J; Rouzina, Ioulia; Musier-Forsyth, Karin

2013-08-01

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

Measuring the binding stoichiometry of HIV-1 Gag to very-low-density oligonucleotide surfaces using surface plasmon resonance spectroscopy.

PubMed

Stephen, Andrew G; Datta, Siddhartha A K; Worthy, Karen M; Bindu, Lakshman; Fivash, Matthew J; Turner, Kevin B; Fabris, Daniele; Rein, Alan; Fisher, Robert J

2007-09-01

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.

A novel type of EWS-CHOP fusion gene in myxoid liposarcoma

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

Matsui, Yoshito; Ueda, Takafumi; Kubo, Takahiro

2006-09-22

The cytogenetic hallmark of myxoid type and round cell type liposarcoma consists of reciprocal translocation of t(12;16)(q13;p11) and t(12;22)(q13;q12), which results in fusion of TLS/FUS and CHOP, and EWS and CHOP, respectively. Nine structural variations of the TLS/FUS-CHOP chimeric transcript have been reported, however, only two types of EWS-CHOP have been described. We describe here a case of myxoid liposarcoma containing a novel EWS-CHOP chimeric transcript and identified the breakpoint occurring in intron 13 of EWS. Reverse transcription-polymerase chain reaction and direct sequence showed that exon 13 of EWS was in-frame fused to exon 2 of CHOP. Genomic analysis revealedmore » that the breaks were located in intron 13 of EWS and intron 1 of CHOP.« less

Age- and gender-related alteration in plasma advanced oxidation protein products (AOPP) and glycosaminoglycan (GAG) concentrations in physiological ageing.

PubMed

Komosinska-Vassev, Katarzyna; Olczyk, Pawel; Winsz-Szczotka, Katarzyna; Kuznik-Trocha, Kornelia; Klimek, Katarzyna; Olczyk, Krystyna

2012-02-13

The authors studied the role of increased oxidative stress in the development of oxidative protein damage and extracellular matrix (ECM) components in ageing. The age- and gender-associated disturbances in connective tissue metabolism were evaluated by the plasma chondroitin sulphated glycosaminoglycans (CS-GAG) and non-sulphated GAG-hyaluronan (HA) measurements. Plasma concentration of advanced oxidation protein products (AOPP) was analysed in order to assess oxidative protein damage and evaluate the possible deleterious role of oxidative phenomenon on tissue proteoglycans' metabolism during the physiological ageing process. Sulphated and non-sulphated GAGs as well as AOPP were quantified in plasma samples from 177 healthy volunteers. A linear age-related decline of plasma CS-GAG level was found in this study (r=-0.46; p<0.05). In contrast, HA concentrations rise gradually with age (r=0.44; p<0.05) in plasma samples. For both ECM components, the observed differences were not gender-specific. A strong age-dependent relationship has been shown in regard to AOPP. AOPP levels significantly increased with age (r=0.63; p<0.05), equally strongly in both men (r=0.69; p<0.05) and women (r=0.57; p<0.05) during physiological ageing. A significant correlation was found between the concentrations of AOPP and both CS-GAG (r=-0.31; p<0.05) and HA (r=0.33; p<0.05). Proceeding with age changes in the ECM are reflected by CS-GAG and HA plasma levels. Strong correlations between AOPP and ECM components indicate that oxidative stress targets protein and non-protein components of the connective tissue matrix during human ageing.

Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production

PubMed Central

Yu, Zheng; Beer, Christiane; Koester, Mario; Wirth, Manfred

2006-01-01

Background Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. Results The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other γ-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. Conclusion This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly. PMID:16956408

ESWR1-CREM Fusion in an Intracranial Myxoid Angiomatoid Fibrous Histiocytoma-Like Tumor: A Case Report and Literature Review.

PubMed

Gareton, Albane; Pierron, Gaëlle; Mokhtari, Karima; Tran, Suzanne; Tauziède-Espariat, Arnault; Pallud, Johan; Louvel, Guillaume; Meary, Eric; Capelle, Laurent; Chrétien, Fabrice; Varlet, Pascale

2018-05-19

Gene fusions of EWSR1 with members of the CREB family of transcription factors (CREB1, ATF1, and CREM) have recently been described in exceptional intracranial myxoid mesenchymal tumors. Although this is a known gene fusion found in various mesenchymal tumors, EWSR1 fusion with CREM has only been observed in 3 intracranial myxoid tumors. In this paper, we present 1 such tumor with in-depth histopathological description and long-term follow-up. There is controversy regarding whether these tumors represent a novel entity or simply an intracranial localization of the myxoid variant of angiomatoid fibrous histiocytoma, a rare soft tissue tumor of the extremities. Out of 11 cases mentioned in the literature, the 3 isolated case reports by Dunham et al, Ochalski et al, and Alshareef et al are designated as angiomatoid fibrous histiocytoma, whereas the others are defined as a novel tumoral entity called intracranial myxoid mesenchymal tumor with EWSR1-CREB fusion. We believe the vast morphological and immunohistochemical spectrum of angiomatoid fibrous histiocytoma makes it difficult to dismiss this diagnosis.

Cytogenetic evidence of metastatic myxoid liposarcoma and therapy-related myelodysplastic syndrome in a bone marrow biopsy.

PubMed

Rossi, Sabrina; Canal, Fabio; Licci, Stefano; Zanatta, Lucia; Laurino, Licia; Gottardi, Michele; Gherlinzoni, Filippo; Dei Tos, Angelo Paolo

2009-07-01

Myxoid liposarcoma exhibits a peculiar clinical behavior, with a tendency to spread to serosal membranes, distant soft tissues, and bones, even in the absence of lung metastases. Therapy-related hematological neoplasms are well-known side effects of cytotoxic chemotherapy. We describe an exceptional case of metastatic myxoid liposarcoma of the spine associated with therapy-related refractory anemia with excess of blasts in a 37-year-old woman who underwent multi-agent chemotherapy for a myxoid liposarcoma of the left thigh. Microscopic examination of the bone marrow biopsy revealed dysplastic features, with abnormal localization of immature precursors and micromegakaryocytes, and islands of undifferentiated oval small/medium-size cells, suggestive of acute myeloid leukemia arising in the setting of a myelodysplastic syndrome. Immunohistochemistry was not discriminant. Cytogenetic analyses of bone marrow aspirate disclosed the presence of 2 different rearrangements, subsequently confirmed by fluorescent in situ hybridization and was crucial in making the correct diagnosis.

An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability

PubMed Central

La Porte, Annalena; Cano, Jennifer; Wu, Xuhong; Mitra, Doyel

2016-01-01

ABSTRACT INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic in trans complementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-state gag RNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown of INI1 reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events. IMPORTANCE Significant gaps exist in our

"Pseudomyxoma Endometrii": Endometrial Deposition of Acellular Mucin from a Low-Grade Appendiceal Mucinous Neoplasm as a Rare Mimic of Myxoid Uterine Tumors.

PubMed

Shaw, Kristin C; Kokh, Dina; Ioffe, Olga B; Staats, Paul N

2015-07-01

Low-grade appendiceal mucinous neoplasms (LAMNs) are commonly associated with deposition of mucin, with or without admixed low-grade epithelium, on peritoneal surfaces (pseudomyxoma peritonei). We describe a very rare presentation of LAMN as extensive mucin deposition in the endometrium of a 43-yr-old woman initially mistaken for a primary uterine myxoid neoplasm. The patient underwent endometrial curettage that demonstrated abundant myxoid/mucoid material interspersed with small vessels, bland eosinophilic spindled cells, scattered foci of typical endometrial stroma, and occasional endometrioid glands. The endometrial stroma was positive for CD10, and the eosinophilic spindled cells were positive for actin. The lesion was interpreted as "myxoid/mucinous neoplasm, most likely of smooth muscle/endometrial stromal origin." Subsequent laparotomy revealed peritoneal mucin in the anterior cul-de-sac and a dilated appendix. Pathologic review confirmed appendiceal LAMN and multifocal peritoneal mucinosis. The uterus contained scant residual mucoid material. On review of all pathologic material at our institution, the endometrial lesion was consistent with organizing mucin derived from the LAMN with entrapped benign endometrium. "Pseudomyxoma endometrii" is readily mistaken for a primary uterine myxoid neoplasm, particularly myxoid endometrial stromal tumor. A key to diagnosis is recognition that the material is mucin rather than myxoid stroma. This is evidenced by the absence of embedded stromal cells and presence of myofibroblastic, vascular, and macrophage infiltration associated with organization. Epithelium containing goblet cells is an important clue if present. The presence of rare endometrial glands within the endometrial stroma suggests that the latter is entrapped rather than neoplastic.

Learning from Synthetic Models of Extracellular Matrix; Differential Binding of Wild Type and Amyloidogenic Human Apolipoprotein A-I to Hydrogels Formed from Molecules Having Charges Similar to Those Found in Natural GAGs.

PubMed

Rosú, Silvana A; Toledo, Leandro; Urbano, Bruno F; Sanchez, Susana A; Calabrese, Graciela C; Tricerri, M Alejandra

2017-08-01

Among other components of the extracellular matrix (ECM), glycoproteins and glycosaminoglycans (GAGs) have been strongly associated to the retention or misfolding of different proteins inducing the formation of deposits in amyloid diseases. The composition of these molecules is highly diverse and a key issue seems to be the equilibrium between physiological and pathological events. In order to have a model in which the composition of the matrix could be finely controlled, we designed and synthesized crosslinked hydrophilic polymers, the so-called hydrogels varying the amounts of negative charges and hydroxyl groups that are prevalent in GAGs. We checked and compared by fluorescence techniques the binding of human apolipoprotein A-I and a natural mutant involved in amyloidosis to the hydrogel scaffolds. Our results indicate that both proteins are highly retained as long as the negative charge increases, and in addition it was shown that the mutant is more retained than the Wt, indicating that the retention of specific proteins in the ECM could be part of the pathogenicity. These results show the importance of the use of these polymers as a model to get deep insight into the studies of proteins within macromolecules.

Incorporation of chimeric gag protein into retroviral particles.

PubMed Central

Weldon, R A; Erdie, C R; Oliver, M G; Wills, J W

1990-01-01

The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells. We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein. Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation. We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag. This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site. Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium. The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions. Images PMID:2166812

Remodelling of the pinna in myxoid degeneration of the ear.

PubMed

Kean, J; Stewart, K J

2010-07-01

Idiopathic deformation of the pinna is not widely reported in the current literature. We present a series of cases in which patients have required surgery for spontaneous thickening and deformation of the auricular cartilage, and a description of a technique for surgical correction. Four cases of idiopathic deformation of the pinna are reported. Our preferred technique of scaphoid rim incision and anterior carving of the cartilage is described, with intra-operative photographs. Each patient reported spontaneous swelling of the upper poles of the ears beginning in the second or third decade of life. In 3 cases the deformity was bilateral, although in each of these cases one side was more severely affected than the other. Histology for these cases was reported as myxoid degeneration of the ear. All of the reported patients were pleased with the aesthetic outcome of their auricular remodelling. Currently, there are no typical patient demographics for idiopathic myxoid degeneration of the ear. We have achieved good aesthetic results by hand carving the anterior aspect of the deformed cartilage via scaphoid rim incisions. Copyright 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Excellent Local Control Rates and Distinctive Patterns of Failure in Myxoid Liposarcoma Treated With Conservation Surgery and Radiotherapy

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

Guadagnolo, B. Ashleigh; Zagars, Gunar K.; Ballo, Matthew T.

2008-03-01

Purpose: To evaluate the local control rates and patterns of metastatic relapse in patients with localized myxoid liposarcoma treated with conservation surgery and radiotherapy (RT). Patients and Methods: Between 1960 and 2003, 127 patients with non-metastatic myxoid liposarcoma were treated with conservation surgery and RT at our institution. The median patient age was 39 years (range, 14-79 years). Of the 127 patients, 46% underwent preoperative RT (median dose, 50 Gy) and 54% underwent postoperative RT (median dose, 60 Gy). Also, 28% received doxorubicin-based chemotherapy as a part of their treatment. Results: The median follow-up was 9.1 years. The overall survivalmore » rate at 5 and 10 years was 87% and 79%, respectively. The corresponding disease-free survival rates were 81% and 73%. The local control rate at {>=}5 years was 97%. The actuarial rate of distant metastases at 5 and 10 years was 15% and 24%, respectively. Of the 27 patients who developed distant metastases, 48% did so in the retroperitoneum, 22% in other extrapulmonary soft tissues, 22% in the lung, 15% in bone, and 4% in the liver. Conclusion: The results of our study have shown that RT and conservation surgery for localized myxoid liposarcoma provide excellent local control. Distant metastatic relapse tended to occur in the retroperitoneum and other nonpulmonary soft tissues. Therefore, staging and surveillance imaging should include the abdomen and pelvis, as well as the thorax, for patients with localized myxoid liposarcoma.« less

In vitro analysis of human immunodeficiency virus particle dissociation: gag proteolytic processing influences dissociation kinetics.

PubMed

Müller, Barbara; Anders, Maria; Reinstein, Jochen

2014-01-01

Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice. In order to measure the influence of individual Gag domains on lattice stability, we established Förster's resonance energy transfer (FRET) reporter virions and employed rapid kinetic FRET and light scatter measurements. This approach allowed us to measure dissociation properties of HIV-1 particles assembled in eukaryotic cells containing Gag proteins in different states of proteolytic processing. While the complex dissociation behavior of the particles prevented an assignment of kinetic rate constants to individual dissociation steps, our analyses revealed characteristic differences in the dissociation properties of the MA layer dependent on the presence of additional domains. The most striking effect observed here was a pronounced stabilization of the MA-CA layer mediated by the presence of the 14 amino acid long spacer peptide SP1 at the CA C-terminus, underlining the crucial role of this peptide for the resolution of the immature particle architecture.

Myxoid malignant fibrous histiocytoma presenting as a midline nasal mass.

PubMed

DeRosa, Jaimie; Smit, Jeffrey R

2012-04-01

Myxoid malignant fibrous histiocytoma is a rare type of pediatric non-rhabdomyosarcoma soft-tissue sarcoma. The case of a 5-year-old girl is presented, highlighting the potential for multiple pitfalls and aberrant differential diagnoses that need to be identified for successful treatment of pediatric myxofibrosarcomas. An awareness of these tumors and a call for standardized postsurgical treatment protocols is necessary in order to successfully treat children with this disease.

Primary dedifferentiated liposarcoma of the axilla arising in a mixed, well-differentiated and myxoid liposarcoma.

PubMed

Campbell, Garth S; Lawrence, Thomas J; Porter, Scott E; Rezeanu, Luminita

2012-01-01

We describe a case of mixed liposarcoma of the axilla presenting as a high grade undifferentiated sarcoma with areas of well-differentiated and myxoid liposarcoma. MRI demonstrated a lobulated, septated intermuscular mass with marked heterogeneous gadolinium enhancement. A small focus of the tumor demonstrated fat suppressed signal more characteristic of well-differentiated liposarcoma. Pathologic analysis following wide local excision revealed a large, high grade sarcomatous component with highly pleomorphic cells with a thin rim of well-differentiated and myxoid liposarcoma on histologic examination. Dedifferentiated liposarcomas arising outside of the retroperitoneum are very rare, as are dedifferentiated liposarcomas arising from a histologically mixed liposarcoma. In this regard, this case illustrates an unusual combination of tumor location and histology which, to our knowledge, has not previously been reported.

Myxoid liposarcoma of the foot: a study of 8 cases.

PubMed

Bekers, Elise M; Song, Wangzhao; Suurmeijer, Albert J H; Bonenkamp, Johannes J; van der Geest, Ingrid C; Braam, Petra M; Ploegmakers, Marieke J M; Desar, Ingrid M E; Tops, Bastiaan B J; van Gorp, Joost M; Creytens, David H; Mentzel, Thomas; Flucke, Uta

2016-12-01

Myxoid liposarcoma is the only translocation-associated liposarcoma subtype. It classically originates in the deep soft tissues of the thigh. At distal sites of the extremities, this tumor is exceedingly rare. We present a series of 8 cases occurring in the foot/ankle. Two female and 6 male patients, aged between 32 and 77 years (mean, 54.3 years), were identified. Tumor size ranged from 1.1 to 10 cm (mean, 6.8 cm). Two lesions eroded bone. All tumors were treated by excision and 7 by (neo)adjuvant radiotherapy. R0 status was reached in 2 cases with 1 case followed by metastasis in the groin. All other cases were documented with R1 (n=2) or R2 (n=4) resection status. In 1 patient, the follow-up status was unknown. All other patients were alive 15-135 (mean, 55.8) months after initial diagnosis. We conclude that myxoid liposarcoma at acral sites are exceedingly rare, and in this series, prognosis was good irrespective of resection status. Clinicians and pathologists have to be aware because this sarcoma type shows a peculiar clinical behavior with high radio- and chemosensitivity and metastatic spread to extrapulmonary sites. Copyright © 2016 Elsevier Inc. All rights reserved.

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

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

Dick, Robert A.; Datta, Siddhartha A. K.; Nanda, Hirsh

2016-05-06

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, whichmore » is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt

Characterization of Glycosaminoglycan (GAG) Sulfatases from the Human Gut Symbiont Bacteroides thetaiotaomicron Reveals the First GAG-specific Bacterial Endosulfatase*

PubMed Central

Ulmer, Jonathan E.; Vilén, Eric Morssing; Namburi, Ramesh Babu; Benjdia, Alhosna; Beneteau, Julie; Malleron, Annie; Bonnaffé, David; Driguez, Pierre-Alexandre; Descroix, Karine; Lassalle, Gilbert; Le Narvor, Christine; Sandström, Corine; Spillmann, Dorothe; Berteau, Olivier

2014-01-01

Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973–25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans. PMID:25002587

Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges

PubMed Central

Khattar, Sunil K.; Manoharan, Vinoth; Bhattarai, Bikash; LaBranche, Celia C.; Montefiori, David C.

2015-01-01

ABSTRACT Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. PMID:26199332

Primary Dedifferentiated Liposarcoma of the Axilla Arising in a Mixed, Well-differentiated and Myxoid Liposarcoma

PubMed Central

Campbell, Garth S.; Lawrence, Thomas J.; Porter, Scott E.; Rezeanu, Luminita

2012-01-01

We describe a case of mixed liposarcoma of the axilla presenting as a high grade undifferentiated sarcoma with areas of well-differentiated and myxoid liposarcoma. MRI demonstrated a lobulated, septated intermuscular mass with marked heterogeneous gadolinium enhancement. A small focus of the tumor demonstrated fat suppressed signal more characteristic of well-differentiated liposarcoma. Pathologic analysis following wide local excision revealed a large, high grade sarcomatous component with highly pleomorphic cells with a thin rim of well-differentiated and myxoid liposarcoma on histologic examination. Dedifferentiated liposarcomas arising outside of the retroperitoneum are very rare, as are dedifferentiated liposarcomas arising from a histologically mixed liposarcoma. In this regard, this case illustrates an unusual combination of tumor location and histology which, to our knowledge, has not previously been reported. PMID:22690275

Nuclear localization of foamy virus Gag precursor protein.

PubMed Central

Schliephake, A W; Rethwilm, A

1994-01-01

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus. Images PMID:8035493

Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice.

PubMed

Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

2016-01-01

Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction.

Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges.

PubMed

Khattar, Sunil K; Manoharan, Vinoth; Bhattarai, Bikash; LaBranche, Celia C; Montefiori, David C; Samal, Siba K

2015-07-21

Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. A safe and effective vaccine that can induce both systemic and mucosal immune responses is needed to control HIV-1. In this study, we showed that coexpression of Env and Gag proteins of HIV-1 performed using a single

HIV-1 subtype A gag variability and epitope evolution.

PubMed

Abidi, Syed Hani; Kalish, Marcia L; Abbas, Farhat; Rowland-Jones, Sarah; Ali, Syed

2014-01-01

The aim of this study was to examine the course of time-dependent evolution of HIV-1 subtype A on a global level, especially with respect to the dynamics of immunogenic HIV gag epitopes. We used a total of 1,893 HIV-1 subtype A gag sequences representing a timeline from 1985 through 2010, and 19 different countries in Africa, Europe and Asia. The phylogenetic relationship of subtype A gag and its epidemic dynamics was analysed through a Maximum Likelihood tree and Bayesian Skyline plot, genomic variability was measured in terms of G → A substitutions and Shannon entropy, and the time-dependent evolution of HIV subtype A gag epitopes was examined. Finally, to confirm observations on globally reported HIV subtype A sequences, we analysed the gag epitope data from our Kenyan, Pakistani, and Afghan cohorts, where both cohort-specific gene epitope variability and HLA restriction profiles of gag epitopes were examined. The most recent common ancestor of the HIV subtype A epidemic was estimated to be 1956 ± 1. A period of exponential growth began about 1980 and lasted for approximately 7 years, stabilized for 15 years, declined for 2-3 years, then stabilized again from about 2004. During the course of evolution, a gradual increase in genomic variability was observed that peaked in 2005-2010. We observed that the number of point mutations and novel epitopes in gag also peaked concurrently during 2005-2010. It appears that as the HIV subtype A epidemic spread globally, changing population immunogenetic pressures may have played a role in steering immune-evolution of this subtype in new directions. This trend is apparent in the genomic variability and epitope diversity of HIV-1 subtype A gag sequences.

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

PubMed Central

Hansen, M S; Barklis, E

1995-01-01

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC. PMID:7815493

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

PubMed Central

Waddington, Lynne; Hijnen, Marcel; Velkov, Tony; McKinstry, William J.

2017-01-01

The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly. PMID:28222188

Modeling the dynamics and kinetics of HIV-1 Gag during viral assembly.

PubMed

Tomasini, Michael D; Johnson, Daniel S; Mincer, Joshua S; Simon, Sanford M

2018-01-01

We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions. These experimental constraints eliminated many classes of potential interactions, and narrowed the model to a single interaction scheme with two non-equivalent interfaces acting to form Gags into a hexamer, and a third interface acting to link hexamers together. This model was able to form into a hexameric structure with correct lattice spacing and reproduced biologically relevant growth rates. We explored the effect of genomic RNA seeding punctum growth, finding that RNA may be a factor in locally concentrating Gags to initiate assembly. The simulation results infer that completion of assembly cannot be governed simply by Gag binding kinetics. However the addition of membrane curvature suggests that budding of the virion from the plasma membrane could factor into slowing incorporation of Gag at an assembly site resulting in virions of the same size and number of Gag molecules independent of Gag concentration or the time taken to complete assembly. To corroborate the results of our simulation model, we developed an analytic model for Gag assembly finding good agreement with the simulation results.

Modeling the dynamics and kinetics of HIV-1 Gag during viral assembly

PubMed Central

Tomasini, Michael D.; Johnson, Daniel S.; Mincer, Joshua S.

2018-01-01

We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions. These experimental constraints eliminated many classes of potential interactions, and narrowed the model to a single interaction scheme with two non-equivalent interfaces acting to form Gags into a hexamer, and a third interface acting to link hexamers together. This model was able to form into a hexameric structure with correct lattice spacing and reproduced biologically relevant growth rates. We explored the effect of genomic RNA seeding punctum growth, finding that RNA may be a factor in locally concentrating Gags to initiate assembly. The simulation results infer that completion of assembly cannot be governed simply by Gag binding kinetics. However the addition of membrane curvature suggests that budding of the virion from the plasma membrane could factor into slowing incorporation of Gag at an assembly site resulting in virions of the same size and number of Gag molecules independent of Gag concentration or the time taken to complete assembly. To corroborate the results of our simulation model, we developed an analytic model for Gag assembly finding good agreement with the simulation results. PMID:29677208

[A giant myxoid leiomyoma mimicking an inguinal hernia].

PubMed

Huszár, Orsolya; Zaránd, Attila; Szántó, Gyöngyi; Juhász, Viktória; Székely, Eszter; Novák, András; Molnár, Béla Ákos; Harsányi, László

2016-03-06

Leiomyoma is a rare, smooth muscle tumour that can occur everywhere in the human body. The authors present the history of a 60-year-old female, who had a giant, Mullerian type myxoid leiomyoma in the inguinal region mimicking acute abdominal symptoms. After examination the authors removed the soft tissue mass in the right femoral region reaching down in supine position to the middle third of the leg measuring 335 × 495 × 437 mm in greatest diameters in weight 33 kg. Reconstruction of the tissue defect was performed using oncoplastic guidelines. During the follow-up time no tumour recurrence was detected and the quality of life of the patient improved significantly.

Interaction of HIV-1 Gag protein components with single DNA molecules

NASA Astrophysics Data System (ADS)

Cruceanu, Margareta; Gorelick, Robert J.; Williams, Mark C.

2003-03-01

The Gag protein of the HIV-1 retrovirus is cleaved into three major proteins as part of viral maturation: nucleocapsid (NC), capsid, and matrix. NC is the first of these proteins to be cleaved, and it is cleaved in three stages into NCp15, followed by NCp9, and finally NCp7. In this study, we use optical tweezers to investigate the capability of these NC proteins to alter the helix-coil transition of single DNA molecules. We have previously shown that the capability to alter the DNA helix-coil transition is an excellent probe of the nucleic acid chaperone activity of NC proteins, in which the secondary structure of nucleic acids is rearranged to facilitate reverse transcription. By examining the capability of NCp15, NCp9, and NCp7 to alter DNA stretching, the current studies will test the role of proteolytic cleavage of Gag in regulating the nucleic acid chaperone activity of NC. Whereas binding studies suggest that NCp9 and NCp15 bind more strongly to DNA than NCp7, our DNA stretching results indicate that these proteins all have similar effects on DNA stretching.

Mobility of human immunodeficiency virus type 1 Pr55Gag in living cells.

PubMed

Gomez, Candace Y; Hope, Thomas J

2006-09-01

Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

PubMed Central

Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

2014-01-01

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

STUMP un"stumped": anti-tumor response to anaplastic lymphoma kinase (ALK) inhibitor based targeted therapy in uterine inflammatory myofibroblastic tumor with myxoid features harboring DCTN1-ALK fusion.

PubMed

Subbiah, Vivek; McMahon, Caitlin; Patel, Shreyaskumar; Zinner, Ralph; Silva, Elvio G; Elvin, Julia A; Subbiah, Ishwaria M; Ohaji, Chimela; Ganeshan, Dhakshina Moorthy; Anand, Deepa; Levenback, Charles F; Berry, Jenny; Brennan, Tim; Chmielecki, Juliann; Chalmers, Zachary R; Mayfield, John; Miller, Vincent A; Stephens, Philip J; Ross, Jeffrey S; Ali, Siraj M

2015-06-11

Recurrent, metastatic mesenchymal myxoid tumors of the gynecologic tract present a management challenge as there is minimal evidence to guide systemic therapy. Such tumors also present a diagnostic dilemma, as myxoid features are observed in leiomyosarcomas, inflammatory myofibroblastic tumors (IMT), and mesenchymal myxoid tumors. Comprehensive genomic profiling was performed in the course of clinical care on a case of a recurrent, metastatic myxoid uterine malignancy (initially diagnosed as smooth muscle tumor of uncertain malignant potential (STUMP)), to guide identify targeted therapeutic options. To our knowledge, this case represents the first report of clinical response to targeted therapy in a tumor harboring a DCTN1-ALK fusion protein. Hybridization capture of 315 cancer-related genes plus introns from 28 genes often rearranged or altered in cancer was applied to >50 ng of DNA extracted from this sample and sequenced to high, uniform coverage. Therapy was given in the context of a phase I clinical trial ClinicalTrials.gov Identifier: ( NCT01548144 ). Immunostains showed diffuse positivity for ALK1 expression and comprehensive genomic profiling identified an in frame DCTN1-ALK gene fusion. The diagnosis of STUMP was revised to that of an IMT with myxoid features. The patient was enrolled in a clinical trial and treated with an anaplastic lymphoma kinase (ALK) inhibitor (crizotinib/Xalkori®) and a multikinase VEGF inhibitor (pazopanib/Votrient®). The patient experienced an ongoing partial response (6+ months) by response evaluation criteria in solid tumors (RECIST) 1.1 criteria. For myxoid tumors of the gynecologic tract, comprehensive genomic profiling can identify clinical relevant genomic alterations that both direct treatment targeted therapy and help discriminate between similar diagnostic entities.

De novo biosynthesis of glycosaminoglycans in the extracellular matrix of skin studied by matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Böhme, Julia; Anderegg, Ulf; Nimptsch, Ariane; Nimptsch, Kathrin; Hacker, Michael; Schulz-Siegmund, Michaela; Huster, Daniel; Schiller, Jürgen

2012-02-15

The self-healing capacity of skin is limited, and medical intervention is often unavoidable. Skin may be generated ex vivo from cultured fibroblasts. Because the molecular composition of de novo formed skin (mostly collagen and glycosaminoglycans [GAGs]) is crucial, analytical methods are required for the quality control of tissue-engineered products. Here, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of fibroblast cultures subsequent to digestion with chondroitinase ABC is a reliable and fast method to monitor the GAG content of native and bioengineered skin. Furthermore, the supplementation of the fibroblast medium with ¹³C-labeled glucose provides insights into the biosynthesis of GAGs. Copyright © 2011 Elsevier Inc. All rights reserved.

[A rare extra-skeletal myxoid chondrosarcoma of the lower leg - is amputation absolutely necessary].

PubMed

Mroczkowski, P; Evert, M; Tautenhahn, J; Meyer, F; Lippert, H

2010-02-01

Sarcomas represent less than 2 % of all malignancies. Special challenges are bone sarcomas in extra-skeletal localisation. The aim of this case report is to show the management of an extraordinary extra-skeletal myxoid chondrosarcoma based on a case report with references from the literature. After a delay in diagnostics for 1.5 years, an MRI scan taken in a 42-year-old male patient with progressive swelling of the left calf showed a soft-tissue tumour in the proximal part of the muscle. Histopathological investigation of a percutaneous biopsy revealed a chondrosarcoma. En-bloc-resection (R 0) of the rear superficial compartment was performed (specimen weight 1 370 g; tumour size 11.5 x 9.5 x 8 cm) leading to the definitive diagnosis of an extra-skeletal myxoid chondrosarcoma. The patient was discharged with a bland wound 8 days after surgery. At 4 weeks postoperatively, the patient received adjuvant radiotherapy with a 56-Gy boost. During the follow-up period of 28 months, there have been neither signs of local tumour recurrence nor distant metastases. The myxoid chondrosarcoma is a rare tumour lesion, and according to the literature, only 2 % occur outside of the skeleton. The accurate diagnostic and therapeutic algorithm allowed a precise preparation for surgery and made amputation obsolete. Compartment resection preserving the main neurovascular bundles as well as enabling an early mobilisation resulted in both sufficient radical resection status and adequate postoperative motor function. Intraoperative clip-marking of the former tumour bed is considered a key point for the focused radiotherapy. Each persistent soft tissue swelling must be appropriately diagnosed using adequate imaging and even biopsy (in case of a doubtful finding), which should be performed with definitive surgery in mind. Georg Thieme Verlag Stuttgart, New York.

Mesenteric Primitive Myxoid Mesenchymal Tumor in a 7-Month-Old Boy.

PubMed

Chacon, Daniella; Correa, Catalina; Luengas, Juan P

2018-05-01

Mesenteric tumors are rare, especially in the pediatric population. We present a case of a 7-month-old boy with an incidental abdominal mass identified as a Primitive Myxoid Mesenchymal Tumor of Infancy derived from the small bowel mesentery. This neoplasm is part of a spectrum of myofibroblastic lesions, with distinct clinical, morphologic, IHQ reactivity, and an aggressive clinical course. There is a paucity of evidence for the management of these tumors, and surgical resection remains the mainstay of treatment.

Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene.

PubMed Central

Schultz, A M; Rabin, E H; Oroszlan, S

1979-01-01

Post-translational modifications of retrovirus gag gene-encoded polyproteins include proteolytic cleavage, phosphorylation, and glycosylation. To study the sequence of these events, we labeled JLS-V9 cells chronically infected with Rauscher murine leukemia virus in pulse-chase experiments with the radioactive precursors [35S]methionine, [14C]mannose, [3H]glucosamine, and [32P]phosphate. Newly synthesized gag polyproteins which incorporated label, and the modified products derived from them, were identified by immunoprecipitation of cell lysates with anti-p30 rabbit serum, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Pulse-chase experiments were carried out in the presence as well as in the absence of tunicamycin, an inhibitor of glycosylation. Among the three major polyproteins synthesized in the absence of tunicamycin, two were found to be glycosylated but not phosphorylated. These were designated gPr80gag and gP94gag. Both shared identical [35S]methionine peptides with Pr65gag and p30. Of the two nonglycosylated precursors, Pr65gag and Pr75gag, only Pr65gag was found to be detectably phosphorylated, and Pr75gag could be readily identified only when glycosylation was inhibited. On the basis of these results, a scheme for the post-translational modification of gag polyproteins is proposed. According to this scheme the gag gene-encoded polyproteins are processed from a common precursor, Pr75gag, by two divergent pathways: one leading through the intermediate Pr65gag to internal virion components via cleavage and phosphorylation and the other via tunicamycin-sensitive mannosylation to the intermediate gPr80gag, which is further glycosylated to yield cell surface polyprotein gP94gag. Images PMID:480454

Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics.

PubMed

Mattson, Jeffrey M; Turcotte, Raphaël; Zhang, Yanhang

2017-02-01

Elastic and collagen fibers are well known to be the major load-bearing extracellular matrix (ECM) components of the arterial wall. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen fibers, and glycosaminoglycans (GAGs) are often neglected. Although GAGs represent only a small component of the vessel wall ECM, they are considerably important because of their diverse functionality and their role in pathological processes. The goal of this study was to study the mechanical and structural contributions of GAGs to the arterial wall. Biaxial tensile testing was paired with multiphoton microscopic imaging of elastic and collagen fibers in order to establish the structure-function relationships of porcine thoracic aorta before and after enzymatic GAG removal. Removal of GAGs results in an earlier transition point of the nonlinear stress-strain curves [Formula: see text]. However, stiffness was not significantly different after GAG removal treatment, indicating earlier but not absolute stiffening. Multiphoton microscopy showed that when GAGs are removed, the adventitial collagen fibers are straighter, and both elastin and collagen fibers are recruited at lower levels of strain, in agreement with the mechanical change. The amount of stress relaxation also decreased in GAG-depleted arteries [Formula: see text]. These findings suggest that the interaction between GAGs and other ECM constituents plays an important role in the mechanics of the arterial wall, and GAGs should be considered in addition to elastic and collagen fibers when studying arterial function.

In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid.

PubMed Central

Schultz, A M; Oroszlan, S

1983-01-01

It has recently been shown by mass spectral analysis (Henderson et al., Proc. Natl. Acad. Sci. U.S.A. 80:339-343, 1983) that the p15gag protein of murine leukemia viruses contains a novel post-translational modification, an amino-terminal myristyl (tetradecanoyl) amide. In this report we show that p15gag is the only structural protein to contain this fatty acid. In addition, the gag precursor polyproteins of type B, C, and D retroviruses have been examined for the presence of myristic acid by metabolic labeling and immunoprecipitation studies. In a panel of mammalian type C retroviruses we found that the precursor polyprotein Pr65gag homologs, but not the glycosylated forms (gPr80gag homologs), were specifically labeled after a 5-min incubation of infected cells with [3H]myristic acid. The gag precursor polyprotein was also labeled in mouse mammary tumor virus and in Mason-Pfizer monkey virus, but Pr76gag of Rous sarcoma virus failed to incorporate [3H]myristate. Under similar conditions, [3H]palmitate was not found to be incorporated into any viral gag proteins. Thus, myristylation appears to be a common feature of mammalian type B, C, and D retroviruses but not of avian retroviruses. Images PMID:6302307

HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

PubMed

Ko, Li-Jung; Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

2015-01-01

Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

PubMed Central

Felsenstein, K M; Goff, S P

1992-01-01

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo. Images PMID:1404606

Structure of glycosylated and unglycosylated gag polyproteins of Rauscher murine leukemia virus: carbohydrate attachment sites.

PubMed Central

Schultz, A M; Lockhart, S M; Rabin, E M; Oroszlan, S

1981-01-01

The structural relationships among the gag polyproteins Pr65gag, Pr75gag, and gPr80gag of Rauscher murine leukemia virus were studied by endoglycosidase H digestion and formic acid cleavage. Fragments were identified by precipitation with specific antisera to constituent virion structural proteins followed by one-dimensional mapping. Endoglycosidase H reduced the size of gPr80gag to that of Pr75gag. By comparing fragments of gPr80gag and the apoprotein Pr75gag, the former was shown to contain two mannose-rich oligosaccharide units. By comparing fragments of Pr65gag and Pr75gag, the latter was shown to differ from Pr65gag at the amino terminus by the presence of a leader peptide approximately 7,000 daltons in size. The internal and carboxyl-terminal peptides of the two unglycosylated polyproteins were not detectably different. The location of the two N-linked carbohydrate chains in gPr80gag has been specified. One occurs in the carboxyl-terminal half of the polyprotein at asparagine177 of the p30 sequence and the other is found in a 23,000-dalton fragment located in the amino-terminal region of gPr80gag and containing the additional amino acid sequences not found in Pr65gag plus a substantial portion of p15. Images PMID:7241663

Genetic diversity in the feline leukemia virus gag gene.

PubMed

Kawamura, Maki; Watanabe, Shinya; Odahara, Yuka; Nakagawa, So; Endo, Yasuyuki; Tsujimoto, Hajime; Nishigaki, Kazuo

2015-06-02

Feline leukemia virus (FeLV) belongs to the Gammaretrovirus genus and is horizontally transmitted among cats. FeLV is known to undergo recombination with endogenous retroviruses already present in the host during FeLV-subgroup A infection. Such recombinant FeLVs, designated FeLV-subgroup B or FeLV-subgroup D, can be generated by transduced endogenous retroviral env sequences encoding the viral envelope. These recombinant viruses have biologically distinct properties and may mediate different disease outcomes. The generation of such recombinant viruses resulted in structural diversity of the FeLV particle and genetic diversity of the virus itself. FeLV env diversity through mutation and recombination has been studied, while gag diversity and its possible effects are less well understood. In this study, we investigated recombination events in the gag genes of FeLVs isolated from naturally infected cats and reference isolates. Recombination and phylogenetic analyses indicated that the gag genes often contain endogenous FeLV sequences and were occasionally replaced by entire endogenous FeLV gag genes. Phylogenetic reconstructions of FeLV gag sequences allowed for classification into three distinct clusters, similar to those previously established for the env gene. Analysis of the recombination junctions in FeLV gag indicated that these variants have similar recombination patterns within the same genotypes, indicating that the recombinant viruses were horizontally transmitted among cats. It remains to be investigated whether the recombinant sequences affect the molecular mechanism of FeLV transmission. These findings extend our understanding of gammaretrovirus evolutionary patterns in the field. Copyright © 2015 Elsevier B.V. All rights reserved.

Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging

PubMed Central

Angert, Isaac; Cao, Sheng; Berk, Serkan; Zhang, Wei; Mueller, Joachim D.

2017-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500–2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag–Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag–Gag interactions that occur

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element

PubMed Central

2011-01-01

Background The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. Results By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

PubMed

George, Maja; Schwecke, Torsten; Beimforde, Nadine; Hohn, Oliver; Chudak, Claudia; Zimmermann, Anja; Kurth, Reinhard; Naumann, Dieter; Bannert, Norbert

2011-05-09

The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and

[Physical restraints and gagging in unnatural death].

PubMed

Grellner, W; Madea, B

1993-01-01

Practices of binding and gagging can be found in different types of unnatural death; three case reports concerning fatal autoeroticism, suicide and homicide are presented. In such cases investigations are usually concentrated on the question whether binding and gagging could be carried out by the persons themselves. Loosely bound final loops, especially of the hands, missing local haemorrhages and the discovery of binding instructions support the assumption of self-binding. Accompanying injuries and the entire situation must be taken into special consideration.

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

PubMed Central

Becker, Jordan T.

2017-01-01

ABSTRACT Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans. In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5′ packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly.

PubMed

Becker, Jordan T; Sherer, Nathan M

2017-03-15

Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis -acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM. IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1

Roles of Gag-RNA interactions in HIV-1 virus assembly deciphered by single-molecule localization microscopy.

PubMed

Yang, Yantao; Qu, Na; Tan, Jie; Rushdi, Muaz N; Krueger, Christopher J; Chen, Antony K

2018-06-11

During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (Gag ZiL ) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly. In this study, we examined the role of vRNA in HIV-1 assembly by characterizing the distribution and mobility of Gag and Gag NC mutants at the PM using photoactivated localization microscopy (PALM) and single-particle tracking PALM (spt-PALM). We showed that both Gag and Gag ZiL assembly involve a similar basic assembly unit, as expected. Unexpectedly, the two proteins underwent different subsequent assembly pathways, with Gag cluster density increasing asymptotically, while Gag ZiL cluster density increased linearly. Additionally, the directed movement of Gag, but not Gag ZiL , was maintained at a constant speed, suggesting that the two proteins experience different external driving forces. Assembly was abolished when Gag was rendered monomeric by NC deletion. Collectively, these results suggest that, beyond inducing Gag to form low-ordered multimer basic assembly units, vRNA is essential in scaffolding and maintaining the stability of the subsequent assembly process. This finding should advance the current understanding of HIV-1 and, potentially, other retroviruses. Copyright © 2018 the Author(s). Published by PNAS.

Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame.

PubMed

Deforges, Jules; de Breyne, Sylvain; Ameur, Melissa; Ulryck, Nathalie; Chamond, Nathalie; Saaidi, Afaf; Ponty, Yann; Ohlmann, Theophile; Sargueil, Bruno

2017-07-07

In the late phase of the HIV virus cycle, the unspliced genomic RNA is exported to the cytoplasm for the necessary translation of the Gag and Gag-pol polyproteins. Three distinct translation initiation mechanisms ensuring Gag production have been described with little rationale for their multiplicity. The Gag-IRES has the singularity to be located within Gag ORF and to directly interact with ribosomal 40S. Aiming at elucidating the specificity and the relevance of this interaction, we probed HIV-1 Gag-IRES structure and developed an innovative integrative modelling strategy to take into account all the gathered information. We propose a novel Gag-IRES secondary structure strongly supported by all experimental data. We further demonstrate the presence of two regions within Gag-IRES that independently and directly interact with the ribosome. Importantly, these binding sites are functionally relevant to Gag translation both in vitro and ex vivo. This work provides insight into the Gag-IRES molecular mechanism and gives compelling evidence for its physiological importance. It allows us to propose original hypotheses about the IRES physiological role and conservation among primate lentiviruses. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

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

López, Claudia S., E-mail: lopezcl@ohsu.edu; Sloan, Rachel; Cylinder, Isabel

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag proteinmore » expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.« less

Physical mapping of chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma, and myxoid liposarcoma

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

Schoenmakers, E.F.P.M.; Kools, P.F.J.; Mols, R.

1994-03-15

The authors report here the physical mapping of recurrent chromosome 12q13-q15 breakpoints in cell lines derived from primary myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands. In fluorescence in situ hybridization (FISH) experiments, they first mapped the position of the chromosome 12 translocation breakpoint in uterine leiomyoma cell line LM-30.1/SV40 relative to loci COL2A1, D12S4, D12S17, D12S6, D12S19, D12S8, and D12S7. It mapped between linkage probes CRI-C86 (D12S19) and p7G11 (D12S8). They then isolated YAC clones using CRI-C86- and p7G11-derived sequence-tagged sites, constructed corresponding YAC contigs of 310 and 800 kb, respectively, and a mixture ofmore » them was used to routinely study the various tumor cell lines by FISH analysis. The chromosome 12 breakpoints of all tumor cell lines tested mapped between cosmids LLNL12NCO1-98C10 and LLNL12NCO1-113D12. None of the breakpoints appeared to map within any of the isolated YAC clones. Furthermore, FISH analysis using cosmid LLNL12-NCO1-144G3, which maps at the CHOP locus, revealed that the chromosome 12 breakpoints in all cell lines of the three benign solid tumors that were tested were located distal to the chromosome 12 translocation breakpoint with the CHOP gene in myxoid liposarcoma cells with t(12;16). In conclusion, the studies seem to indicate that the chromosome 12 breakpoints of myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands are all clustered within the 7-cM interval between D12S19 and D12S8, with those of the benign solid tumors distal to CHOP. Finally, the MYF5 gene mapped telomeric to LLNL12NCO1-113D12, and the MIP gene mapped centromeric to the chromosome 12 translocation breakpoint in myxoid liposarcoma cells. 56 refs., 5 figs., 3 tabs.« less

Management of exaggerated gag reflex in dental patients using intravenous sedation with dexmedetomidine.

PubMed

Reshetnikov, Aleksei P; Kasatkin, Anton A; Urakov, Aleksandr L; Baimurzin, Dmitrii Y

2017-01-01

Pharmacological sedation is one of the effective ways of prevention of gag reflex development in patients experiencing anxiety and fright before dental treatment. We are reporting a case where we could successfully eliminate exaggerated gag reflex (intravenous [IV] Gagging Severity Index) in a dental patient using IV sedation with dexmedetomidine. IV administration of dexmedetomidine provided elimination of gag reflex at a depth of sedation for the patient with the Richmond Agitation-Sedation Scale score of -2 and -1. The patient received dexmedetomidine 1.0 μg/kg for 10 min and then a continuous infusion of dexmedetomidine 0.4 μg/kg/h. The use of dexmedetomidine for sedation may be an alternative to other pharmacological agents in patients with dental anxiety accompanied by exaggerated gag reflex.

How Can Hypnodontics Manage Severe Gag Reflex for Root Canal Therapy? A Case Report

PubMed Central

Ramazani, Mohsen; zarenejad, Nafiseh; Parirokh, Masoud; Zahedpasha, Samir

2016-01-01

In endodontics, severe involuntary gagging can have a severe impact on treatment procedure. There are many ways to ease the gag reflex, one of which is hypnosis. A 34-year-old male was referred for root canal treatment of a molar tooth. He had not received any dental treatments for the past nine years due to fear of severe gag reflex. Three hypnotic sessions based upon eye fixation, progressive muscle relaxation and guided imagery techniques were spent for psychosomatic management. The gag reflex was controlled and reduced to a normal level, and the required dental treatments including root canal therapy and restoration were performed successfully. This report shows that hypnosis can control gag reflex for dental treatments. PMID:27141226

Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae.

PubMed

Tomo, Naoki; Goto, Toshiyuki; Morikawa, Yuko

2013-03-26

Yeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system. The nearly full-length HIV-1 genome containing the entire 5' long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells

Adipokines induce catabolism of newly synthesized matrix in cartilage and meniscus tissues.

PubMed

Nishimuta, James F; Levenston, Marc E

Altered synovial levels of various adipokines (factors secreted by fat as well as other tissues) have been associated with osteoarthritis (OA) onset and progression. However, the metabolic effects of adipokines on joint tissues, in particular the fibrocartilaginous menisci, are not well understood. This study investigated effects of several adipokines on release of recently synthesized extracellular matrix in bovine cartilage and meniscus tissue explants. After labeling newly synthesized proteins and sulfated glycosaminoglycans (sGAGs) with 3 H-proline and 35 S-sulfate, respectively; bovine cartilage and meniscus tissue explants were cultured for 6 days in basal medium (control) or media supplemented with adipokines (1 µg/ml of leptin, visfatin, adiponectin, or resistin) or 20 ng/ml interleukin-1 (IL-1). Release of radiolabel and sGAG to the media during culture and the final explant water, DNA, sGAG, and retained radiolabel were measured. Matrix metalloproteinase (MMP-2) and MMP-3 activities were assessed using gelatin and casein zymography, respectively. Water and DNA contents were not significantly altered by any treatment. Visfatin, adiponectin, resistin, and IL-1 stimulated sGAG release from meniscus, whereas only IL-1 stimulated sGAG release from cartilage. Release of 3 H and 35 S was stimulated not only by resistin and IL-1 in meniscus but also by IL-1 in cartilage. Retained 3 H was unaltered by any treatment, while retained 35 S was reduced by visfatin, resistin, and IL-1 in meniscus and by only IL-1 in cartilage. Resistin and IL-1 elevated active MMP-2 and total MMP-3 in meniscus, whereas cartilage MMP-3 activity was elevated by only IL-1. Resistin stimulated rapid and extensive catabolism of meniscus tissue, similar to IL-1, whereas adipokines minimally affected cartilage. Release of newly synthesized matrix was similar to overall release in both tissues. These observations provide further indications that meniscal tissue is more sensitive to pro

Primary extraskeletal myxoid chondrosarcoma of the vulva.

PubMed

Sawada, Morio; Tochigi, Naobumi; Sasajima, Yuko; Hasegawa, Tadashi; Kasamatsu, Takahiro; Kitawaki, Jo

2011-11-01

Primary extraskeletal myxoid chondrosarcoma (EMC) of the vulva is extremely rare. There is little available information about the biological behavior and treatment strategy for primary EMC of the vulva. We report a rare case of primary EMC of the vulva treated surgically. A 24-year-old Japanese woman had demonstrated a small and elastic mass of the vulva and underwent enucleation of the mass at a previous hospital, but a definitive histopathological diagnosis was not obtained. Therefore, the patient was referred to our hospital for further evaluation and treatment. We histopathologically diagnosed the tumor as primary EMC of the vulva and performed vulvectomy with vulvoperineal reconstruction. Microscopic examination of the resected specimens demonstrated residual tumor nodules of EMC. However, there were no viable tumor cells at the surgical margin. Approximately two years after wide local excision was performed, the patient is doing well and there is no apparent recurrence of EMC. © 2011 The Authors. Journal of Obstetrics and Gynaecology Research © 2011 Japan Society of Obstetrics and Gynecology.

Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

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

Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett

Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specificmore » antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.« less

Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome.

PubMed Central

Huang, M; Jolicoeur, P

1990-01-01

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus. Images PMID:2243376

Human Foamy Virus Capsid Formation Requires an Interaction Domain in the N Terminus of Gag

PubMed Central

Tobaly-Tapiero, Joelle; Bittoun, Patricia; Giron, Marie-Lou; Neves, Manuel; Koken, Marcel; Saïb, Ali; de Thé, Hugues

2001-01-01

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly. PMID:11287585

Transport and processing of the Rous sarcoma virus Gag protein in the endoplasmic reticulum.

PubMed Central

Krishna, N K; Weldon, R A; Wills, J W

1996-01-01

The Gag proteins of replication-competent retroviruses direct budding at the plasma membrane and are cleaved by the viral protease (PR) just before or very soon after particle release. In contrast, defective retroviruses that bud into the endoplasmic reticulum (ER) have been found, and morphologically these appear to contain uncleaved Gag proteins. From this, it has been proposed that activation of PR may depend upon a host factor found only at the plasma membrane. However, if Gag proteins were cleaved by PR before the particle could pinch off the ER membrane, then the only particles that would remain visible are those that packaged smaller-than-normal amounts of PR, and these would have an immature morphology. To distinguish between these two hypotheses, we made use of the Rous sarcoma virus (RSV) Gag protein, the PR of RSV IS included on each Gag molecule. To target Gag to the ER, a signal peptide was installed at its amino terminus in place of the plasma membrane-binding domain. An intervening, hydrophobic, transmembrane anchor was included to keep Gag extended into the cytoplasm. We found that PR-mediated processing occurred, although the cleavage products were rapidly degraded. When the anchor was removed, allowing the entire protein to be inserted into the lumen of the ER, Gag processing occurred with a high level of efficiency, and the cleavage products were quite stable. Thus, PR activation does not require targeting of Gag molecules to the plasma membrane. Unexpectedly, molecules lacking the transmembrane anchor were rapidly secreted from the cell in a nonmembrane-enclosed form and in a manner that was very sensitive to brefeldin A and monensin. In contrast, the wild-type RSV and Moloney murine leukemia virus Gag proteins were completely insensitive to these inhibitors, suggesting that the normal mechanism of transport to the plasma membrane does not require interactions with the secretory pathway. PMID:8627676

Tsg101 regulates PI(4,5)P2/Ca2+ signaling for HIV-1 Gag assembly

PubMed Central

Ehrlich, Lorna S.; Medina, Gisselle N.; Photiadis, Sara; Whittredge, Paul B.; Watanabe, Susan; Taraska, Justin W.; Carter, Carol A.

2014-01-01

Our previous studies identified the 1,4,5-inositol trisphosphate receptor (IP3R), a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC)-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2. Raising cytosolic Ca2+ promotes viral particle production and our studies indicate that IP3R and the ER Ca2+ store are the physiological providers of Ca2+ for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca2+ level originating from the ER store than control cells, suggesting that Gag induced release of store Ca2+. This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca2+ elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence (TIRF) and immunoelectron microscopy (IEM) or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5)P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag with an

[A rare tumor of the parapharyngeal space: myxoid chondrosarcoma].

PubMed

Bahri, I; Boudawara, T; Sellami, A; Khabir, A; Ghorgel, M; Drira, M; Daoud, J; Jlidi, R

2002-01-01

Extrasqueletal myxoid chondrosarcoma (EMC) is an uncommon soft tissue malignant tumor, locally aggressive with a high incidence of distant metastasis. It has distinctive clinical, immunophenotypic, cytogenetic and ultrastructural features. Most EMC are associated with the translocation t(9;22) (q22;q12). Their occurrence in the parapharyngeal space is extremely rare. Our objective is to discuss the difficulties of the histological diagnosis of EMC and to describe its immunophenotypic, cytogenetic features and clinical behavior. We report a case of a 67 years old woman who presented with a five months history of dysphagia. The oral examination found a mass displacing the posterior and left walls of the pharynx. Surgical resection of the tumor was undertaken. The pathologic examination concluded to the diagnosis of an EMC of the left parapharygeal space. Now, the patient is receiving an adjuvant radiotherapy.

How HIV-1 Gag assembles in cells: putting together pieces of the puzzle

PubMed Central

Lingappa, Jaisri R; Reed, Jonathan C; Tanaka, Motoko; Chutiraka, Kasana; Robinson, Bridget A

2014-01-01

During the late stage of the viral life cycle, HIV-1 Gag assembles into a spherical immature capsid, and undergoes budding, release, and maturation. Here we review events involved in immature capsid assembly from the perspective of five different approaches used to study this process: mutational analysis, structural studies, assembly of purified recombinant Gag, assembly of newly-translated Gag in a cell-free system, and studies in cells using biochemical and imaging techniques. We summarize key findings obtained using each approach, point out where there is consensus, and highlight unanswered questions. Particular emphasis is placed on reconciling data suggesting that Gag assembles by two different paths, depending on the assembly environment. Specifically, in assembly systems that lack cellular proteins, high concentrations of Gag can spontaneously assemble using purified nucleic acid as a scaffold. However, in the more complex intracellular environment, barriers that limit self-assembly are present in the form of cellular proteins, organelles, host defenses, and the absence of free nucleic acid. To overcome these barriers and promote efficient immature capsid formation in an unfavorable environment, Gag appears to utilize an energy-dependent, host-catalyzed, pathway of assembly intermediates in cells. Overall, we show how data obtained using a variety of techniques has led to our current understanding of HIV assembly. PMID:25066606

Selection of HLA-B57-associated Gag A146P mutant by HLA-B∗48:01-restricted Gag140-147-specific CTLs in chronically HIV-1-infected Japanese.

PubMed

Naruto, Takuya; Murakoshi, Hayato; Chikata, Takayuki; Koyanagi, Madoka; Kawashima, Yuka; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

2011-08-01

We previously showed the possibility that Gag A146P, which is an escape mutant from HLA-B∗57-restricted CTLs, was selected by HLA-B∗48:01-restricted Gag138-147(LI10)-specific CTLs in a Japanese cohort in which HLA-B∗57 individuals were not detected. We herein demonstrated Gag140-147(GI8) to be the optimal epitope rather than LI10 and that GI8-specific T cells failed to recognize the A146P mutant virus-infected cells. The sequence analysis of Gag146 in 261 chronically HIV-1-infected Japanese showed the accumulation of the A146P mutation in HLA-B∗48:01(+) individuals. These findings together indicate that the A146P mutant is accumulating in Japanese by selection by GI8-specific CTLs. Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

The Use of an Alternative Extraoral Periapical Technique for Patients with Severe Gag Reflex

PubMed Central

e Silva, Mauro Henrique Chagas; Santos, Mariane Floriano Lopes; de Lima, Carolina Oliveira; Campos, Celso Neiva

2016-01-01

Gag reflex is a physiologic mechanism that promotes contraction of the muscles of the tongue and pharyngeal walls. Different factors, including intraoral radiographic films and sensors, may trigger this reflex. Patients with severe gag reflex may not be able to tolerate the presence of intraoral radiographic films or sensors during root canal therapy (RCT). This factor may prevent an appropriate intraoral radiograph, which is important in RCT. Different approaches have been used to facilitate dental procedures in patients suffering from severe gag reflex. The use of an extraoral radiographic technique is an alternative method to obtain working length confirmation in patients with severe gag reflex. In this report of 2 cases, the use of an extraoral radiographic technique as an alternative approach during RCT in patients with severe gag reflex associated with phobic behavior and trismus was successfully demonstrated. PMID:27547474

Bag the gag rule. Poll indicates most Americans think global gag rule is wrong.

PubMed

Ernst, J; Farmer, A

2000-09-01

According to studies conducted by the Rand Corporation and the District of Columbia-based Center for Development and Population Activities, conservative and liberal Americans alike overwhelmingly support foreign assistance to international family planning programs. In addition, the 1998 poll shows that 92% of Americans believe that couples have the right to family planning, and a slight majority support government funding of legal overseas abortion services. Despite such evidence, members of the House Representative voted to restrict foreign family planning organizations that receive federal money from using their own non-US funds to provide abortion services overseas. To this effect, foreign family planning organizations and other concerned agencies argued that such a restriction undermines the objectives of the US and effectively denies access to desperately needed support to millions of women worldwide. In particular, organizations like the Center for Reproductive Law and Policy are lobbying lawmakers to strike the gag rule language from the final appropriations bill that will reach US President Clinton's desk, as well as to increase family planning funding levels. However, Clinton has indicated a veto if congress does pass gag rule legislation for the second year in a row.

CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

PubMed Central

Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

2011-01-01

Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

Terminal-Repeat Retrotransposons with GAG Domain in Plant Genomes: A New Testimony on the Complex World of Transposable Elements

PubMed Central

Chaparro, Cristian; Gayraud, Thomas; de Souza, Rogerio Fernandes; Domingues, Douglas Silva; Akaffou, Sélastique; Laforga Vanzela, Andre Luis; de Kochko, Alexandre; Rigoreau, Michel; Crouzillat, Dominique; Hamon, Serge; Hamon, Perla; Guyot, Romain

2015-01-01

A novel structure of nonautonomous long terminal repeat (LTR) retrotransposons called terminal repeat with GAG domain (TR-GAG) has been described in plants, both in monocotyledonous, dicotyledonous and basal angiosperm genomes. TR-GAGs are relatively short elements in length (<4 kb) showing the typical features of LTR-retrotransposons. However, they carry only one open reading frame coding for the GAG precursor protein involved for instance in transposition, the assembly, and the packaging of the element into the virus-like particle. GAG precursors show similarities with both Copia and Gypsy GAG proteins, suggesting evolutionary relationships of TR-GAG elements with both families. Despite the lack of the enzymatic machinery required for their mobility, strong evidences suggest that TR-GAGs are still active. TR-GAGs represent ubiquitous nonautonomous structures that could be involved in the molecular diversities of plant genomes. PMID:25573958

Vaginal para-urethral myxoid leiomyoma: case report and review of the literature.

PubMed

Costantini, Elisabetta; Cochetti, Giovanni; Porena, Massimo

2008-08-01

Vaginal leiomyoma is a rare solid tumour of unknown aetiology, which usually develops in women between the ages of 35 and 50. As it presents with a wide range of symptoms, diagnosis is based on histological findings. Here we present the case of a 27-year-old white woman with a painless vaginal mass associated with dyspareunia, dysmenorrhea and foul-smelling vaginal discharge. She underwent surgery by vaginal route, and the histological findings confirmed a myxoid leiomyoma. After 17 months, the patient was disease-free, and symptoms disappeared. Vaginal leiomyomas are rare, local recurrence and transformation into sarcoma are rare, and complete surgical excision as soon as possible is recommended.

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

PubMed

Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea; Burdick, Ryan C; Levine, Louis; Li, Kelvin; Rein, Alan; Pathak, Vinay K; Hu, Wei-Shau

2017-08-15

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly

Inconspicuous Insertion 22;12 in Myxoid/Round Cell Liposarcoma Accompanied by the Secondary Structural Abnormality der(16)t(1;16)

PubMed Central

Birch, Nathan C.; Antonescu, Cristina R.; Nelson, Marilu; Sarran, Lisa; Neff, James R.; Seemayer, Thomas; Bridge, Julia A.

2003-01-01

In myxoid/round cell liposarcoma, the t(12;16)(q13;p11) and its associated fusion transcript, FUS-CHOP, characterize greater than 95% of cases. The variant translocation t(12;22)(q13;q12) and associated EWS-CHOP fusion transcript are rare. A second non-random aberration observed in roughly 20% of Ewing’s sarcomas, and to a lesser extent other select sarcomas, is the unbalanced 1;16 translocation. Recognition of this secondary aberration in the absence of an obvious primary karyotypic abnormality strongly suggests that the use of other genetic approaches will be informative in uncovering a clinically suspected primary anomaly. The following case illustrates the utility of molecular cytogenetic and reverse transcriptase-polymerase chain reaction techniques in diagnosing an ins(22;12)(q12;q13q14) and associated EWS-CHOP fusion transcript in a myxoid/round cell liposarcoma exhibiting a der(16)t(1;16)(q11;q11). PMID:12876210

Inconspicuous insertion 22;12 in myxoid/round cell liposarcoma accompanied by the secondary structural abnormality der(16)t(1;16).

PubMed

Birch, Nathan C; Antonescu, Cristina R; Nelson, Marilu; Sarran, Lisa; Neff, James R; Seemayer, Thomas; Bridge, Julia A

2003-08-01

In myxoid/round cell liposarcoma, the t(12;16)(q13;p11) and its associated fusion transcript, FUS-CHOP, characterize greater than 95% of cases. The variant translocation t(12;22)(q13;q12) and associated EWS-CHOP fusion transcript are rare. A second non-random aberration observed in roughly 20% of Ewing's sarcomas, and to a lesser extent other select sarcomas, is the unbalanced 1;16 translocation. Recognition of this secondary aberration in the absence of an obvious primary karyotypic abnormality strongly suggests that the use of other genetic approaches will be informative in uncovering a clinically suspected primary anomaly. The following case illustrates the utility of molecular cytogenetic and reverse transcriptase-polymerase chain reaction techniques in diagnosing an ins(22;12)(q12;q13q14) and associated EWS-CHOP fusion transcript in a myxoid/round cell liposarcoma exhibiting a der(16)t(1;16)(q11;q11).

Preparation of BFV Gag antiserum and preliminary study on cellular distribution of BFV.

PubMed

Wang, Jian; Guo, Hong-yan; Jia, Rui; Xu, Xuan; Tan, Juan; Geng, Yun-qi; Qiao, Wen-tao

2010-04-01

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Synchronized HIV assembly by tunable PIP2 changes reveals PIP2 requirement for stable Gag anchoring

PubMed Central

Mücksch, Frauke; Laketa, Vibor; Müller, Barbara; Schultz, Carsten; Kräusslich, Hans-Georg

2017-01-01

HIV-1 assembles at the plasma membrane (PM) of infected cells. PM association of the main structural protein Gag depends on its myristoylated MA domain and PM PI(4,5)P2. Using a novel chemical biology tool that allows rapidly tunable manipulation of PI(4,5)P2 levels in living cells, we show that depletion of PI(4,5)P2 completely prevents Gag PM targeting and assembly site formation. Unexpectedly, PI(4,5)P2 depletion also caused loss of pre-assembled Gag lattices from the PM. Subsequent restoration of PM PI(4,5)P2 reinduced assembly site formation even in the absence of new protein synthesis, indicating that the dissociated Gag molecules remained assembly competent. These results reveal an important role of PI(4,5)P2 for HIV-1 morphogenesis beyond Gag recruitment to the PM and suggest a dynamic equilibrium of Gag-lipid interactions. Furthermore, they establish an experimental system that permits synchronized induction of HIV-1 assembly leading to induced production of infectious virions by targeted modulation of Gag PM targeting. DOI: http://dx.doi.org/10.7554/eLife.25287.001 PMID:28574338

Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

PubMed

Mullins, Christina S; Hühns, Maja; Krohn, Mathias; Peters, Sven; Cheynet, Valérie; Oriol, Guy; Guillotte, Michèle; Ducrot, Sandrine; Mallet, François; Linnebacher, Michael

2016-01-01

A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals.

PubMed

Mendiratta, Sanjay; Vajpayee, Madhu; Malhotra, Uma; Kaushik, Shweta; Dar, Lalit; Mojumdar, Kamalika; Chauhan, Neeraj Kumar; Sreenivas, Vishnubhatla

2009-02-01

Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-gamma ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = -0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.

Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid

PubMed Central

Dutta, Moumita; Pollard, Dominic J.; Goldstone, David C.; Ramos, Andres; Müllers, Erik; Stirnnagel, Kristin; Stanke, Nicole; Lindemann, Dirk; Taylor, William R.; Rosenthal, Peter B.

2016-01-01

The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold. PMID:27829070

Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro.

PubMed Central

Sakalian, M; Parker, S D; Weldon, R A; Hunter, E

1996-01-01

The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly. PMID:8648705

Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA.

PubMed

Bartels, Hanni; Luban, Jeremy

2014-09-12

All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated. The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag-only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto

Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells.

PubMed

Llewellyn, G Nicholas; Hogue, Ian B; Grover, Jonathan R; Ono, Akira

2010-10-28

T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS), are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs.

Vaccination directed against the human endogenous retrovirus-K (HERV-K) gag protein slows HERV-K gag expressing cell growth in a murine model system.

PubMed

Kraus, Benjamin; Fischer, Katrin; Sliva, Katja; Schnierle, Barbara S

2014-03-26

Human endogenous retroviruses (HERVs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. However enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed inter-alia in HIV-infected individuals and tumor patients. Therefore HERV-K might serve as a tumor-specific antigen or even as a constant target for the development of an HIV vaccine. To verify our hypothesis, we tested the immunogenicity of HERV-K Gag by using a recombinant vaccinia virus (MVA-HKcon) expressing the HERV-K Gag protein and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) and the HERV-K Gag protein (RLZ-HKGag cells). Subcutaneous application of RLZ-HKGag cells into syngenic BALB/c mice resulted in the formation of local tumors in MVA vaccinated mice. MVA-HKcon vaccination reduced the tumor growth. Furthermore, intravenous injection of RLZ-HKGag cells led to the formation of pulmonary metastases. Vaccination of tumor-bearing mice with MVA-HKcon drastically reduced the number of pulmonary RLZ-HKGag tumor nodules compared to vaccination with wild-type MVA. The data demonstrate that HERV-K Gag is a useful target for vaccine development and might offer new treatment opportunities for cancer patients.

Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples

PubMed Central

Mullins, Christina S.; Hühns, Maja; Krohn, Mathias; Peters, Sven; Cheynet, Valérie; Oriol, Guy; Guillotte, Michèle; Ducrot, Sandrine; Mallet, François; Linnebacher, Michael

2016-01-01

Introduction A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). Results The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. Conclusion Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut. PMID:27119520

Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag.

PubMed

Grewe, Bastian; Hoffmann, Bianca; Ohs, Inga; Blissenbach, Maik; Brandt, Sabine; Tippler, Bettina; Grunwald, Thomas; Uberla, Klaus

2012-03-01

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

NASA Technical Reports Server (NTRS)

Spooner, B. S.; Bassett, K. E.; Spooner, B. S. Jr

1993-01-01

The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.

Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.

PubMed

Comas-Garcia, Mauricio; Datta, Siddhartha Ak; Baker, Laura; Varma, Rajat; Gudla, Prabhakar R; Rein, Alan

2017-07-20

Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis -acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.

Analysis of Bovine Leukemia Virus Gag Membrane Targeting and Late Domain Function

PubMed Central

Wang, Huating; Norris, Kendra M.; Mansky, Louis M.

2002-01-01

Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the Deltaretrovirus genus of the Retroviridae family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain). PMID:12134053

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

PubMed Central

Enssle, J; Jordan, I; Mauer, B; Rethwilm, A

1996-01-01

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses. Images Fig. 3 PMID:8633029

Primary extraskeletal myxoid chondrosarcoma of bone: Report of three cases and review of the literature.

PubMed

Finos, L; Righi, A; Frisoni, T; Gambarotti, M; Ghinelli, C; Benini, S; Vanel, D; Picci, P

2017-05-01

Extraskeletal myxoid chondrosarcoma is a rare neoplasm of soft tissue. The usual location is in deep parts of the proximal extremities and limb girdles in middle-aged adults. The bone location as primary location is extremely rare and few cases are reported. We present three cases arising in bone with molecular confirmation using both RT-PCR and FISH analysis. Patients include two men and one woman with an age of 62, 69 and 73 years old. The mean size of the lesion was 13cm (range 8-18cm). Tumors arose in the iliac bone in two cases and in the proximal humerus in the other case. At time of diagnosis the three cases show bone cortex and soft tissue involvement. On imaging, lesions have a lobular pattern, are purely lytic, but take up contrast medium after injection. Two patients are alive with disease (local recurrence and lung metastasis) after five years and five years and six months, respectively and one patient died of disease two years after the diagnosis. The primary extraskeletal myxoid chondrosarcoma of bone seems to have a more aggressive behavior than the soft tissue counterpart. The molecular confirmation of diagnosis using RT-PCR is necessary to do the differential diagnosis with other entities, in particular with myoepithelioma that shows similar morphological features and EWSR1 and FUS genes rearrangement. Copyright © 2017 Elsevier GmbH. All rights reserved.

Matrix expansion and syncytial aggregation of syndecan-1+ cells underpin villous atrophy in coeliac disease.

PubMed

Salvestrini, Camilla; Lucas, Mark; Lionetti, Paolo; Torrente, Franco; James, Sean; Phillips, Alan D; Murch, Simon H

2014-01-01

We studied the expression of sulphated glycosaminoglycans (GAGs) in coeliac disease (CD) mucosa, as they are critical determinants of tissue volume, which increases in active disease. We also examined mucosal expression of IL-6, which stimulates excess GAG synthesis in disorders such as Grave's ophthalmopathy. We stained archival jejunal biopsies from 5 children with CD at diagnosis, on gluten-free diet and challenge for sulphated GAGs. We then examined duodenal biopsies from 9 children with CD compared to 9 histological normal controls, staining for sulphated GAGs, heparan sulphate proteoglycans (HSPG), short-chain HSPG (Δ-HSPG) and the proteoglycan syndecan-1 (CD138), which is expressed on epithelium and plasma cells. We confirmed findings with a second monoclonal in another 12 coeliac children. We determined mucosal IL-6 expression by immunohistochemistry and PCR in 9 further cases and controls, and used quantitative real time PCR for other Th17 pathway cytokines in an additional 10 cases and controls. In CD, HSPG expression was lost in the epithelial compartment but contrastingly maintained within an expanded lamina propria. Within the upper lamina propria, clusters of syndecan-1(+) plasma cells formed extensive syncytial sheets, comprising adherent plasma cells, lysed cells with punctate cytoplasmic staining and shed syndecan ectodomains. A dense infiltrate of IL-6(+) mononuclear cells was detected in active coeliac disease, also localised to the upper lamina propria, with significantly increased mRNA expression of IL-6 and IL-17A but not IL-23 p19. Matrix expansion, through syndecan-1(+) cell recruitment and lamina propria GAG increase, underpins villous atrophy in coeliac disease. The syndecan-1(+) cell syncytia and excess GAG production recapitulate elements of the invertebrate encapsulation reaction, itself dependent on insect transglutaminase and glutaminated early response proteins. As in other matrix expansion disorders, IL-6 is upregulated and

Matrix Expansion and Syncytial Aggregation of Syndecan-1+ Cells Underpin Villous Atrophy in Coeliac Disease

PubMed Central

Salvestrini, Camilla; Lucas, Mark; Lionetti, Paolo; Torrente, Franco; James, Sean; Phillips, Alan D.; Murch, Simon H.

2014-01-01

Background We studied the expression of sulphated glycosaminoglycans (GAGs) in coeliac disease (CD) mucosa, as they are critical determinants of tissue volume, which increases in active disease. We also examined mucosal expression of IL-6, which stimulates excess GAG synthesis in disorders such as Grave's ophthalmopathy. Methods We stained archival jejunal biopsies from 5 children with CD at diagnosis, on gluten-free diet and challenge for sulphated GAGs. We then examined duodenal biopsies from 9 children with CD compared to 9 histological normal controls, staining for sulphated GAGs, heparan sulphate proteoglycans (HSPG), short-chain HSPG (Δ-HSPG) and the proteoglycan syndecan-1 (CD138), which is expressed on epithelium and plasma cells. We confirmed findings with a second monoclonal in another 12 coeliac children. We determined mucosal IL-6 expression by immunohistochemistry and PCR in 9 further cases and controls, and used quantitative real time PCR for other Th17 pathway cytokines in an additional 10 cases and controls. Results In CD, HSPG expression was lost in the epithelial compartment but contrastingly maintained within an expanded lamina propria. Within the upper lamina propria, clusters of syndecan-1+ plasma cells formed extensive syncytial sheets, comprising adherent plasma cells, lysed cells with punctate cytoplasmic staining and shed syndecan ectodomains. A dense infiltrate of IL-6+ mononuclear cells was detected in active coeliac disease, also localised to the upper lamina propria, with significantly increased mRNA expression of IL-6 and IL-17A but not IL-23 p19. Conclusions Matrix expansion, through syndecan-1+ cell recruitment and lamina propria GAG increase, underpins villous atrophy in coeliac disease. The syndecan-1+ cell syncytia and excess GAG production recapitulate elements of the invertebrate encapsulation reaction, itself dependent on insect transglutaminase and glutaminated early response proteins. As in other matrix expansion disorders

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

PubMed Central

Kean, Thomas J.; Dennis, James E.

2015-01-01

Background Current tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential. Methods Porcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically. Results Human chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions. Conclusions

HIV-1 Gag as an Antiviral Target: Development of Assembly and Maturation Inhibitors.

PubMed

Spearman, Paul

2016-01-01

HIV-1 Gag is the master orchestrator of particle assembly. The central role of Gag at multiple stages of the HIV lifecycle has led to efforts to develop drugs that directly target Gag and prevent the formation and release of infectious particles. Until recently, however, only the catalytic site protease inhibitors have been available to inhibit late stages of HIV replication. This review summarizes the current state of development of antivirals that target Gag or disrupt late events in the retrovirus lifecycle such as maturation of the viral capsid. Maturation inhibitors represent an exciting new series of antiviral compounds, including those that specifically target CA-SP1 cleavage and the allosteric integrase inhibitors that inhibit maturation by a completely different mechanism. Numerous small molecules and peptides targeting CA have been studied in attempts to disrupt steps in assembly. Efforts to target CA have recently gained considerable momentum from the development of small molecules that bind CA and alter capsid stability at the post-entry stage of the lifecycle. Efforts to develop antivirals that inhibit incorporation of genomic RNA or to inhibit late budding events remain in preliminary stages of development. Overall, the development of novel antivirals targeting Gag and the late stages in HIV replication appears much closer to success than ever, with the new maturation inhibitors leading the way.

Gag grouper larvae pathways on the West Florida Shelf

NASA Astrophysics Data System (ADS)

Weisberg, Robert H.; Zheng, Lianyuan; Peebles, Ernst

2014-10-01

A numerical circulation model, quantitatively assessed against in situ observations, is used to describe the circulation on the West Florida Continental Shelf during spring 2007 when pre-settlement gag (Mycteroperca microlepis) were present in the surf zone near Tampa Bay, Florida. The pre-settlement fish were found to be isotopically distinct from settled juveniles in the area, which is consistent with recent arrival at near shore nursery habitats from offshore spawning grounds. Simulated particle trajectories are employed to test hypotheses relating to either a surface or a near-bottom route of across-shelf transport. The surface-route hypothesis is rejected, whereas the bottom-route hypothesis is found to be consistent with the location of pre-settlement fish and their co-occurrence with macroalgae of offshore, hard-bottom origin. We conclude that gag larvae are transported to the near shore via the bottom Ekman layer and that such transport is facilitated by remote forcing associated with Gulf of Mexico Loop Current interactions with the shelf slope near the Dry Tortugas. Being that such remote forcing occurs inter-annually and not always in phase with the preferred spawning months (late winter through early spring), gag recruitment success should similarly vary with year and location.

Power and politics in international funding for reproductive health: the US Global Gag Rule.

PubMed

Crane, Barbara B; Dusenberry, Jennifer

2004-11-01

Since 2001, the US government has used its power as a leading donor to family planning programmes to pursue policies in conflict with global agreements on reproductive rights. Prominent among these policies is the Mexico City Policy (or Global Gag Rule), which restricts non-governmental organisations (NGOs) in developing countries that receive USAID family planning funding from engaging in most abortion-related activities, even with their own funds. This paper reviews the history and political origins of the Gag Rule under several Republican party presidents. The Gag Rule has not achieved an overall reduction in abortions; rather, where it has disrupted family planning services, the policy is more likely to have increased the number of abortions. This paper concludes that the Gag Rule is a radical intrusion on the rights and autonomy of recipients of US funding. Regardless of whether or not it is rescinded in the future, the underlying issues in the politics of US reproductive health assistance are likely to persist. NGOs that wish to free themselves from the constraints it imposes must find the means to end their dependence on USAID funding, including turning to other donors. NGOs should also take the lead in opposing policies such as the Gag Rule that violate global agreements.

A Temporospatial Map That Defines Specific Steps at Which Critical Surfaces in the Gag MA and CA Domains Act during Immature HIV-1 Capsid Assembly in Cells

PubMed Central

Robinson, Bridget A.; Reed, Jonathan C.; Geary, Clair D.; Swain, J. Victor

2014-01-01

ABSTRACT During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known. Here, we analyzed how mutations in these four critical surfaces affect the formation and stability of assembly intermediates in cells expressing the HIV-1 provirus. The resulting temporospatial map reveals that critical MA residues act during membrane targeting, residues in the C-terminal CA subdomain (CA-CTD) dimer interface are needed for the stability of the first membrane-bound assembly intermediate, CA-CTD base residues are necessary for progression past the first membrane-bound intermediate, and residues in the N-terminal CA subdomain (CA-NTD) stabilize the last membrane-bound intermediate. Importantly, we found that all four critical surfaces act while Gag is associated with the cellular facilitators of assembly ABCE1 and DDX6. When correlated with existing structural data, our findings suggest the following model: Gag dimerizes via the CA-CTD dimer interface just before or during membrane targeting, individual CA-CTD hexamers form soon after membrane targeting, and the CA-NTD hexameric lattice forms just prior to capsid release. This model adds an important new dimension to current structural models by proposing the potential order in which key contacts within the immature capsid lattice are made during assembly in cells. IMPORTANCE While much is known about the structure of the completed HIV-1 immature capsid and domains of its component Gag proteins, less is known about the sequence of events leading to formation of the HIV-1 immature capsid. Here we used

Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression.

PubMed

Kiguoya, Marion W; Mann, Jaclyn K; Chopera, Denis; Gounder, Kamini; Lee, Guinevere Q; Hunt, Peter W; Martin, Jeffrey N; Ball, T Blake; Kimani, Joshua; Brumme, Zabrina L; Brockman, Mark A; Ndung'u, Thumbi

2017-07-01

There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates ( r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases ( P < 0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C < D < intersubtype recombinants ( P < 0.0029), which is consistent with reported intersubtype differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes. IMPORTANCE HIV-1 subtypes

Lack of a significant impact of Gag-Protease-mediated HIV-1 replication capacity on clinical parameters in treatment-naive Japanese individuals.

PubMed

Sakai, Keiko; Chikata, Takayuki; Brumme, Zabrina L; Brumme, Chanson J; Gatanaga, Hiroyuki; Gatanag, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

2015-11-19

HLA class I-associated escape mutations in HIV-1 Gag can reduce viral replication, suggesting that associated fitness costs could impact HIV-1 disease progression. Previous studies in North American and African cohorts have reported reduced Gag-Protease mediated viral replication capacity (Gag-Pro RC) in individuals expressing protective HLA class I alleles including HLA-B*57:01, B*27:05, and B*81:01. These studies also reported significant positive associations between Gag-Pro RCs and plasma viral load (pVL). However, these HLA alleles are virtually absent in Japan, and the importance of Gag as an immune target is not clearly defined in this population. We generated chimeric NL4-3 viruses carrying patient-derived Gag-Protease from 306 treatment-naive Japanese individuals chronically infected with HIV-1 subtype B. We analyzed associations between Gag-Pro RC and clinical markers of HIV-1 infection and host HLA expression. We observed no significant correlation between Gag-Pro RC and pVL in Japan in the overall cohort. However, upon exclusion of individuals expressing Japanese protective alleles HLA-B*52:01 and B*67:01, Gag-Pro RC correlated positively with pVL and negatively with CD4 T-cell count. Our results thus contrast with studies from other global cohorts reporting significantly lower Gag-Pro RC among persons expressing protective HLA alleles, and positive relationships between Gag-Pro RC and pVL in the overall study populations. We also identified five amino acids in Gag-Protease significantly associated with Gag-Pro RC, whose effects on RC were confirmed by site-directed mutagenesis. However, of the four mutations that decreased Gag-Pro RC, none were associated with reductions in pVL in Japan though two were associated with lower pVL in North America. These data indicate that Gag fitness does not affect clinical outcomes in subjects with protective HLA class I alleles as well as the whole Japanese population. Moreover, the impact of Gag fitness costs on HIV

The Foreseeable Harms of Trump's Global Gag Rule.

PubMed

Bingenheimer, Jeffrey B; Skuster, Patty

2017-09-01

As one of his first acts as President of the United States, Donald Trump signed an executive order reinstating a version of the global gag rule. Under this rule, US grantees are barred from receiving global health funding if they engage in abortion-related work: not only abortion services, but also abortion referrals and counseling or advocacy for the liberalization of abortion laws. Critics of the Trump global gag rule generally raise three classes of objections: (1) that the rule fails to accomplish its presumed objective of reducing the number of abortions; (2) that it negatively affects the health and well-being of individuals and populations in affected countries; and (3) that it interferes with governments' ability to meet their international obligations. In this commentary, we examine the scientific and policy bases for these criticisms. © 2017 The Population Council, Inc.

Maternal LAMP/p55gagHIV-1 DNA immunization induces in utero priming and a long-lasting immune response in vaccinated neonates.

PubMed

Rigato, Paula Ordonhez; Maciel, Milton; Goldoni, Adriana Letícia; Piubelli, Orlando Guerra; Orii, Noemia Mie; Marques, Ernesto Torres; August, Joseph Thomas; Duarte, Alberto José da Silva; Sato, Maria Notomi

2012-01-01

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.

Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

PubMed Central

Rigato, Paula Ordonhez; Maciel, Milton; Goldoni, Adriana Letícia; Piubelli, Orlando Guerra; Orii, Noemia Mie; Marques, Ernesto Torres; August, Joseph Thomas; Duarte, Alberto José da Silva; Sato, Maria Notomi

2012-01-01

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods. PMID:22355381

The long terminal repeat-containing retrotransposon Tf1 possesses amino acids in gag that regulate nuclear localization and particle formation.

PubMed

Kim, Min-Kyung; Claiborn, Kathryn C; Levin, Henry L

2005-08-01

Tf1 is a long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe that is studied to further our understanding of retrovirus propagation. One important application is to examine Tf1 as a model for how human immunodeficiency virus type 1 proteins enter the nucleus. The accumulation of Tf1 Gag in the nucleus requires an N-terminal nuclear localization signal (NLS) and the nuclear pore factor Nup124p. Here, we report that NLS activity is regulated by adjacent residues. Five mutant transposons were made, each with sequential tracts of four amino acids in Gag replaced by alanines. All five versions of Tf1 transposed with frequencies that were significantly lower than that of the wild type. Although all five made normal amounts of Gag, two of the mutations did not make cDNA, indicating that Gag contributed to reverse transcription. The localization of the Gag in the nucleus was significantly reduced by mutations A1, A2, and A3. These results identified residues in Gag that contribute to the function of the NLS. The Gags of A4 and A5 localized within the nucleus but exhibited severe defects in the formation of virus-like particles. Of particular interest was that the mutations in Gag-A4 and Gag-A5 caused their nuclear localization to become independent of Nup124p. These results suggested that Nup124p was only required for import of Tf1 Gag because of its extensive multimerization.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

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

Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia

2010-10-10

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses,more » as measured by the breadth of the Gag peptide-specific IFN-{gamma}, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.« less

Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

PubMed Central

Urata, Shuzo; Yokosawa, Hideyoshi; Yasuda, Jiro

2007-01-01

Background HTLV-1 Gag protein is a matrix protein that contains the PTAP and PPPY sequences as L-domain motifs and which can be released from mammalian cells in the form of virus-like particles (VLPs). The cellular factors Tsg101 and Nedd4.1 interact with PTAP and PPPY, respectively, within the HTLV-1 Gag polyprotein. Tsg101 forms a complex with Vps28 and Vps37 (ESCRT-I complex) and plays an important role in the class E Vps pathway, which mediates protein sorting and invagination of vesicles into multivesicular bodies. Nedd4.1 is an E3 ubiquitin ligase that binds to the PPPY motif through its WW motif, but its function is still unknown. In the present study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using dominant negative (DN) forms of the class E proteins. Results Here, we report that DN forms of Vps4A, Vps4B, and AIP1 inhibit HTLV-1 budding. Conclusion These findings suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be targets for prevention of mother-to-infant vertical transmission of the virus. PMID:17601348

Replication-defective Friend murine leukemia virus particles containing uncleaved gag polyproteins and decreased levels of envelope glycoprotein.

PubMed Central

Collins, J K; Chesebro, B

1981-01-01

An erythroleukemia cell clone, 7C, which failed to produce reverse transcriptase-containing virions or infectious virus, was found to produce noninfectious virus particles by gradient banding of [3H]leucine- and [3H]uridine-labeled virions. The RNA from the 7C virus was shown to consist of the normal 70S size component, which converted to 35S upon heat denaturation. In contrast, the 7C virion proteins showed multiple defects. Analysis of the virion proteins by gel electrophoresis demonstrated that the pr65 gag precursor was incorporated into the 7C virus and that the processing of this precursor was severely diminished. Polymerase proteins pr180gag-pol and pr120pol were also detected in virions, and a third possible polymerase protein, p70, was reduced in size compared to its normal counterpart, p80. Incorporation of the viral gp70 glycoprotein into particles was also reduced 10-fold, despite synthesis and incorporation of gp70 into the 7C cell membrane in normal amounts. Pulse-chase analysis of the synthesis of the viral gag and env proteins in 7C cells showed greatly reduced amounts of pr180gag-pol, pr65gag, p80gag, and p42gag, whereas pr90env, gp70, and spleen focus-forming virus-specific gp55 were synthesized and processed normally. These results suggested that at least one defect in 7C virus was impaired cleavage of gag or pol proteins or both, most likely due to a lack of the appropriate viral protease, and that this lack of cleavage might affect incorporation of gp70 into virus particles. Images PMID:6163868

Heparin (GAG-hed) inhibits LCR activity of human papillomavirus type 18 by decreasing AP1 binding.

PubMed

Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Angel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

2006-08-31

High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data suggest that GAG-hed could have antitumoral

Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release.

PubMed Central

Weldon, R A; Wills, J W

1993-01-01

Retroviral Gag proteins have the ability to induce budding and particle release from the plasma membrane when expressed in the absence of all of the other virus-encoded components; however, the locations of the functional domains within the Gag protein that are important for this process are poorly understood. It was shown previously that the protease sequence of the Rous sarcoma virus (RSV) Gag protein can be replaced with a foreign polypeptide, iso-1-cytochrome c from a yeast, without disrupting particle assembly (R. A. Weldon, Jr., C. R. Erdie, M. G. Oliver, and J. W. Wills, J. Virol. 64:4169-4179, 1990). An unexpected product of the chimeric gag gene is a small, Gag-related protein named p25C. This product was of interest because of its high efficiency of packaging into particles. The goal of the experiments described here was to determine the mechanism by which p25C is synthesized and packaged into particles. The results demonstrate that it is not the product of proteolytic processing of the Gag-cytochrome precursor but is derived from an unusual spliced mRNA. cDNA clones of the spliced mRNA were obtained, and each expressed a product of approximately 25 kDa, designated p25M1, which was released into the growth medium in membrane-enclosed particles that were much lighter than authentic retrovirions as measured in sucrose density gradients. DNA sequencing revealed that the clones encode the first 180 of the 701 amino acids of the RSV Gag protein and no residues from iso-1-cytochrome c. This suggested that a domain in the carboxy-terminal half of Gag is important for the packaging of Gag proteins into dense arrays within the particles. In support of this hypothesis, particles of the correct density were obtained when a small segment from the carboxy terminus of the RSV Gag protein (residues 417 to 584) was included on the end of p25. Images PMID:8394460

Preferential Ty1 retromobility in mother cells and nonquiescent stationary phase cells is associated with increased concentrations of total Gag or processed Gag and is inhibited by exposure to a high concentration of calcium.

PubMed

Peifer, Andrew C; Maxwell, Patrick H

2018-03-21

Retrotransposons are abundant mobile DNA elements in eukaryotic genomes that are more active with age in diverse species. Details of the regulation and consequences of retrotransposon activity during aging remain to be determined. Ty1 retromobility in Saccharomyces cerevisiae is more frequent in mother cells compared to daughter cells, and we found that Ty1 was more mobile in nonquiescent compared to quiescent subpopulations of stationary phase cells. This retromobility asymmetry was absent in mutant strains lacking BRP1 that have reduced expression of the essential Pma1p plasma membrane proton pump, lacking the mRNA decay gene LSM1 , and in cells exposed to a high concentration of calcium. Mother cells had higher levels of Ty1 Gag protein than daughters. The proportion of protease-processed Gag decreased as cells transitioned to stationary phase, processed Gag was the dominant form in nonquiescent cells, but was virtually absent from quiescent cells. Treatment with calcium reduced total Gag levels and the proportion of processed Gag, particularly in mother cells. We also found that Ty1 reduced the fitness of proliferating but not stationary phase cells. These findings may be relevant to understanding regulation and consequences of retrotransposons during aging in other organisms, due to conserved impacts and regulation of retrotransposons.

Preferential Ty1 retromobility in mother cells and nonquiescent stationary phase cells is associated with increased concentrations of total Gag or processed Gag and is inhibited by exposure to a high concentration of calcium

PubMed Central

Peifer, Andrew C.

2018-01-01

Retrotransposons are abundant mobile DNA elements in eukaryotic genomes that are more active with age in diverse species. Details of the regulation and consequences of retrotransposon activity during aging remain to be determined. Ty1 retromobility in Saccharomyces cerevisiae is more frequent in mother cells compared to daughter cells, and we found that Ty1 was more mobile in nonquiescent compared to quiescent subpopulations of stationary phase cells. This retromobility asymmetry was absent in mutant strains lacking BRP1 that have reduced expression of the essential Pma1p plasma membrane proton pump, lacking the mRNA decay gene LSM1, and in cells exposed to a high concentration of calcium. Mother cells had higher levels of Ty1 Gag protein than daughters. The proportion of protease-processed Gag decreased as cells transitioned to stationary phase, processed Gag was the dominant form in nonquiescent cells, but was virtually absent from quiescent cells. Treatment with calcium reduced total Gag levels and the proportion of processed Gag, particularly in mother cells. We also found that Ty1 reduced the fitness of proliferating but not stationary phase cells. These findings may be relevant to understanding regulation and consequences of retrotransposons during aging in other organisms, due to conserved impacts and regulation of retrotransposons. PMID:29562219

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

PubMed

Atwood, A; Lin, J H; Levin, H L

1996-01-01

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Acid glycosaminoglycan (aGAG) excretion is increased in children with autism spectrum disorder, and it can be controlled by diet.

PubMed

Endreffy, Ildikó; Bjørklund, Geir; Dicső, Ferenc; Urbina, Mauricio A; Endreffy, Emőke

2016-04-01

Autism research continues to receive considerable attention as the options for successful management are limited. The understanding of the autism spectrum disorder (ASD) etiology has now progressed to encompass genetic, epigenetic, neurological, hormonal, and environmental factors that affect outcomes for patients with ASD. Glycosaminoglycans (GAGs) are a family of linear, sulfated polysaccharides that are associated with central nervous system (CNS) development, maintenance, and disorders. Proteoglycans (PG) regulate diverse functions in the central nervous system. Heparan sulfate (HS) and chondroitin sulfate (CS) are two major GAGs present in the PGs of the CNS. As neuroscience advances, biochemical treatments to correct brain chemistry become better defined. Nutrient therapy can be very potent and has minimal to no side effects, since no molecules foreign to the body are needed. Given GAGs are involved in several neurological functions, and that its level can be somewhat modulated by the diet, the present study aimed to evaluate the role of GAGs levels in ASD symptoms. Both tGAG and its different fractions were evaluated in the urine of ASD and healthy control childrens. As levels differed between groups, a second trial was conduted evaluating if diet could reduce tGAG levels and if this in turn decrease ASD symptoms. The present study found that tGAG concentration was significantly higher in the urine of children with ASD compared to healthy control children and this was also evident in all GAG fractions. Within groups (controls and ASD), no gender differences in GAG excretion were found. The use of a 90 days elimination diet (casein-free, special carbohydrates, multivitamin/mineral supplement), had major effects in reducing urinary tGAG excretion in children with ASD.

Maturation of the Gag core decreases the stability of retroviral lipid membranes.

PubMed

Davidoff, Candice; Payne, Riley J; Willis, Sharon H; Doranz, Benjamin J; Rucker, Joseph B

2012-11-25

To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core. Copyright © 2012 Elsevier Inc. All rights reserved.

Maturation of the Gag core decreases the stability of retroviral lipid membranes

PubMed Central

Davidoff, Candice; Payne, Riley; Willis, Sharon H.; Doranz, Benjamin J.; Rucker, Joseph B.

2012-01-01

To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core. PMID:22995186

The Effect of Various Concentrations of Nitrous Oxide and Oxygen on the Hypersensitive Gag Reflex.

PubMed

De Veaux, Candace K E; Montagnese, Thomas A; Heima, Masahiro; Aminoshariae, Anita; Mickel, Andre

2016-01-01

The purpose of this study was to compare the effectiveness of various concentrations of N 2 O/O 2 on obtunding a hypersensitive gag reflex. We hypothesized that the administration of nitrous oxide and oxygen would obtund a hypersensitive gag reflex enough to allow a patient to tolerate the placement and holding of a digital x-ray sensor long enough to obtain a dental radiograph. Volunteers claiming to have a hypersensitive gag reflex were first screened to validate their claim and then tested by placing a size 2 digital x-ray sensor in the position for a periapical radiograph of the right mandibular molar area and holding it in place for 10 seconds. Subjects were first tested using room air only, then 30%, 50%, or 70% nitrous oxide until they were able to tolerate the sensor without gagging or discomfort. A visual analog scale was used for subjective responses, and other statistical tests were used to analyze the results. We found that for some subjects, 30% nitrous oxide was sufficient; for others, 50% was needed; and for the remainder of the subjects, 70% was sufficient to tolerate the test. Using a combination of 70% nitrous oxide and 30% oxygen allowed all patients claiming to have a hypersensitive gag reflex to tolerate the placement and holding of a digital x-ray sensor long enough to take a periapical radiograph.

Comparison of the up-conversion photoluminescence for GAP, GAG and GAM phosphors

NASA Astrophysics Data System (ADS)

Deng, Taoli; Jiang, Xianbang

2018-04-01

GdAlO3:Er3+/Yb3+, Gd3Al5O12:Er3+/Yb3+ and Gd4Al2O9:Er3+/Yb3+ phosphors were prepared by co-precipitation. The effects for Gd2O3-Al2O3 composite oxides as the host materials with different crystal structures such as GdAlO3, Gd3Al5O12 and Gd4Al2O9 were investigated. It was found that the perovskite structured GdAlO3:Er3+/Yb3+ (GAP phosphor) could be obtained from the precursor when the calcination temperature was 1000 °C, while the garnet structured Gd3Al5O12:Er3+/Yb3+ (GAG phosphor) could be formed when the calcination temperature was 1300 °C, but the monoclinic-structured Gd4Al2O9:Er3+/Yb3+ (GAM phosphor) could be formed only when the calcination temperature was raised up to 1500 °C. The difference of the up-conversion photoluminescence (UCPL) spectra under 980 nm between the GAP, GAG and GAM phosphors was studied. The result showed that the UCPL intensity of the GAP phosphor was close to that of the GAM phosphor with much higher red-to-green intensity ratio than that of GAP phosphor. The UCPL intensity of GAG phosphor was the weakest among them. Finally, the factors which influenced on the UCPL of the GAP, GAG and GAM phosphors were discussed.

Interactions between nattokinase and heparin/GAGs.

PubMed

Zhang, Fuming; Zhang, Jianhua; Linhardt, Robert J

2015-12-01

Nattokinase (NK) is a serine protease extracted from a traditional Japanese food called natto. Due to its strong fibrinolytic and thrombolytic activity, NK is regarded as a valuable dietary supplement or nutraceutical for the oral thrombolytic therapy. In addition, NK has been investigated for some other medical applications including treatment of hypertension, Alzheimer's disease, and vitreoretinal disorders. The most widely used clinical anticoagulants are heparin and low molecular weight heparins. The interactions between heparin and proteins modulate diverse patho-physiological processes and heparin modifies the activity of serine proteases. Indeed, heparin plays important roles in almost all of NK's potential therapeutically applications. The current report relies on surface plasmon resonance spectroscopy to examine NK interacting with heparin as well as other glycosaminoglycans (GAGs). These studies showed that NK is a heparin binding protein with an affinity of ~250 nM. Examination with differently sized heparin oligosaccharides indicated that the interaction between NK and heparin is chain-length dependent and the minimum size for heparin binding is a hexasaccharide. Studies using chemically modified heparin showed the 6-O-sulfo as well as the N-sulfo groups but not the 2-O-sulfo groups within heparin, are essential for heparin's interaction with NK. Other GAGs (including HS, DS, and CSE) displayed modest binding affinity to NK. NK also interfered with other heparin-protein interactions, including heparin's interaction with antithrombin and fibroblast growth factors.

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

PubMed Central

Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

2016-01-01

The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane. PMID:27120610

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

PubMed

Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

2016-04-25

The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

Primary extraskeletal myxoid chondrosarcoma in cerebellum: A case report with literature review.

PubMed

Qin, You; Zhang, Hai-Bo; Ke, Chang-Shu; Huang, Jing; Wu, Bian; Wan, Chao; Yang, Chen-Su; Yang, Kun-Yu

2017-11-01

Extraskeletal myxoid chondrosarcoma (EMC) is a rare malignant neoplasm of which intracranial EMC is the rarest. We present an unusual case report of a 41-year-old woman who was sent to the emergency department for a sudden headache and other symptoms related to increased intracranial pressure. Emergent CT revealed an occupying lesion in the left cerebellum with surrounding edema. A complete surgical excision of the lesion through a transcortical approach was performed. After the operation, this patient received adjuvant radiotherapy and temozolomide treatment. Pathology diagnosis was an intracranial EMC. The patient survives with no tumor recurrence as of the last follow-up. Progression-free survival exceeded 20 months. We have reviewed the literature and here summarize the diagnosis and treatment options for intracranial EMC. Diagnosis and treatment options of this rare disease are discussed. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging.

PubMed

Nikolaitchik, Olga A; Hu, Wei-Shau

2014-04-01

A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5' untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5' end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1

Deciphering the Role of the Gag-Pol Ribosomal Frameshift Signal in HIV-1 RNA Genome Packaging

PubMed Central

Nikolaitchik, Olga A.

2014-01-01

ABSTRACT A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5′ untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. IMPORTANCE To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5′ end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important

Palateless custom bar supported overdenture: a treatment modality to treat patient with severe gag reflex.

PubMed

Singh, Kunwarjeet; Gupta, Nidhi

2012-01-01

To suggest a custom bar supported overdenture treatment modality for prosthodontic management of patients with severe gag reflex. Some patients have a severe gag reflex and cannot tolerate conventional maxillary complete dentures with maximum palatal coverage and extensions of all borders. The condition further gets complicated in patients suffering from respiratory problems along with severe gag reflex. Severe gagging acts as a barrier to treat such patients with accepted clinical procedures and prevent patients from wearing the prosthesis. By saving some of the remaining natural teeth and fabricating, a horse shoe shape palateless simple tooth or bar supported overdenture can be successfully used for treating such patients. The remaining maxillary right and left canines were prepared with the tapered round end diamond bur to receive copings of custom bar after intentional root canal treatment of same teeth. Impression was made with light body and putty of the polyvinyl siloxane elastomer with double step putty wash technique. Impression was poured with die stone. Wax pattern of copings with bar was fabricated with inlay wax which was invested and casted. After retrieving the bar, it was finished and its fit was evaluated. The coping-bar assembly was finally cemented with the glass ionomer cement. Palateless overdenture was fabricated by conventional technique used for the fabrication of complete denture. Palateless custom bar supported overdenture procedure can be successfully used for the management of patients with severe gag reflex with improved denture retention, stability, chewing efficiency and comfort of the patient.

Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1).

PubMed

Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

2013-10-01

In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.

PubMed

Barajas, Brook C; Tanaka, Motoko; Robinson, Bridget A; Phuong, Daryl J; Chutiraka, Kasana; Reed, Jonathan C; Lingappa, Jaisri R

2018-04-01

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging

PubMed Central

Barajas, Brook C.; Tanaka, Motoko; Robinson, Bridget A.; Phuong, Daryl J.; Reed, Jonathan C.

2018-01-01

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing

Evaluation of humoral, mucosal, and cellular immune responses following co-immunization of HIV-1 Gag and Env proteins expressed by Newcastle disease virus

PubMed Central

Khattar, Sunil K; Palaniyandi, Senthilkumar; Samal, Sweety; LaBranche, Celia C; Montefiori, David C; Zhu, Xiaoping; Samal, Siba K

2015-01-01

The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In this study, we used NDV vaccine strain LaSota to generate rNDV (rLaSota/optGag) expressing human codon optimized p55 Gag protein of HIV-1. We examined the effect of co-immunization of rLaSota/optGag with rNDVs expressing different forms of Env protein gp160, gp120, gp140L [a version of gp140 that lacked cytoplasmic tail and contained complete membrane-proximal external region (MPER)] and gp140S (a version of gp140 that lacked cytoplasmic tail and distal half of MPER) on magnitude and breadth of humoral, mucosal and cellular immune responses in guinea pigs and mice. Our results showed that inclusion of rLaSota/optGag with rNDVs expressing different forms of Env HIV Gag did not affect the Env-specific humoral and mucosal immune responses in guinea pigs and that the potent immune responses generated against Env persisted for at least 13 weeks post immunization. The highest Env-specific humoral and mucosal immune responses were observed with gp140S+optGag group. The neutralizing antibody responses against HIV strains BaL.26 and MN.3 induced by gp140S+optGag and gp160+optGag were higher than those elicited by other groups. Inclusion of Gag with gp160, gp140S and gp140L enhanced the level of Env-specific IFN-γ-producing CD8+ T cells in mice. Inclusion of Gag with gp160 and gp140L also resulted in increased Env-specific CD4+ T cells. The level of Gag-specific CD8+ and CD4+ T cells was also enhanced in mice immunized with Gag along with gp140S and gp120. These results indicate lack of antigen interference in a vaccine containing rNDVs expressing Env and Gag proteins. PMID:25695657

Next generation sequencing of extraskeletal myxoid chondrosarcoma.

PubMed

Davis, Elizabeth J; Wu, Yi-Mi; Robinson, Dan; Schuetze, Scott M; Baker, Laurence H; Athanikar, Jyoti; Cao, Xuhong; Kunju, Lakshmi P; Chinnaiyan, Arul M; Chugh, Rashmi

2017-03-28

Extraskeletal myxoid chondrosarcoma (EMC) is an indolent translocation-associated soft tissue sarcoma with a high propensity for metastases. Using a clinical sequencing approach, we genomically profiled patients with metastatic EMC to elucidate the molecular biology and identify potentially actionable mutations. We also evaluated potential predictive factors of benefit to sunitinib, a multi-targeted tyrosine kinase inhibitor with reported activity in a subset of EMC patients. Between January 31, 2012 and April 15, 2016, six patients with EMC participated in the clinical sequencing research study. High quality DNA and RNA was isolated and matched normal samples underwent comprehensive next generation sequencing (whole or OncoSeq capture exome of tumor and normal, tumor PolyA+ and capture transcriptome). The expression levels of sunitinib targeted-kinases were measured by transcriptome sequencing for KDR, PDGFRA/B, KIT, RET, FLT1, and FLT4. The previously reported EWSR1-NR4A3 translocation was identified in all patient tumors; however, other recurring genomic abnormalities were not detected. RET expression was significantly greater in patients with EMC relative to other types of sarcomas except for liposarcoma (p<0.0002). The folate receptor was overexpressed in two patients. Our study demonstrated that similar to other translocation-associated sarcomas, the mutational profile of metastatic EMC is limited beyond the pathognomonic translocation. The clinical significance of RET expression in EMC should be explored. Additional pre-clinical investigations of EMC may help elucidate molecular mechanisms contributing to EMC tumorigenesis that could be translated to the clinical setting.

Interactions between nattokinase and heparin/GAGs

PubMed Central

Zhang, Fuming

2015-01-01

Nattokinase (NK) is a serine protease extracted from a traditional Japanese food called natto. Due to its strong fibrinolytic and thrombolytic activity, NK is regarded as a valuable dietary supplement or nutraceutical for the oral thrombolytic therapy. In addition, NK has been investigated for some other medical applications including treatment of hypertension, Alzheimer’s disease, and vitreoretinal disorders. The most widely used clinical anticoagulants are heparin and low molecular weight heparins. The interactions between heparin and proteins modulate a diverse patho-physiological processes and heparin modifies the activity of serine proteases. Indeed, heparin plays important roles in almost all of NK’s potential therapeutically applications. The current report relies on surface plasmon resonance spectroscopy to examine NK interacting with heparin as well as other glycosaminoglycans (GAGs). These studies showed that NK is a heparin binding protein with an affinity of ~250 nM. Examination with differently sized heparin oligosaccharides indicated that the interaction between NK and heparin is chain-length dependent and the minimum size for heparin binding is a hexasaccharide. Studies using chemically modified heparin showed the 6-O-sulfo as well as the N-sulfo groups but not the 2-O-sulfo groups within heparin, are essential for heparin’s interaction with NK. Other GAGs (including HS, DS, and CSE) displayed modest binding affinity to NK. NK also interfered with other heparin-protein interactions, including heparin’s interaction with antithrombin and fibroblast growth factors. PMID:26412225

Drug evaluation: bevirimat--HIV Gag protein and viral maturation inhibitor.

PubMed

Temesgen, Zelalem; Feinberg, Judith E

2006-08-01

Panacos Pharmaceuticals Inc is developing the HIV Gag protein and viral maturation inhibitor bevirimat for the potential oral treatment of HIV infection. Phase II clinical trials are underway and phase III trials expected to commence in 2007.

Modification of the dingman mouth gag for better visibility and access in the management of cleft palate.

PubMed

Rao, Latha P; Peter, Sherry

2015-03-01

Palatal and pharyngeal surgeries often require wide visibility and access. Various mouth gags and retractors have been devised and many modifications suggested to optimize these surgeries. The Dingman mouth gag, one of the commonly used retractors, offers a lot of advantages in terms of good mouth opening, tongue retraction, self-retaining cheek retractors, and anchorage for sutures, but it has a main limitation in that it allows only limited visibility of the anterior palate and alveolus. Hence, a modification of the Dingman mouth gag is presented for better visibility of and accessibility to the anterior palate.

Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus.

PubMed Central

Maurer, B; Bannert, H; Darai, G; Flügel, R M

1988-01-01

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae. Images PMID:2451755

Does granisetron eliminate the gag reflex? A crossover, double-blind, placebo-controlled pilot study.

PubMed

Barenboim, Silvina Friedlander; Dvoyris, Vladislav; Kaufman, Eliezer

2009-01-01

Although gagging is a frequent problem that, when severe, can jeopardize the dental procedure, no single protocol is used to alleviate this phenomenon. Selective 5-HT3 antagonists, such as granisetron, may attenuate gagging. In this study, granisetron and placebo were administered intravenously, in a crossover, double-blind manner, to 25 healthy volunteers in 2 different sessions. Gagging levels were recorded before and after administration, as were BP, pulse, and O2 saturation. Recorded results were analyzed with the use of tests for nonparametric values (P = .05). A significant increase in the depth of swab insertion was noted after administration of both placebo and drug. The increase in drug effectiveness correlated with decreased body weight. The true efficacy of granisetron in gagger patients with this treatment protocol has yet to be fully established, although it has been theorized that an increased dosage of granisetron may have a better effect.

Does Granisetron Eliminate the Gag Reflex? A Crossover, Double-Blind, Placebo-Controlled Pilot Study

PubMed Central

Friedlander Barenboim, Silvina; Dvoyris, Vladislav; Kaufman, Eliezer

2009-01-01

Although gagging is a frequent problem that, when severe, can jeopardize the dental procedure, no single protocol is used to alleviate this phenomenon. Selective 5-HT3 antagonists, such as granisetron, may attenuate gagging. In this study, granisetron and placebo were administered intravenously, in a crossover, double-blind manner, to 25 healthy volunteers in 2 different sessions. Gagging levels were recorded before and after administration, as were BP, pulse, and O2 saturation. Recorded results were analyzed with the use of tests for nonparametric values (P = .05). A significant increase in the depth of swab insertion was noted after administration of both placebo and drug. The increase in drug effectiveness correlated with decreased body weight. The true efficacy of granisetron in gagger patients with this treatment protocol has yet to be fully established, although it has been theorized that an increased dosage of granisetron may have a better effect. PMID:19562886

Primary Human Immunodeficiency Virus Type 1 (HIV-1) Infection during HIV-1 Gag Vaccination▿

PubMed Central

Balamurugan, Arumugam; Lewis, Martha J.; Kitchen, Christina M. R.; Robertson, Michael N.; Shiver, John W.; Daar, Eric S.; Pitt, Jacqueline; Ali, Ayub; Ng, Hwee L.; Currier, Judith S.; Yang, Otto O.

2008-01-01

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016. PMID:18199650

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

PubMed

Bauer, Asli; Podola, Lilli; Mann, Philipp; Missanga, Marco; Haule, Antelmo; Sudi, Lwitiho; Nilsson, Charlotta; Kaluwa, Bahati; Lueer, Cornelia; Mwakatima, Maria; Munseri, Patricia J; Maboko, Leonard; Robb, Merlin L; Tovanabutra, Sodsai; Kijak, Gustavo; Marovich, Mary; McCormack, Sheena; Joseph, Sarah; Lyamuya, Eligius; Wahren, Britta; Sandström, Eric; Biberfeld, Gunnel; Hoelscher, Michael; Bakari, Muhammad; Kroidl, Arne; Geldmacher, Christof

2017-09-15

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gag p37 and two vaccinations with MVA-CMDR encoding subtype A Gag p55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ + ) Gag-specific T-cell responses were dominated by CD4 + T cells ( P < 0.001 compared to CD8 + T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gag p24 regions, more conserved between prime and boost, compared to those of regions within Gag p15 (not primed) and Gag p17 (less conserved; P < 0.0001 for both). Four regions within Gag p24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens ( P = 0.04, r 2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4 + T cells and that targeted multiple antigenic regions within the conserved Gag p24 protein. IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a

Aging-associated modifications of collagen affect its degradation by matrix metalloproteinases.

PubMed

Panwar, Preety; Butler, Georgina S; Jamroz, Andrew; Azizi, Pouya; Overall, Christopher M; Brömme, Dieter

2018-01-01

The natural aging process and various pathologies correlate with alterations in the composition and the structural and mechanical integrity of the connective tissue. Collagens represent the most abundant matrix proteins and provide for the overall stiffness and resilience of tissues. The structural changes of collagens and their susceptibility to degradation are associated with skin wrinkling, bone and cartilage deterioration, as well as cardiovascular and respiratory malfunctions. Here, matrix metalloproteinases (MMPs) are major contributors to tissue remodeling and collagen degradation. During aging, collagens are modified by mineralization, accumulation of advanced glycation end-products (AGEs), and the depletion of glycosaminoglycans (GAGs), which affect fiber stability and their susceptibility to MMP-mediated degradation. We found a reduced collagenolysis in mineralized and AGE-modified collagen fibers when compared to native fibrillar collagen. GAGs had no effect on MMP-mediated degradation of collagen. In general, MMP digestion led to a reduction in the mechanical strength of native and modified collagen fibers. Successive fiber degradation with MMPs and the cysteine-dependent collagenase, cathepsin K (CatK), resulted in their complete degradation. In contrast, MMP-generated fragments were not or only poorly cleaved by non-collagenolytic cathepsins such as cathepsin V (CatV). In conclusion, our data indicate that aging and disease-associated collagen modifications reduce tissue remodeling by MMPs and decrease the structural and mechanic integrity of collagen fibers, which both may exacerbate extracellular matrix pathology. Copyright © 2017 Elsevier B.V. All rights reserved.

A Case of Myxoid Liposarcoma of the Retroperitoneum: A Challenging Tumour for Diagnosis and Treatment

PubMed Central

Marino, Fabio; Simone, Michele

2014-01-01

Retroperitoneal sarcomas are rare neoplasms that account for only 1%-2% of all solid tumors and liposarcomas represent the most frequent histological type. We describe the case of a 44-year-old female with a retroperitoneal myxoid liposarcoma of 22 × 19 × 8 cm in size. The only symptoms were persistent pain and progressive tenderness of the abdomen lasting for two months. The mass was radically excised during laparotomy. CT and MRI were useful to clarify the site of origin of the tumor, relationships with other organs, and planning surgery but final diagnosis was based on histological findings. Here we review the literature about the challenging diagnosis, treatment, and prognostic factors of this disease. PMID:25024863

Decorin GAG synthesis and TGF-β signaling mediate Ox-LDL-induced mineralization of human vascular smooth muscle cells.

PubMed

Yan, Jianyun; Stringer, Sally E; Hamilton, Andrew; Charlton-Menys, Valentine; Götting, Christian; Müller, Benjamin; Aeschlimann, Daniel; Alexander, M Yvonne

2011-03-01

Decorin and oxidized low-density lipoprotein (Ox-LDL) independently induce osteogenic differentiation of vascular smooth muscle cells (VSMCs). We aimed to determine whether decorin glycosaminoglycan (GAG) chain synthesis contributes to Ox-LDL-induced differentiation and calcification of human VSMCs in vitro. Human VSMCs treated with Ox-LDL to induce oxidative stress showed increased alkaline phosphatase (ALP) activity, accelerated mineralization, and a difference in both decorin GAG chain biosynthesis and CS/DS structure compared with untreated controls. Ox-LDL increased mRNA abundance of both xylosyltransferase (XT)-I, the key enzyme responsible for GAG chain biosynthesis and Msx2, a marker of osteogenic differentiation. Furthermore, downregulation of XT-I expression using small interfering RNA blocked Ox-LDL-induced VSMC mineralization. Adenoviral-mediated overexpression of decorin, but not a mutated unglycanated form, accelerated mineralization of VSMCs, suggesting GAG chain addition on decorin is crucial for the process of differentiation. The decorin-induced VSMC osteogenic differentiation involved activation of the transforming growth factor (TGF)-β pathway, because it was attenuated by blocking of TGF-β receptor signaling and because decorin overexpression potentiated phosphorylation of the downstream signaling molecule smad2. These studies provide direct evidence that oxidative stress-mediated decorin GAG chain synthesis triggers TGF-β signaling and mineralization of VSMCs in vitro.

Induction of strong anti-HIV cellular immunity by a combination of Clostridium perfringens expressing HIV gag and virus like particles.

PubMed

Pegu, Poonam; Helmus, Ruth; Gupta, Phalguni; Tarwater, Patrick; Caruso, Lori; Shen, Chengli; Ross, Ted; Chen, Yue

2011-12-01

The lower gastrointestinal tract is a major mucosal site of HIV entry and initial infection. Thus, the induction of strong cellular immune responses at this mucosal site will be an important feature of an effective HIV vaccine. We have used a novel prime-boost vaccination approach to induce immune responses at mucosal sites. Orally delivered recombinant Clostridium perfringens expressing HIV-1 gag (Cp-Gag) was evaluated for induction of HIV-1 Gag specific T cell responses in a prime-boost model with intranasal inoculation of HIV-1 virus like particles (VLP). HIV-1 specific cellular immune responses in both the effector (Lamina propria) and inductive sites (Peyer's patches) of the gastrointestinal (GI) tract were significantly higher in mice immunized using Cp-Gag and VLPs in a prime-boost approach compared to mice immunized with either Cp-Gag or VLPs alone. Such cellular immune response was found to be mediated by both CD8(+) and CD4(+) T cells. Such a strong mucosal immune response could be very useful in developing a mucosal vaccine against HIV-1.

Resveratrol increases nucleus pulposus matrix synthesis through activating the PI3K/Akt signaling pathway under mechanical compression in a disc organ culture.

PubMed

Han, Xiaorui; Leng, Xiaoming; Zhao, Man; Wu, Mei; Chen, Amei; Hong, Guoju; Sun, Ping

2017-12-22

Disc nucleus pulposus (NP) matrix homeostasis is important for normal disc function. Mechanical overloading seriously decreases matrix synthesis and increases matrix degradation. The present study aims to investigate the effects of resveratrol on disc NP matrix homeostasis under a relatively high-magnitude mechanical compression and the potential mechanism underlying this process. Porcine discs were perfusion-cultured and subjected to a relatively high-magnitude mechanical compression (1.3 MPa at a frequency of 1.0 Hz for 2 h once per day) for 7 days in a mechanically active bioreactor. The non-compressed discs were used as controls. Resveratrol was added along with culture medium to observe the effects of resveratrol on NP matrix synthesis under mechanical load respectively. NP matrix synthesis was evaluated by histology, biochemical content (glycosaminoglycan (GAG) and hydroxyproline (HYP)), and expression of matrix macromolecules (aggrecan and collagen II). Results showed that this high-magnitude mechanical compression significantly decreased NP matrix content, indicated by the decreased staining intensity of Alcian Blue and biochemical content (GAG and HYP), and the down-regulated expression of NP matrix macromolecules (aggrecan and collagen II). Further analysis indicated that resveratrol partly stimulated NP matrix synthesis and increased activity of the PI3K/Akt pathway in a dose-dependent manner under mechanical compression. Together, resveratrol is beneficial for disc NP matrix synthesis under mechanical overloading, and the activation of the PI3K/Akt pathway may participate in this regulatory process. Resveratrol may be promising to regenerate mechanical overloading-induced disc degeneration. © 2017 The Author(s).

NMR structure of the myristylated feline immunodeficiency virus matrix protein.

PubMed

Brown, Lola A; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G; Kuo, Lillian; Freed, Eric O; Summers, Michael F

2015-04-30

Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag's N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

Vulvar Myxoid Liposarcoma and Well Differentiated Liposarcoma With Molecular Cytogenetic Confirmation: Case Reports With Review of Malignant Lipomatous Tumors of the Vulva.

PubMed

Schoolmeester, John Kenneth; Leifer, Aaron J; Wang, Lu; Hameed, Meera R

2015-07-01

Malignant lipomatous tumors of the vulva are uncommon. We present 2 cases of liposarcoma arising in the vulva: a myxoid liposarcoma harboring DDIT3 and FUS rearrangements and a well differentiated liposarcoma/atypical lipomatous tumor harboring MDM2 amplification detected by fluorescence in situ hybridization. Both cases are the first liposarcomas of this site to be confirmed by molecular cytogenetic analysis. We also review the literature's cases of liposarcoma to further examine the clinicopathologic features of these tumors.

A new avenue to the synthesis of GAG-mimicking polymers highly promoting neural differentiation of embryonic stem cells.

PubMed

Wang, Mengmeng; Lyu, Zhonglin; Chen, Gaojian; Wang, Hongwei; Yuan, Yuqi; Ding, Kaiguo; Yu, Qian; Yuan, Lin; Chen, Hong

2015-10-28

A new strategy for the fabrication of glycosaminoglycan (GAG) analogs was proposed by copolymerizing the sulfonated unit and the glyco unit, 'splitted' from the sulfated saccharide building blocks of GAGs. The synthetic polymers can promote cell proliferation and neural differentiation of embryonic stem cells with the effects even better than those of heparin.

Strategies for Enhancing the Accumulation and Retention of Extracellular Matrix in Tissue-Engineered Cartilage Cultured in Bioreactors

PubMed Central

Shahin, Kifah; Doran, Pauline M.

2011-01-01

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min−1) and gradually increasing (0.075–0.2 mL min−1) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0–4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8–5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min−1. GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention. PMID:21858004

A trans-Dominant Form of Gag Restricts Ty1 Retrotransposition and Mediates Copy Number Control

PubMed Central

Saha, Agniva; Mitchell, Jessica A.; Nishida, Yuri; Hildreth, Jonathan E.; Ariberre, Joshua A.; Gilbert, Wendy V.

2015-01-01

ABSTRACT Saccharomyces cerevisiae and Saccharomyces paradoxus lack the conserved RNA interference pathway and utilize a novel form of copy number control (CNC) to inhibit Ty1 retrotransposition. Although noncoding transcripts have been implicated in CNC, here we present evidence that a truncated form of the Gag capsid protein (p22) or its processed form (p18) is necessary and sufficient for CNC and likely encoded by Ty1 internal transcripts. Coexpression of p22/p18 and Ty1 decreases mobility more than 30,000-fold. p22/p18 cofractionates with Ty1 virus-like particles (VLPs) and affects VLP yield, protein composition, and morphology. Although p22/p18 and Gag colocalize in the cytoplasm, p22/p18 disrupts sites used for VLP assembly. Glutathione S-transferase (GST) affinity pulldowns also suggest that p18 and Gag interact. Therefore, this intrinsic Gag-like restriction factor confers CNC by interfering with VLP assembly and function and expands the strategies used to limit retroelement propagation. IMPORTANCE Retrotransposons dominate the chromosomal landscape in many eukaryotes, can cause mutations by insertion or genome rearrangement, and are evolutionarily related to retroviruses such as HIV. Thus, understanding factors that limit transposition and retroviral replication is fundamentally important. The present work describes a retrotransposon-encoded restriction protein derived from the capsid gene of the yeast Ty1 element that disrupts virus-like particle assembly in a dose-dependent manner. This form of copy number control acts as a molecular rheostat, allowing high levels of retrotransposition when few Ty1 elements are present and inhibiting transposition as copy number increases. Thus, yeast and Ty1 have coevolved a form of copy number control that is beneficial to both “host and parasite.” To our knowledge, this is the first Gag-like retrotransposon restriction factor described in the literature and expands the ways in which restriction proteins modulate

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

PubMed

Datta, Siddhartha A K; Clark, Patrick K; Fan, Lixin; Ma, Buyong; Harvin, Demetria P; Sowder, Raymond C; Nussinov, Ruth; Wang, Yun-Xing; Rein, Alan

2016-02-15

HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

PubMed Central

Clark, Patrick K.; Fan, Lixin; Ma, Buyong; Harvin, Demetria P.; Sowder, Raymond C.; Nussinov, Ruth; Wang, Yun-Xing

2015-01-01

ABSTRACT HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a “spacer” between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly

Fiber Development and Matrix Production in Tissue Engineered Menisci using Bovine Mesenchymal Stem Cells and Fibrochondrocytes

PubMed Central

McCorry, Mary Clare; Bonassar, Lawrence J.

2017-01-01

Mesenchymal stem cells (MSCs) have been investigated with promising results for meniscus healing and tissue engineering. While MSCs are known to contribute to ECM production, less is known about how MSCs produce and align large organized fibers for application to tissue engineering the meniscus. The goal of this study was to investigate the capability of MSCs to produce and organize extracellular matrix molecules compared to meniscal fibrochondrocytes (FCCs). Bovine FCCs and MSCs were encapsulated in an anatomically accurate collagen meniscus using mono-culture and co-culture of each cell type. Each meniscus was mechanically anchored at the horns to mimic the physiological fixation by the meniscal entheses. Mechanical fixation generates a static mechanical boundary condition previously shown to induce formation of oriented fiber by FCCs. Samples were cultured for 4 weeks and then evaluated for biochemical composition and fiber development. MSCs increased the GAG and collagen production in both co-culture and mono-culture groups compared to FCC mono-culture. Collagen organization was greatest in the FCC mono-culture group. While MSCs had increased matrix production they lacked the fiber organization capabilities of FCCs. This study suggests that GAG production and fiber formation are linked. Co-culture can be used as a means of balancing the synthetic properties of MSCs and the matrix remodeling capabilities of FCCs for tissue engineering applications. PMID:27925474

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

PubMed Central

Enssle, J; Fischer, N; Moebes, A; Mauer, B; Smola, U; Rethwilm, A

1997-01-01

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked. PMID:9311808

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

PubMed

Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

1994-12-01

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

Central nervous system-specific consequences of simian immunodeficiency virus Gag escape from major histocompatability complex class I-mediated control

PubMed Central

Beck, Sarah E.; Queen, Suzanne E.; Viscidi, Raphael; Johnson, Darius; Kent, Stephen J.; Adams, Robert J.; Tarwater, Patrick M.; Mankowski, Joseph L.

2016-01-01

In the fourth decade of the HIV epidemic, the relationship between host immunity and HIV central nervous system (CNS) disease remains incompletely understood. Using a simian immunodeficiency virus (SIV)/macaque model, we examined CNS outcomes in pigtailed macaques expressing the MHC class I allele Mane-A1*084:01 which confers resistance to SIV-induced CNS disease and induces the prototypic viral escape mutation Gag K165R. Insertion of gag K165R into the neurovirulent clone SIV/17E-Fr reduced viral replication in vitro compared to SIV/17E-Fr. We also found lower CSF, but not plasma, viral loads in macaques inoculated with SIV/17E-Fr K165R versus those inoculated with wildtype. Although escape mutation K165R was genotypically stable in plasma, it rapidly reverted to wildtype Gag KP9 in both CSF and in microglia cultures. We induced robust Gag KP9-specific CTL tetramer responses by vaccinating Mane-A*084:01-positive pigtailed macaques with a Gag KP9 virus-like particle (VLP) vaccine. Upon SIV/17E-Fr challenge, vaccinated animals had lower SIV RNA in CSF compared to unvaccinated controls, but showed no difference in plasma viral loads. These data clearly demonstrate that viral fitness in the CNS is distinct from the periphery and underscores the necessity of understanding the consequences of viral escape in CNS disease with the advent of new therapeutic vaccination strategies. PMID:26727909

Central nervous system-specific consequences of simian immunodeficiency virus Gag escape from major histocompatibility complex class I-mediated control.

PubMed

Beck, Sarah E; Queen, Suzanne E; Viscidi, Raphael; Johnson, Darius; Kent, Stephen J; Adams, Robert J; Tarwater, Patrick M; Mankowski, Joseph L

2016-08-01

In the fourth decade of the HIV epidemic, the relationship between host immunity and HIV central nervous system (CNS) disease remains incompletely understood. Using a simian immunodeficiency virus (SIV)/macaque model, we examined CNS outcomes in pigtailed macaques expressing the MHC class I allele Mane-A1*084:01 which confers resistance to SIV-induced CNS disease and induces the prototypic viral escape mutation Gag K165R. Insertion of gag K165R into the neurovirulent clone SIV/17E-Fr reduced viral replication in vitro compared to SIV/17E-Fr. We also found lower cerebrospinal fluid (CSF), but not plasma, viral loads in macaques inoculated with SIV/17E-Fr K165R versus those inoculated with wildtype. Although escape mutation K165R was genotypically stable in plasma, it rapidly reverted to wildtype Gag KP9 in both CSF and in microglia cultures. We induced robust Gag KP9-specific CTL tetramer responses by vaccinating Mane-A*084:01-positive pigtailed macaques with a Gag KP9 virus-like particle (VLP) vaccine. Upon SIV/17E-Fr challenge, vaccinated animals had lower SIV RNA in CSF compared to unvaccinated controls, but showed no difference in plasma viral loads. These data clearly demonstrate that viral fitness in the CNS is distinct from the periphery and underscores the necessity of understanding the consequences of viral escape in CNS disease with the advent of new therapeutic vaccination strategies.

Genetic analysis of the major homology region of the Rous sarcoma virus Gag protein.

PubMed Central

Craven, R C; Leure-duPree, A E; Weldon, R A; Wills, J W

1995-01-01

The mature cores of all retroviruses contain a major structural protein known as the CA (capsid) protein. Although it appears to form a shell around the ribonucleoprotein complex that contains the viral RNA, its function in viral replication is largely unknown. Little sequence similarity exists between the CA proteins of different retroviruses, except for a region of about 20 amino acids termed the major homology region (MHR). To examine the role of the CA protein in particle assembly and release, mutants of Rous sarcoma virus were created in which segments of CA were deleted or single conserved residues in the MHR were altered. The ability of the deletion mutants to release particles at rates similar to the wild-type protein demonstrated that the CA domain of Gag is not an essential component of the minimal budding machinery. Certain point mutations in the MHR region did block assembly and release in certain cell types, presumably by perturbing the global structure of the Gag precursor. Another group of MHR substitutions produced noninfectious or poorly infectious particles that were normal in their content of gag and pol gene products and viral RNA. The mutants were capable of initiating reverse transcription in vitro; however, the association of CA protein with the core was compromised, as indicated by its sensitivity to extraction with nonionic detergent. Prominent blebs on the virion envelope also indicated a disturbance at the membrane. Finally, an anti-peptide serum directed against MHR was found to react with the uncleaved Gag protein but not with mature CA, suggesting that MHR undergoes a dynamic rearrangement upon liberation from the polyprotein. We conclude that the MHR is involved in the very late steps in maturation of the virion (i.e., ones that occur after budding is initiated) and is essential for proper function of the core upon entry into a new host cell. PMID:7769681

Fullerene-like organization of HIV gag-protein shell in virus-like particles produced by recombinant baculovirus.

PubMed

Nermut, M V; Hockley, D J; Jowett, J B; Jones, I M; Garreau, M; Thomas, D

1994-01-01

Virus-like particles produced by a recombinant baculovirus containing the HIV gag gene were examined by negative staining after delipidization. This technique demonstrated that the gag-protein shell consisted of radially arranged short rods which formed a network of ring-like structures. Similar structures were observed at the plasma membrane of infected cells which had been opened by wet-cleaving. Occasionally five or six subunits were observed forming a ring. These findings suggest that the gag-encoded precursor (pr55) is a rod-like molecule about 34 A in diameter and 85 A in length. A protein cylinder of such dimensions would have a molecular weight of 56K. The center-to-center distance of two neighboring rings formed by the rods was 66 +/- 8 A (N = 200) by direct measurements and 65 A as obtained from averaged images. This morphology and these dimensions indicate that the virus-like particles contain the gag precursor in the form of a near-spherical "fullerene-like" icosahedral shell. Our data indicate that the triangulation number of the rings equals 63. However, since one rod of pr55 is shared by two rings, the number of copies of the precursor will be 1890 as opposed to 2522 if the molecules were closely packed. The particle diameter of 102 nm deduced from the proposed model was close to the diameter obtained from thin sections of low-temperature-embedded specimens (103-108 nm).

Modeling the full length HIV-1 Gag polyprotein reveals the role of its p6 subunit in viral maturation and the effect of non-cleavage site mutations in protease drug resistance.

PubMed

Su, Chinh Tran-To; Kwoh, Chee-Keong; Verma, Chandra Shekhar; Gan, Samuel Ken-En

2017-12-27

HIV polyprotein Gag is increasingly found to contribute to protease inhibitor resistance. Despite its role in viral maturation and in developing drug resistance, there remain gaps in the knowledge of the role of certain Gag subunits (e.g. p6), and that of non-cleavage mutations in drug resistance. As p6 is flexible, it poses a problem for structural experiments, and is hence often omitted in experimental Gag structural studies. Nonetheless, as p6 is an indispensable component for viral assembly and maturation, we have modeled the full length Gag structure based on several experimentally determined constraints and studied its structural dynamics. Our findings suggest that p6 can mechanistically modulate Gag conformations. In addition, the full length Gag model reveals that allosteric communication between the non-cleavage site mutations and the first Gag cleavage site could possibly result in protease drug resistance, particularly in the absence of mutations in Gag cleavage sites. Our study provides a mechanistic understanding to the structural dynamics of HIV-1 Gag, and also proposes p6 as a possible drug target in anti-HIV therapy.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

PubMed Central

Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José

2015-01-01

ABSTRACT During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCE During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

PubMed Central

Lim, Kwang-il; Klimczak, Ryan; Yu, Julie H.; Schaffer, David V.

2010-01-01

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 109 possible human genome locations (P = 4.6 × 10-29). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS. PMID:20616052

Role of Gag and lipids during HIV-1 assembly in CD4+ T cells and macrophages

PubMed Central

Mariani, Charlotte; Desdouits, Marion; Favard, Cyril; Benaroch, Philippe; Muriaux, Delphine M.

2014-01-01

HIV-1 is an RNA enveloped virus that preferentially infects CD4+ T lymphocytes and also macrophages. In CD4+ T cells, HIV-1 mainly buds from the host cell plasma membrane. The viral Gag polyprotein targets the plasma membrane and is the orchestrator of the HIV assembly as its expression is sufficient to promote the formation of virus-like particles carrying a lipidic envelope derived from the host cell membrane. Certain lipids are enriched in the viral membrane and are thought to play a key role in the assembly process and the envelop composition. A large body of work performed on infected CD4+ T cells has provided important knowledge about the assembly process and the membrane virus lipid composition. While HIV assembly and budding in macrophages is thought to follow the same general Gag-driven mechanism as in T-lymphocytes, the HIV cycle in macrophage exhibits specific features. In these cells, new virions bud from the limiting membrane of seemingly intracellular compartments, where they accumulate while remaining infectious. These structures are now often referred to as Virus Containing Compartments (VCCs). Recent studies suggest that VCCs represent intracellularly sequestered regions of the plasma membrane, but their precise nature remains elusive. The proteomic and lipidomic characterization of virions produced by T cells or macrophages has highlighted the similarity between their composition and that of the plasma membrane of producer cells, as well as their enrichment in acidic lipids, some components of raft lipids and in tetraspanin-enriched microdomains. It is likely that Gag promotes the coalescence of these components into an assembly platform from which viral budding takes place. How Gag exactly interacts with membrane lipids and what are the mechanisms involved in the interaction between the different membrane nanodomains within the assembly platform remains unclear. Here we review recent literature regarding the role of Gag and lipids on HIV-1

HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates

NASA Astrophysics Data System (ADS)

Wille-Reece, Ulrike; Flynn, Barbara J.; Loré, Karin; Koup, Richard A.; Kedl, Ross M.; Mattapallil, Joseph J.; Weiss, Walter R.; Roederer, Mario; Seder, Robert A.

2005-10-01

Induction and maintenance of antibody and T cell responses will be critical for developing a successful vaccine against HIV. A rational approach for generating such responses is to design vaccines or adjuvants that have the capacity to activate specific antigen-presenting cells. In this regard, dendritic cells (DCs) are the most potent antigen-presenting cells for generating primary T cell responses. Here, we report that Toll-like receptor (TLR) agonists and ligands that activate DCs in vitro influence the magnitude and quality of the cellular immune response in nonhuman primates (NHPs) when administered with HIV Gag protein. NHPs immunized with HIV Gag protein and a TLR7/8 agonist or a TLR9 ligand [CpG oligodeoxynucleotides (CpG ODN)] had significantly increased Gag-specific T helper 1 and antibody responses, compared with animals immunized with HIV Gag protein alone. Importantly, conjugating the HIV Gag protein to the TLR7/8 agonist (Gag-TLR7/8 conjugate) dramatically enhanced the magnitude and altered the quality of the T helper 1 response, compared with animals immunized with HIV Gag protein and the TLR7/8 agonist or CpG ODN. Furthermore, immunization with the Gag-TLR7/8 conjugate vaccine elicited Gag-specific CD8+ T responses. Collectively, our results show that conjugating HIV Gag protein to a TLR7/8 agonist is an effective way to elicit broad-based adaptive immunity in NHPs. This type of vaccine formulation should have utility in preventive or therapeutic vaccines in which humoral and cellular immunity is required. vaccine | dendritic cell | cross-presentation | cellular immunity

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells.

PubMed

Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José; Muriaux, Delphine

2015-08-01

During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly

Scaffold composition affects cytoskeleton organization, cell-matrix interaction and the cellular fate of human mesenchymal stem cells upon chondrogenic differentiation.

PubMed

Li, Yuk Yin; Choy, Tze Hang; Ho, Fu Chak; Chan, Pui Barbara

2015-06-01

The stem cell niche, or microenvironment, consists of soluble, matrix, cell and mechanical factors that together determine the cellular fates and/or differentiation patterns of stem cells. Collagen and glycosaminoglycans (GAGs) are important scaffolding materials that can mimic the natural matrix niche. Here, we hypothesize that imposing changes in the scaffold composition or, more specifically, incorporating GAGs into the collagen meshwork, will affect the morphology, cytoskeletal organization and integrin expression profiles, and hence the fate of human mesenchymal stem cells (MSCs) upon the induction of differentiation. Using chondrogenesis as an example, we microencapsulated MSCs in three scaffold systems that had varying matrix compositions: collagen alone (C), aminated collagen (AC) and aminated collagen with GAGs (ACG). We then induced the MSCs to differentiate toward a chondrogenic lineage, after which, we characterized the cell viability and morphology, as well as the level of cytoskeletal organization and the integrin expression profile. We also studied the fate of the MSCs by evaluating the major chondrogenic markers at both the gene and protein level. In C, MSC chondrogenesis was successfully induced and MSCs that spread in the scaffolds had a clear actin cytoskeleton; they expressed integrin α2β1, α5 and αv; promoted sox9 nuclear localization transcription activation; and upregulated the expression of chondrogenic matrix markers. In AC, MSC chondrogenesis was completely inhibited but the scaffold still supported cell survival. The MSCs did not spread and they had no actin cytoskeleton; did not express integrin α2 or αv; they failed to differentiate into chondrogenic lineage cells even on chemical induction; and there was little colocalization or functional interaction between integrin α5 and fibronectin. In ACG, although the MSCs did not express integrin α2, they did express integrin αv and there was strong co-localization and hence functional

Relationships of gag-pol diversity between Ty3/Gypsy and Retroviridae LTR retroelements and the three kings hypothesis

PubMed Central

2008-01-01

Background The origin of vertebrate retroviruses (Retroviridae) is yet to be thoroughly investigated, but due to their similarity and identical gag-pol (and env) genome structure, it is accepted that they evolve from Ty3/Gypsy LTR retroelements the retrotransposons and retroviruses of plants, fungi and animals. These 2 groups of LTR retroelements code for 3 proteins rarely studied due to the high variability – gag polyprotein, protease and GPY/F module. In relation to 3 previously proposed Retroviridae classes I, II and II, investigation of the above proteins conclusively uncovers important insights regarding the ancient history of Ty3/Gypsy and Retroviridae LTR retroelements. Results We performed a comprehensive study of 120 non-redundant Ty3/Gypsy and Retroviridae LTR retroelements. Phylogenetic reconstruction inferred based on the concatenated analysis of the gag and pol polyproteins shows a robust phylogenetic signal regarding the clustering of OTUs. Evaluation of gag and pol polyproteins separately yields discordant information. While pol signal supports the traditional perspective (2 monophyletic groups), gag polyprotein describes an alternative scenario where each Retroviridae class can be distantly related with one or more Ty3/Gypsy lineages. We investigated more in depth this evidence through comparative analyses performed based on the gag polyprotein, the protease and the GPY/F module. Our results indicate that contrary to the traditional monophyletic view of the origin of vertebrate retroviruses, the Retroviridae class I is a molecular fossil, preserving features that were probably predominant among Ty3/Gypsy ancestors predating the split of plants, fungi and animals. In contrast, classes II and III maintain other phenotypes that emerged more recently during Ty3/Gypsy evolution. Conclusion The 3 Retroviridae classes I, II and III exhibit phenotypic differences that delineate a network never before reported between Ty3/Gypsy and Retroviridae LTR

Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

PubMed Central

Chen, W; Qin, H; Chesebro, B; Cheever, M A

1996-01-01

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV. PMID:8892898

Compositional analysis and structural elucidation of glycosaminoglycans in chicken eggs

PubMed Central

Liu, Zhangguo; Zhang, Fuming; Li, Lingyun; Li, Guoyun; He, Wenqing; Linhardt, Robert J.

2014-01-01

Glycosaminoglycans (GAGs) have numerous applications in the fields of pharmaceuticals, cosmetics, nutraceuticals, and foods. GAGs are also critically important in the developmental biology of all multicellular animals. GAGs were isolated from chicken egg components including yolk, thick egg white, thin egg white, membrane, calcified shell matrix supernatant, and shell matrix deposit. Disaccharide compositional analysis was performed using ultra high-performance liquid chromatography-mass spectrometry. The results of these analyses showed that all four families of GAGs were detected in all egg components. Keratan sulfate was found in egg whites (thick and thin) and shell matrix (calcified shell matrix supernatant and deposit) with high level. Chondroitin sulfates were much more plentiful in both shell matrix components and membrane. Hyaluronan was plentiful in both shell matrix components and membrane, but were only present in a trace of quantities in the yolk. Heparan sulfate was plentiful in the shell matrix deposit but was present in a trace of quantities in the egg content components (yolk, thick and thin egg whites). Most of the chondroitin and heparan sulfate disaccharides were present in the GAGs found in chicken eggs with the exception of chondroitin and heparan sulfate 2,6-disulfated disaccharides. Both CS and HS in the shell matrix deposit contained the most diverse chondroitin and heparan sulfate disaccharide compositions. Eggs might provide a potential new source of GAGs. PMID:25218438

Mother-to-Child HIV Transmission Bottleneck Selects for Consensus Virus with Lower Gag-Protease-Driven Replication Capacity

PubMed Central

Naidoo, Vanessa L.; Mann, Jaclyn K.; Noble, Christie; Adland, Emily; Carlson, Jonathan M.; Thomas, Jake; Brumme, Chanson J.; Thobakgale-Tshabalala, Christina F.; Brumme, Zabrina L.; Goulder, Philip J. R.

2017-01-01

ABSTRACT In the large majority of cases, HIV infection is established by a single variant, and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease-driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma-derived Gag-protease from 53 nontransmitter mothers, 48 transmitter mothers, and 47 infected infants were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacities between the nontransmitter (n = 53) and transmitter (n = 44) mothers (P = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses (n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than that of viruses derived from the mothers (P < 0.0001 by a paired t test). High percent similarities to consensus subtype C Gag, p17, p24, and protease sequences were also found in the infants (n = 28) in comparison to their mothers (P = 0.07, P = 0.002, P = 0.03, and P = 0.02, respectively, as determined by a paired t test). These data suggest that of the viral quasispecies found in mothers, the HIV mother-to-child transmission bottleneck favors the transmission of consensus-like viruses with lower viral replication capacities. IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag, a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response, in the MTCT

Generation and characterization of Dyt1 DeltaGAG knock-in mouse as a model for early-onset dystonia.

PubMed

Dang, Mai T; Yokoi, Fumiaki; McNaught, Kevin St P; Jengelley, Toni-Ann; Jackson, Tehone; Li, Jianyong; Li, Yuqing

2005-12-01

A trinucleotide deletion of GAG in the DYT1 gene that encodes torsinA protein is implicated in the neurological movement disorder of Oppenheim's early-onset dystonia. The mutation removes a glutamic acid in the carboxy region of torsinA, a member of the Clp protease/heat shock protein family. The function of torsinA and the role of the mutation in causing dystonia are largely unknown. To gain insight into these unknowns, we made a gene-targeted mouse model of Dyt1 DeltaGAG to mimic the mutation found in DYT1 dystonic patients. The mutated heterozygous mice had deficient performance on the beam-walking test, a measure of fine motor coordination and balance. In addition, they exhibited hyperactivity in the open-field test. Mutant mice also showed a gait abnormality of increased overlap. Mice at 3 months of age did not display deficits in beam-walking and gait, while 6-month mutant mice did, indicating an age factor in phenotypic expression as well. While striatal dopamine and 4-dihydroxyphenylacetic acid (DOPAC) levels in Dyt1 DeltaGAG mice were similar to that of wild-type mice, a 27% decrease in 4-hydroxy, 3-methoxyphenacetic acid (homovanillic acid) was detected in mutant mice. Dyt1 DeltaGAG tissues also have ubiquitin- and torsinA-containing aggregates in neurons of the pontine nuclei. A sex difference was noticed in the mutant mice with female mutant mice exhibiting fewer alterations in behavioral, neurochemical, and cellular changes. Our results show that knocking in a Dyt1 DeltaGAG allele in mouse alters their motor behavior and recapitulates the production of protein aggregates that are seen in dystonic patients. Our data further support alterations in the dopaminergic system as a part of dystonia's neuropathology.

Metastatic patterns of myxoid/round cell liposarcoma: a review of a 25-year experience.

PubMed

Asano, Naofumi; Susa, Michiro; Hosaka, Seiichi; Nakayama, Robert; Kobayashi, Eisuke; Takeuchi, Katsuhito; Horiuchi, Keisuke; Suzuki, Yoshihisa; Anazawa, Ukei; Mukai, Makio; Toyama, Yoshiaki; Yabe, Hiroo; Morioka, Hideo

2012-01-01

Myxoid/round cell liposarcoma (MRCL), unlike other soft tissue sarcomas, has been associated with unusual pattern of metastasis to extrapulmonary sites. In an attempt to elucidate the clinical features of MRCL with metastatic lesions, 58 cases, from the medical database of Keio University Hospital were used for the evaluation. 47 patients (81%) had no metastases, whereas 11 patients (11%) had metastases during their clinical course. Among the 11 patients with metastatic lesions, 8 patients (73%) had extrapulmonary metastases and 3 patients (27%) had pulmonary metastases. Patients were further divided into three groups; without metastasis, with extrapulmonary metastasis, and with pulmonary metastasis. When the metastatic patterns were stratified according to tumor size, there was statistical significance between the three groups (P = 0.028). The 8 cases with extrapulmonary metastases were all larger than 10 cm. Similarly, histological grading had a significant impact on metastatic patterns (P = 0.027). 3 cases with pulmonary metastatic lesions were all diagnosed as high grade. In conclusion, large size and low histological grade were significantly associated with extrapulmonary metastasis.

Sedation-analgesia with propofol and remifentanil: concentrations required to avoid gag reflex in upper gastrointestinal endoscopy.

PubMed

Borrat, Xavier; Valencia, José Fernando; Magrans, Rudys; Gimenez-Mila, Marc; Mellado, Ricard; Sendino, Oriol; Perez, Maria; Nunez, Matilde; Jospin, Mathieu; Jensen, Erik Weber; Troconiz, Inaki; Gambus, Pedro L

2015-07-01

The purpose of this study was to identify optimal target propofol and remifentanil concentrations to avoid a gag reflex in response to insertion of an upper gastrointestinal endoscope. Patients presenting for endoscopy received target-controlled infusions (TCI) of both propofol and remifentanil for sedation-analgesia. Patients were randomized to 4 groups of fixed target effect-site concentrations: remifentanil 1 ng•mL (REMI 1) or 2 ng•mL (REMI 2) and propofol 2 μg•mL (PROP 2) or 3 μg•mL (PROP 3). For each group, the other drug (propofol for the REMI groups and vice versa) was increased or decreased using the "up-down" method based on the presence or absence of a gag response in the previous patient. A modified isotonic regression method was used to estimate the median effective Ce,50 from the up-down method in each group. A concentration-effect (sigmoid Emax) model was built to estimate the corresponding Ce,90 for each group. These data were used to estimate propofol bolus doses and remifentanil infusion rates that would achieve effect-site concentrations between Ce,50 and Ce,90 when a TCI system is not available for use. One hundred twenty-four patients were analyzed. To achieve between a 50% and 90% probability of no gag response, propofol TCIs were between 2.40 and 4.23 μg•mL (that could be achieved with a bolus of 1 mg•kg) when remifentanil TCI was fixed at 1 ng•mL, and target propofol TCIs were between 2.15 and 2.88 μg•mL (that could be achieved with a bolus of 0.75 mg•kg) when remifentanil TCI was fixed at 2 ng•mL. Remifentanil ranges were 1.00 to 4.79 ng•mL and 0.72 to 3.19 ng•mL when propofol was fixed at 2 and 3 μg•mL, respectively. We identified a set of propofol and remifentanil TCIs that blocked the gag response to endoscope insertion in patients undergoing endoscopy. Propofol bolus doses and remifentanil infusion rates designed to achieve similar effect-site concentrations can be used to prevent gag response when TCI is

Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection

PubMed Central

French, Martyn A.; Abudulai, Laila N.; Fernandez, Sonia

2013-01-01

The development of vaccines to treat and prevent human immunodeficiency virus (HIV) infection has been hampered by an incomplete understanding of “protective” immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8+ T-cell responses restricted by “protective” HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-α-dependant natural killer (NK) cell responses and plasmacytoid dendritic cell (pDC) responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection. PMID:26344116

Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection.

PubMed

French, Martyn A; Abudulai, Laila N; Fernandez, Sonia

2013-08-09

The development of vaccines to treat and prevent human immunodeficiency virus (HIV) infection has been hampered by an incomplete understanding of "protective" immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8⁺ T-cell responses restricted by "protective" HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-a-dependant natural killer (NK) cell responses and plasmacytoid dendritic cell (pDC) responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

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

Meador, Lydia R.

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viralmore » vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. - Highlights: • We devised a prime/boost anti HIV-1 vaccination strategy modeled after RV144. • We used plant-derived virus-like particles (VLPs) consisting of Gag and dgp41. • We used attenuated, replicating vaccinia virus vectors expressing the same antigens. • The immunogens elicited strong cellular and humoral immune responses.« less

The baculovirus-integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase.

PubMed Central

Lerch, R A; Friesen, P D

1992-01-01

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus. Images PMID:1371168

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

PubMed

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-05-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. Copyright © 2012 Elsevier B.V. All rights reserved.

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

PubMed Central

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-01-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. PMID:22391119

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles.

PubMed

Nguyen, Albert T; Feasley, Christa L; Jackson, Ken W; Nitz, Theodore J; Salzwedel, Karl; Air, Gillian M; Sakalian, Michael

2011-12-07

Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Safety and immunogenicity of adenovirus-vectored near-consensus HIV type 1 clade B gag vaccines in healthy adults.

PubMed

Harro, Clayton D; Robertson, Michael N; Lally, Michelle A; O'Neill, Lori D; Edupuganti, Srilatha; Goepfert, Paul A; Mulligan, Mark J; Priddy, Frances H; Dubey, Sheri A; Kierstead, Lisa S; Sun, Xiao; Casimiro, Danilo R; DiNubile, Mark J; Shiver, John W; Leavitt, Randi Y; Mehrotra, Devan V

2009-01-01

Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-gamma ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus > or =200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus > or = 200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen

Safety and Immunogenicity of Adenovirus-Vectored Near-Consensus HIV Type 1 Clade B gag Vaccines in Healthy Adults

PubMed Central

Robertson, Michael N.; Lally, Michelle A.; O'Neill, Lori D.; Edupuganti, Srilatha; Goepfert, Paul A.; Mulligan, Mark J.; Priddy, Frances H.; Dubey, Sheri A.; Kierstead, Lisa S.; Sun, Xiao; Casimiro, Danilo R.; DiNubile, Mark J.; Shiver, John W.; Leavitt, Randi Y.; Mehrotra, Devan V.

2009-01-01

Abstract Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-γ ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers <200 versus ≥200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers <200 versus ≥200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers <200. Preexistent and/or vaccine-induced immunity to the Ad5 vector may dampen the

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

PubMed Central

Gherghe, Cristina; Lombo, Tania; Leonard, Christopher W.; Datta, Siddhartha A. K.; Bess, Julian W.; Gorelick, Robert J.; Rein, Alan; Weeks, Kevin M.

2010-01-01

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs—only four nucleotides per genomic RNA—reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs. PMID:20974908

Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method.

PubMed

Kim, Eun-Young; Stanton, Jennifer; Korber, Bette T M; Krebs, Kendall; Bogdan, Derek; Kunstman, Kevin; Wu, Samuel; Phair, John P; Mirkin, Chad A; Wolinsky, Steven M

2008-06-01

Detection of HIV-1 in patients is limited by the sensitivity and selectivity of available tests. The nanotechnology-based bio-barcode-amplification method offers an innovative approach to detect specific HIV-1 antigens from diverse HIV-1 subtypes. We evaluated the efficacy of this protein-detection method in detecting HIV-1 in men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). The method relies on magnetic microparticles with antibodies that specifically bind the HIV-1 p24 Gag protein and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the microparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes (hundreds per target) were identified by a nanoparticle-based detection method that does not rely on PCR. Of 112 plasma samples from HIV-1-infected subjects, 111 were positive for HIV-1 p24 Gag protein (range: 0.11-71.5 ng/ml of plasma) by the bio-barcode-amplification method. HIV-1 p24 Gag protein was detected in only 23 out of 112 men by the conventional ELISA. A total of 34 uninfected subjects were negative by both tests. Thus, the specificity of the bio-barcode-amplification method was 100% and the sensitivity 99%. The bio-barcode-amplification method detected HIV-1 p24 Gag protein in plasma from all study subjects with less than 200 CD4(+) T cells/microl of plasma (100%) and 19 out of 20 (95%) HIV-1-infected men who had less than 50 copies/ml of plasma of HIV-1 RNA. In a separate group of 60 diverse international isolates, representative of clades A, B, C and D and circulating recombinant forms CRF01_AE and CRF02_AG, the bio-barcode-amplification method identified the presence of virus correctly. The bio-barcode-amplification method was superior to the conventional ELISA assay for the detection of HIV-1 p24 Gag protein in plasma with a breadth of coverage for diverse

Amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences

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

Wheeler, E.F.; Roussel, M.F.; Hampe, A.

1986-08-01

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180/sup gag-fms/ encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180/sup gag-fms/) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence ofmore » the resulting v-fms-coded glycoprotein, gp120/sup v-fms/, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. The authors conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180/gag-fms/ is mediated by signal peptidase and that the amino termini of gp140/sup v-fms/ and the c-fms gene product are identical.« less

Poorly expressed endogenous ecotropic provirus of DBA/2 mice encodes a mutant Pr65gag protein that is not myristylated.

PubMed Central

Copeland, N G; Jenkins, N A; Nexø, B; Schultz, A M; Rein, A; Mikkelsen, T; Jørgensen, P

1988-01-01

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus designated Emv-3. Although this provirus appears to be nondefective by genomic restriction enzyme mapping, weanling mice do not produce virus and only about one-third of adult mice ever express virus. 5-Iododeoxyuridine and 5-azacytidine, two potent inducers of ecotropic virus expression, are relatively ineffective at inducing Emv-3 expression. However, the chemical carcinogen 7,12-dimethylbenz(a)anthracene can induce ecotropic virus expression in approximately 95% of treated DBA/2 mice. Previous experiments involving DNA transfection and marker rescue analysis of molecularly cloned Emv-3 DNA suggested that Emv-3 carries a small defect(s) in the gag gene, not detectable by restriction enzyme mapping, that inhibits virus expression in vivo and in vitro. Using a combination of approaches, including DNA sequencing, peptide mapping, and metabolic labeling of cells with [3H]myristate, we have demonstrated that the defect in Emv-3 most likely results from a single nucleotide substitution within the gene for p15gag that inhibits myristylation of the Pr65gag N terminus. Myristylation of Pr65gag is thought to be required for this protein to associate with the plasma membrane and is essential for virus particle formation. These results provide a conceptual framework for understanding how Emv-3 expression is regulated during development and after chemical induction. Images PMID:2826810

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix

PubMed Central

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-01-01

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPBKKK−, a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of α4β1 and α4β7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB–GAG interaction in the chemokine-like activity of this protein. PMID:11867726

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.

PubMed

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-03-05

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPB(KKK-), a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of alpha4beta1 and alpha4beta7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB-GAG interaction in the chemokine-like activity of this protein.

Improved genetic stability of recombinant yellow fever 17D virus expressing a lentiviral Gag gene fragment.

PubMed

de Santana, Marlon G Veloso; Neves, Patrícia C C; dos Santos, Juliana Ribeiro; Lima, Noemia S; dos Santos, Alexandre A C; Watkins, David I; Galler, Ricardo; Bonaldo, Myrna C

2014-03-01

We have previously designed a method to construct viable recombinant Yellow Fever (YF) 17D viruses expressing heterologous polypeptides including part of the Simian Immunodeficiency Virus (SIV) Gag protein. However, the expressed region, encompassing amino acid residues from 45 to 269, was genetically unstable. In this study, we improved the genetic stability of this recombinant YF 17D virus by introducing mutations in the IRES element localized at the 5' end of the SIV gag gene. The new stable recombinant virus elicited adaptive immune responses similar to those induced by the original recombinant virus. It is, therefore, possible to increase recombinant stability by removing functional motifs from the insert that may have deleterious effects on recombinant YF viral fitness. Copyright © 2014 Elsevier Inc. All rights reserved.

Differences in Env and Gag protein expression patterns and epitope availability in feline immunodeficiency virus infected PBMC compared to infected and transfected feline model cell lines.

PubMed

Roukaerts, Inge D M; Grant, Chris K; Theuns, Sebastiaan; Christiaens, Isaura; Acar, Delphine D; Van Bockstael, Sebastiaan; Desmarets, Lowiese M B; Nauwynck, Hans J

2017-01-02

Env and Gag are key components of the FIV virion that are targeted to the plasma membrane for virion assembly. They are both important stimulators and targets of anti-FIV immunity. To investigate and compare the expression pattern and antigenic changes of Gag and Env in various research models, infected PBMC (the natural FIV host cells) and GFox, and transfected CrFK were stained over time with various Env and Gag specific MAbs. In FIV infected GFox and PBMC, Env showed changes in epitope availability for antibody binding during processing and trafficking, which was not seen in transfected CrFK. Interestingly, epitopes exposed on intracellular Env and Env present on the plasma membrane of CrFK and GFox seem to be hidden on plasma membrane expressed Env of FIV infected PBMC. A kinetic follow up of Gag and Env expression showed a polarization of both Gag and Env expression to specific sites at the plasma membrane of PBMC, but not in other cell lines. In conclusion, mature trimeric cell surface expressed Env might be antigenically distinct from intracellular monomeric Env in PBMC and might possibly be unrecognizable by feline humoral immunity. In addition, Env expression is restricted to a small area on the plasma membrane and co-localizes with a large moiety of Gag, which may represent a preferred FIV budding site, or initiation of virological synapses with direct cell-to-cell virus transmission. Copyright © 2016. Published by Elsevier B.V.

Infrared irradiation alters the expression of matrix metalloproteinases and glycosaminoglycans in the cornea and crystalline lens.

PubMed

Dadoukis, Panagiotis; Klagas, Ioannis; Komnenou, Anastasia; Karakiulakis, George; Karoutis, Athanasios; Karampatakis, Vassilios; Papakonstantinou, Eleni

2013-08-01

Prolonged exposure to infrared (IR) radiation is associated with different types of damage to cornea and lens. The aim of our study was to investigate the effect of acute and chronic exposure to IR radiation on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 and on the expression of glycosaminoglycans (GAG) in the rabbit cornea and crystalline lens. New Zealand rabbits were subjected to IR radiation for 4 months (chronic exposure to IR) or to normal light (control group). In experiments regarding acute exposure, animals were subjected to IR radiation or normal light for 12 h, in the presence of 0.1% diclofenac sodium (eye drops instilled in the right eye of animals) or saline (instilled in the left eye of animals). The cornea and lens were dissected away and homogenized. The activity of MMP-2 and MMP-9 was assayed by gelatine zymography. Total GAG were isolated from tissue specimens after lipid extraction and extensive digestion with pronase and DNase and characterized by treatment with GAG-degrading enzymes, followed by electrophoresis on cellulose acetate membranes. Acute or chronic exposure to IR radiation induced the activity of MMP-2 in cornea and lens, whereas only acute IR radiation increased the content of heparan sulphate in crystalline lens. Local administration of diclofenac sodium did not prevent the above effects of acute IR radiation. The detrimental effects of excessive or prolonged exposure of the eyes to IR radiation are associated with induced activity of MMP-2 in cornea and lens and alterations in the content of heparan sulphate in lens. Thus, MMP and GAG may offer alternative targets for pharmacological intervention to confront ocular damages associated with IR radiation.

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

Susceptibility of human immunodeficiency virus type 1 to the maturation inhibitor bevirimat is modulated by baseline polymorphisms in Gag spacer peptide 1.

PubMed

Van Baelen, Kurt; Salzwedel, Karl; Rondelez, Evelien; Van Eygen, Veerle; De Vos, Stephanie; Verheyen, Ann; Steegen, Kim; Verlinden, Yvan; Allaway, Graham P; Stuyver, Lieven J

2009-05-01

In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

Development of extracellular matrix in chick paravertebral sympathetic ganglia.

PubMed

Luckenbill-Edds, L

1986-08-01

Alcian blue staining coupled with enzyme digestion or critical electrolyte staining revealed differences in the development of extracellular matrix (ECM) within sympathetic ganglia compared with the surrounding capsule. On day 5 of chick development (Hamburger-Hamilton stage 26) only hyaluronic acid (HA) could be detected in the ECM surrounding condensing primary ganglia. By day 7 (st 30) the ganglionic capsule contained HA, as well as sulfated glycosaminoglycans (GAGs), and this pattern continued into the adult stage. During the later stages of embryonic life (st 41-45) satellite cells appear, showing fine structural characteristics that point to their role in the secretion of intraganglionic ECM. Only during these stages could ECM be detected histochemically within ganglia, the same stages (days 15-19) when routine electron microscopic methods reveal collagen fibrils embedded in a granular ground substance. Thus, the intraganglionic environment appears as a separate compartment free of detectable amounts of GAG until late embryonic stages when ECM is secreted around satellite cells. This developmental pattern could represent a role of ECM in the histological stabilization of ganglia during the late stages of differentiation, since the appearance of intraganglionic ECM is correlated with the appearance of small dense-cored vesicles characteristic of adult neurons. The developmental pattern of ECM in differentiating sympathetic ganglia is compared with that of other tissues that undergo condensation and morphogenesis.

From Shelf to Shelf: Assessing Historical and Contemporary Genetic Differentiation and Connectivity across the Gulf of Mexico in Gag, Mycteroperca microlepis

PubMed Central

Jue, Nathaniel K.; Brulé, Thierry; Coleman, Felicia C.; Koenig, Christopher C.

2015-01-01

Describing patterns of connectivity among populations of species with widespread distributions is particularly important in understanding the ecology and evolution of marine species. In this study, we examined patterns of population differentiation, migration, and historical population dynamics using microsatellite and mitochondrial loci to test whether populations of the epinephelid fish, Gag, Mycteroperca microlepis, an important fishery species, are genetically connected across the Gulf of Mexico and if so, whether that connectivity is attributable to either contemporary or historical processes. Populations of Gag on the Campeche Bank and the West Florida Shelf show significant, but low magnitude, differentiation. Time since divergence/expansion estimates associated with historical population dynamics indicate that any population or spatial expansions indicated by population genetics would have likely occurred in the late Pleistocene. Using coalescent-based approaches, we find that the best model for explaining observed spatial patterns of contemporary genetic variation is one of asymmetric gene flow, with movement from Campeche Bank to the West Florida Shelf. Both estimated migration rates and ecological data support the hypothesis that Gag populations throughout the Gulf of Mexico are connected via present day larval dispersal. Demonstrating this greatly expanded scale of connectivity for Gag highlights the influence of “ghost” populations (sensu Beerli) on genetic patterns and presents a critical consideration for both fisheries management and conservation of this and other species with similar genetic patterns. PMID:25856095

C-Terminal HIV-1 Transframe p6* Tetrapeptide Blocks Enhanced Gag Cleavage Incurred by Leucine Zipper Replacement of a Deleted p6* Domain.

PubMed

Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

2017-05-15

HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation. IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6

Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage.

PubMed

Nowicka-Sans, Beata; Protack, Tricia; Lin, Zeyu; Li, Zhufang; Zhang, Sharon; Sun, Yongnian; Samanta, Himadri; Terry, Brian; Liu, Zheng; Chen, Yan; Sin, Ny; Sit, Sing-Yuen; Swidorski, Jacob J; Chen, Jie; Venables, Brian L; Healy, Matthew; Meanwell, Nicholas A; Cockett, Mark; Hanumegowda, Umesh; Regueiro-Ren, Alicia; Krystal, Mark; Dicker, Ira B

2016-07-01

BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1

Amino- and carboxyl-terminal amino acid sequences of proteins coded by gag gene of murine leukemia virus

PubMed Central

Oroszlan, Stephen; Henderson, Louis E.; Stephenson, John R.; Copeland, Terry D.; Long, Cedric W.; Ihle, James N.; Gilden, Raymond V.

1978-01-01

The amino- and carboxyl-terminal amino acid sequences of proteins (p10, p12, p15, and p30) coded by the gag gene of Rauscher and AKR murine leukemia viruses were determined. Among these proteins, p15 from both viruses appears to have a blocked amino end. Proline was found to be the common NH2 terminus of both p30s and both p12s, and alanine of both p10s. The amino-terminal sequences of p30s are identical, as are those of p10s, while the p12 sequences are clearly distinctive but also show substantial homology. The carboxyl-terminal amino acids of both viral p30s and p12s are leucine and phenylalanine, respectively. Rauscher leukemia virus p15 has tyrosine as the carboxyl terminus while AKR virus p15 has phenylalanine in this position. The compositional and sequence data provide definite chemical criteria for the identification of analogous gag gene products and for the comparison of viral proteins isolated in different laboratories. On the basis of amino acid sequences and the previously proposed H-p15-p12-p30-p10-COOH peptide sequence in the precursor polyprotein, a model for cleavage sites involved in the post-translational processing of the precursor coded for by the gag gene is proposed. PMID:206897

Performance of EAG and GAG Award Recipients Based on Length and Amount of Aid

ERIC Educational Resources Information Center

Battaglini, Janis K.

2004-01-01

The purpose of this analysis was to examine the persistence, graduation and transfer rates of full-time students who received an EAG (Educational Assistance Grant) or GAG (Guaranteed Access Grant) and matriculated during the 1996-1997 academic year. The performance of these students was examined on the basis of the number of years in which…

Glycosaminoglycan-Mimetic Signals Direct the Osteo/Chondrogenic Differentiation of Mesenchymal Stem Cells in a Three-Dimensional Peptide Nanofiber Extracellular Matrix Mimetic Environment.

PubMed

Arslan, Elif; Guler, Mustafa O; Tekinay, Ayse B

2016-04-11

Recent efforts in bioactive scaffold development focus strongly on the elucidation of complex cellular responses through the use of synthetic systems. Designing synthetic extracellular matrix (ECM) materials must be based on understanding of cellular behaviors upon interaction with natural and artificial scaffolds. Hence, due to their ability to mimic both the biochemical and mechanical properties of the native tissue environment, supramolecular assemblies of bioactive peptide nanostructures are especially promising for development of bioactive ECM-mimetic scaffolds. In this study, we used glycosaminoglycan (GAG) mimetic peptide nanofiber gel as a three-dimensional (3D) platform to investigate how cell lineage commitment is altered by external factors. We observed that amount of fetal bovine serum (FBS) presented in the cell media had synergistic effects on the ability of GAG-mimetic nanofiber gel to mediate the differentiation of mesenchymal stem cells into osteogenic and chondrogenic lineages. In particular, lower FBS concentration in the culture medium was observed to enhance osteogenic differentiation while higher amount FBS promotes chondrogenic differentiation in tandem with the effects of the GAG-mimetic 3D peptide nanofiber network, even in the absence of externally administered growth factors. We therefore demonstrate that mesenchymal stem cell differentiation can be specifically controlled by the combined influence of growth medium components and a 3D peptide nanofiber environment.

Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism.

PubMed Central

Spearman, P; Horton, R; Ratner, L; Kuli-Zade, I

1997-01-01

The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction. PMID:9261380

Gag-Positive Reservoir Cells Are Susceptible to HIV-Specific Cytotoxic T Lymphocyte Mediated Clearance In Vitro and Can Be Detected In Vivo

PubMed Central

Graf, Erin H.; Pace, Matthew J.; Peterson, Bennett A.; Lynch, Lindsay J.; Chukwulebe, Steve B.; Mexas, Angela M.; Shaheen, Farida; Martin, Jeffrey N.; Deeks, Steven G.; Connors, Mark; Migueles, Stephen A.; O’Doherty, Una

2013-01-01

Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy. PMID:23951263

NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

PubMed Central

Brown, Lola A.; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G.; Kuo, Lillian; Freed, Eric O.; Summers, Michael F.

2015-01-01

Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag’s N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly. PMID:25941825

Proinflammatory cytokine activities, matrix metalloproteinase-3 activity, and sulfated glycosaminoglycan content in synovial fluid of dogs with naturally acquired cranial cruciate ligament rupture.

PubMed

Fujita, Yukihiro; Hara, Yasushi; Nezu, Yoshinori; Schulz, Kurt S; Tagawa, Masahiro

2006-06-01

To measure and compare activities of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinase-3 (MMP-3); as well as sulfated glycosaminoglycan (S-GAG) content in synovial fluid from dogs with cranial cruciate ligament rupture (CCLR) and dogs with clinically normal stifles. To determine whether correlations exist between demographic and disease-related variables and these synovial markers. Prospective clinical study. Dogs with CCLR (n=23) and Beagles with normal stifle joints (n=21). Synovial fluid activities of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) were determined by bioassay. MMP-3 activity was measured using fluorogenic substrate. S-GAG contents were determined by dimethylmethylene blue dye-binding assay. Mann-Whitney U-test was used to compare results from CCLR joints with normal controls. Spearman's rank correlation test was used to evaluate associations between demographic and disease-related markers and synovial markers. Mean values for synovial markers were significantly higher in CCLR joints compared with controls. IL-1beta and MMP-3 were positively correlated with lameness duration. Activities of proinflammatory cytokines, MMP-3 activity and S-GAG contents were significantly elevated in synovial fluid from canine stifle joints with naturally acquired CCLR. These results indicate that there is joint inflammation and increased release of GAGs into synovial fluid, suggesting that these inflammatory changes are associated with depletion of proteoglycan from articular cartilage. Medical and surgical treatments designed to decrease joint inflammation and breakdown of proteoglycans may be of value in the management of CCLR in the dog.

New methods to study the composition and structure of the extracellular matrix in natural and bioengineered tissues

PubMed Central

Schiller, Jürgen; Huster, Daniel

2012-01-01

The extracellular matrix (ECM) comprises a gel of numerous biopolymers that occurs in a multitude of biological tissues. The ECM provides the basic support and mechanical strength of skeletal tissue and is responsible for shape retention. At the same time, the ECM is responsible for the viscoelastic properties and the elasticity of soft tissues. As expected, there are several important diseases that affect and degenerate the ECM with severe consequences for its properties. Bioengineering is a promising approach to support the regenerative capacity of the body. Unfortunately, the biomechanical properties of bioengineered ECM often only poorly meet the standards of their native counterparts. Many bioengineered tissues are characterized by an increased glycosaminoglycan (GAG) but decreased collagen content. This leads to an enhanced water content that strongly alters the viscoelastic and thus the biomechanical properties. Therefore, compositional analysis is important to estimate the tissue quality. We will show that nuclear magnetic resonance (NMR) spectroscopy and soft-ionization mass spectrometry (MS) represent useful techniques for ECM research both in natural and bioengineered tissues. Both methods are strongly complimentary: while MS techniques such as matrix-assisted laser desorption and ionization (MALDI) are excellent and very sensitive analytical tools to determine the collagen and the GAG contents of tissues, NMR spectroscopy provides insight into the molecular architecture of the ECM, its dynamics and other important parameters such as the water content of the tissue as well as the diffusion of molecules within the ECM. PMID:23507863

Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice

PubMed Central

Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D.

2014-01-01

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation. PMID:24614057

Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

PubMed

Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D

2014-01-01

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

Replication-Competent Simian Immunodeficiency Virus (SIV) Gag Escape Mutations Archived in Latent Reservoirs during Antiretroviral Treatment of SIV-Infected Macaques▿

PubMed Central

Queen, Suzanne E.; Mears, Brian M.; Kelly, Kathleen M.; Dorsey, Jamie L.; Liao, Zhaohao; Dinoso, Jason B.; Gama, Lucio; Adams, Robert J.; Zink, M. Christine; Clements, Janice E.; Kent, Stephen J.; Mankowski, Joseph L.

2011-01-01

In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8+ T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8+ T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4+ T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8+ T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted. PMID:21715484

Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis.

PubMed

Müllers, Erik; Uhlig, Tobias; Stirnnagel, Kristin; Fiebig, Uwe; Zentgraf, Hanswalter; Lindemann, Dirk

2011-02-01

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

Glucosamine Supplementation Demonstrates a Negative Effect On Intervertebral Disc Matrix in an Animal Model of Disc Degeneration

PubMed Central

Jacobs, Lloydine; Vo, Nam; Coehlo, J. Paulo; Dong, Qing; Bechara, Bernard; Woods, Barrett; Hempen, Eric; Hartman, Robert; Preuss, Harry; Balk, Judith; Kang, James; Sowa, Gwendolyn

2013-01-01

Study Design Laboratory based controlled in vivo study Objective To determine the in vivo effects of oral glucosamine sulfate on intervertebral disc degeneration Summary of Background Data Although glucosamine has demonstrated beneficial effect in articular cartilage, clinical benefit is uncertain. A CDC report from 2009 reported that many patients are using glucosamine supplementation for low back pain (LBP), without significant evidence to support its use. Because disc degeneration is a major contributor of LBP, we explored the effects of glucosamine on disc matrix homeostasis in an animal model of disc degeneration. Methods Eighteen skeletally mature New Zealand White rabbits were divided into four groups: control, annular puncture, glucosamine, and annular puncture+glucosamine. Glucosamine treated rabbits received daily oral supplementation with 107mg/day (weight based equivalent to human 1500mg/day). Annular puncture surgery involved puncturing the annulus fibrosus (AF) of 3 lumbar discs with a 16G needle to induce degeneration. Serial MRIs were obtained at 0, 4, 8, 12, and 20 weeks. Discs were harvested at 20 weeks for determination of glycosaminoglycan(GAG) content, relative gene expression measured by RT-PCR, and histological analyses. Results The MRI index and NP area of injured discs of glucosamine treated animals with annular puncture was found to be lower than that of degenerated discs from rabbits not supplemented with glucosamine. Consistent with this, decreased glycosaminoglycan was demonstrated in glucosamine fed animals, as determined by both histological and GAG content. Gene expression was consistent with a detrimental effect on matrix. Conclusions These data demonstrate that the net effect on matrix in an animal model in vivo, as measured by gene expression, MRI, histology, and total proteoglycan is anti-anabolic. This raises concern over this commonly used supplement, and future research is needed to establish the clinical relevance of these

Correlation between urinary GAG and anti-idursulfase ERT neutralizing antibodies during treatment with NICIT immune tolerance regimen: A case report☆,☆☆

PubMed Central

Kim, Sarah; Whitley, Chester B.; Jarnes Utz, Jeanine R.

2018-01-01

Introduction Antibodies to intravenous idursulfase enzyme replacement therapy (ERT) for patients with Hunter syndrome (mucopolysaccharidosis type II, MPS II) can have a harmful clinical impact, including both increasing risk of infusion reactions and inhibiting therapeutic activity. Thus, failure to monitor anti-idursulfase antibodies and neutralizing antibodies, and delays in reporting results, may postpone critical clinical decisions. Hypothesis Urinary glycosaminoglycan (GAG) levels may be used as a biomarker for anti-idursulfase antibodies and neutralizing antibodies to improve timeliness in monitoring and managing ERT. Methods This is a case report describing a patient with MPS II with high levels of neutralizing antibodies and worsened clinical status who was treated for five years with a non-immunosuppressive and non-cytotoxic immune tolerance (NICIT) regimen, consisting of intravenous immune globulin and frequent infusions of idursulfase. Neutralizing antibodies and total anti-idursulfase antibodies were measured by two different methods, the direct 1,9-dimethylmethylene blue (DMB) assay and cetylpyridinium chloride carbazole-borate (CPC) assay. Results Neutralizing antibodies, measured as percent inhibition of enzyme activity and also by total neutralizing antibody titer, were correlated with quantitative urinary GAG measured by DMB assay (p = 0.026, p = 0.0067), and quantitative urinary GAG by CPC assay with percent inhibition of enzyme activity by neutralizing antibodies (p = 0.0475). The NICIT regimen showed a sustained immune tolerance after five years and was well-tolerated. Conclusions Urinary GAG, measured by DMB assay, may be a biomarker for anti-idursulfase neutralizing antibodies and is useful for managing immune tolerance regimens for patients with MPS II who have high levels of anti-idursulfase neutralizing antibodies. This study highlights the importance of regular and frequent monitoring of urinary GAG in patients with MPS II who are

Three-Dimensional Coculture of Meniscal Cells and Mesenchymal Stem Cells in Collagen Type I Hydrogel on a Small Intestinal Matrix-A Pilot Study Toward Equine Meniscus Tissue Engineering.

PubMed

Kremer, Antje; Ribitsch, Iris; Reboredo, Jenny; Dürr, Julia; Egerbacher, Monika; Jenner, Florien; Walles, Heike

2017-05-01

Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for their qualification to be applied for meniscus tissue engineering. To investigate cell attachment of primary MC and MSC on SIS-muc matrix SEM pictures were performed. For molecular analysis, lyophilized samples of coculture constructs with different cell ratios (100% MC, 100% MSC, and 50% MC and 50% MSC, 20% MC, and 80% MSC) were digested and analyzed for DNA and GAG content. Active matrix remodeling of 3D coculture models was indicated by matrix metalloproteinases detection. For comparison of tissue-engineered constructs with the histologic architecture of natural equine menisci, paired lateral and medial menisci of 15 horses representing different age groups were examined. A meniscus phenotype with promising similarity to native meniscus tissue in its GAG/DNA expression in addition to Col I, Col II, and Aggrecan production was achieved using a scaffold composed of Col I gel on SIS-muc combined with a coculture of MC and MSC. The results encourage further development of this scaffold-cell combination for meniscus tissue engineering.

In the absence of overt urothelial damage, chondroitinase ABC digestion of the GAG layer increases bladder permeability in ovariectomized female rats

PubMed Central

Van Gordon, Samuel; Tyler, Karl; Kropp, Bradley; Towner, Rheal; Lin, HsuehKung; Marentette, John O.; McHowat, Jane; Mohammedi, Ehsan; Greenwood-Van Meerveld, Beverley

2016-01-01

Loss of integrity of the protective impermeability barrier in the urothelium has been identified as significant in bladder dysfunction. In this study, we tested the theory that the luminal layer of glycosaminoglycans (GAG) serves as an important component of barrier function. The peptide polycation protamine sulfate (PS), 1 mg/ml, was instilled intravesically for 10 min into rat bladders. Chondroitinase ABC (ChABC), 63 IU/ml, was instilled into an additional six rats for 30 min to digest the GAG layer. Unmanipulated controls and sham-injected controls were also performed. After 24 h, the rats were euthanized, the bladders were removed, and permeability was assessed in the Ussing chamber and by diffusion of FITC-labeled dextran (4 kDa) to measure macromolecular permeability. The status of tight junctions was assessed by immunofluorescence and electron microscopy. In control and sham treated rat bladders, the transepithelial electrical resistance were means of 2.5 ± 1.1 vs. 2.6 ± 1.1 vs 1.2 ± 0.5 and 1.01 ± 0.7 kΩ·cm2 in the PS-treated and ChABC-treated rat bladders (P = 0.0016 and P = 0.0039, respectively). Similar differences were seen in dextran permeability. Histopathology showed a mild inflammation following PS treatment, but the ChABC-treated bladders were indistinguishable from controls. Tight junctions generally remained intact. ChABC digestion alone induced bladder permeability, confirming the importance of the GAG layer to bladder barrier function and supports that loss of the GAG layer seen in bladder biopsies of interstitial cystitis patients could be a significant factor producing symptoms for at least some interstitial cystitis/painful bladder syndrome patients. PMID:26911855

Intersubtype Differences in the Effect of a Rare p24 Gag Mutation on HIV-1 Replicative Fitness

PubMed Central

Chopera, Denis R.; Cotton, Laura A.; Zawaira, Alexander; Mann, Jaclyn K.; Ngandu, Nobubelo K.; Ntale, Roman; Carlson, Jonathan M.; Mlisana, Koleka; Woodman, Zenda; de Assis Rosa, Debra; Martin, Eric; Miura, Toshiyuki; Pereyra, Florencia; Walker, Bruce D.; Gray, Clive M.; Martin, Darren P.; Ndung'u, Thumbi; Brockman, Mark A.; Karim, Salim Abdool

2012-01-01

Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness. PMID:23015721

Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA

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

LeBlanc, Jason J.; Uddowla, Sabena; Abraham, Benjamin

2007-07-05

All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had amore » diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5.« less

Dynamic Changes in Cervical Glycosaminoglycan Composition during Normal Pregnancy and Preterm Birth

PubMed Central

Akgul, Yucel; Holt, Roxane; Mummert, Mark; Word, Ann

2012-01-01

Glycosaminoglycans (GAG) have diverse functions that regulate macromolecular assembly in the extracellular matrix. During pregnancy, the rigid cervix transforms to a pliable structure to allow birth. Quantitative assessment of cervical GAG is a prerequisite to identify GAG functions in term and preterm birth. In the current study, total GAG levels increased at term, yet the abundance, chain length, and sulfation levels of sulfated GAG remained constant. The increase in total GAG resulted exclusively from an increase in hyaluronan (HA). HA can form large structures that promote increased viscosity, hydration, and matrix disorganization as well as small structures that have roles in inflammation. HA levels increased from 19% of total GAG in early pregnancy to 71% at term. Activity of the HA-metabolizing enzyme, hyaluronidase, increased in labor, resulting in metabolism of large to small HA. Similar to mice, HA transitions from high to low molecular weight in term human cervix. Mouse preterm models were also characterized by an increase in HA resulting from differential expression of the HA synthase (Has) genes, with increased Has1 in preterm in contrast to Has2 induction at term. The Has2 gene but not Has1 is regulated in part by estrogen. These studies identify a shift in sulfated GAG dominance in the early pregnant cervix to HA dominance in term and preterm ripening. Increased HA synthesis along with hyaluronidase-induced changes in HA size in mice and women suggest diverse contributions of HA to macromolecular changes in the extracellular matrix, resulting in loss of tensile strength during parturition. PMID:22529214

Influence of residual composition on the structure and properties of extracellular matrix derived hydrogels.

PubMed

Claudio-Rizo, Jesús A; Rangel-Argote, Magdalena; Castellano, Laura E; Delgado, Jorge; Mata-Mata, José L; Mendoza-Novelo, Birzabith

2017-10-01

In this work, hydrolysates of extracellular matrix (hECM) were obtained from rat tail tendon (TR), bovine Achilles tendon (TAB), porcine small intestinal submucosa (SIS) and bovine pericardium (PB), and they were polymerized to generate ECM hydrogels. The composition of hECM was evaluated by quantifying the content of sulphated glycosaminoglycans (sGAG), fibronectin and laminin. The polymerization process, structure, physicochemical properties, in vitro degradation and biocompatibility were studied and related to their composition. The results indicated that the hECM derived from SIS and PB were significantly richer in sGAG, fibronectin and laminin, than those derived from TAB and TR. These differences in hECM composition influenced the polymerization and the structural characteristics of the fibrillar gel network. Consequently, the swelling, mechanics and degradation of the hydrogels showed a direct relationship with the remaining composition. Moreover, the cytocompatibility and the secretion of transforming growth factor beta-1 (TGF-β1) by macrophages were enhanced in hydrogels with the highest residual content of ECM biomolecules. The results of this work evidenced the role of the ECM molecules remaining after both decellularization and hydrolysis steps to produce tissue derived hydrogels with structure and properties tailored to enhance their performance in tissue engineering and regenerative medicine applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

PubMed

Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

2005-11-01

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

PubMed

Rizvi, Tahir A; Kenyon, Julia C; Ali, Jahabar; Aktar, Suriya J; Phillip, Pretty S; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M L

2010-10-15

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV

Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.

PubMed

Hammer, Diana; Wild, Jens; Ludwig, Christine; Asbach, Benedikt; Notka, Frank; Wagner, Ralf

2008-06-01

Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

Effectiveness of Radiotherapy in Myxoid Sarcomas Is Associated With a Dense Vascular Pattern

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

Vreeze, Ronald S.A. de; Jong, Daphne de; Haas, Rick L.

2008-12-01

Purpose: Surgery and adjuvant radiotherapy (RT) have long been the standard treatment for most deep-seated sarcomas; however, since the randomized trial from the National Cancer Institute of Canada, which described similar local control for pre- vs. postoperative RT, both modalities are now widely accepted. As a group, sarcomas are classified as radiation resistant. The subgroup of myxoid liposarcoma (MLS), a sarcoma with a typical vascular crow's feet pattern, is highly radiosensitive, but a mechanism for this phenomenon is unknown. Here we describe our results with preoperative RT and propose a mechanism explaining the high sensitivity based on the distinctive vascularizationmore » pattern of MLS. Methods and Materials: Between 2002 and 2006, 31 sarcoma patients, including 10 with MLS, underwent preoperative RT at our institute. Resected specimens were histologically evaluated, focusing on classification, grade, and vascularization patterns. Results: Twenty sarcomas showed more than 80% pathologic response after preoperative RT. A pathologic complete response was found in all 'pure' MLS specimens after preoperative RT (n = 8). There were no pathologic complete responses in the remaining sarcoma patients (n = 23), although 12 showed 80% to 90% pathologic response. In contrast to the remaining RT-resistant sarcomas, the highly responding specimens contained branching vasculature, partial thrombus formation and inflammation of medium sized arterioles, similar to the vascular changes in MLS. Conclusions: Both MLS and sarcomas with MLS-like vasculature are highly radiosensitive. Radiation sensitivity may be explained by changes in medium-sized arterioles, obstructing the specific crow's feet vascularization and inducing hypoxia with secondary tumor cell death.« less

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges

PubMed Central

Lakhashe, Samir K.; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B.; DiPasquale, Janet M.; Hemashettar, Girish; Yoon, John K.; Rasmussen, Robert A.; Yang, Feng; Lee, Sandra J.; Montefiori, David C.; Novembre, Francis J.; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R.; Robert-Guroff, Marjorie; Johnson, Welkin E.; Lieberman, Judy; Ruprecht, Ruth M.

2011-01-01

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus >90%; these RM also had strong SIV Gag-specific proliferation of CD8+ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4+ T cells; the latter have been implicated as preferential virus targets in-vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. PMID:21693155

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

PubMed

Lakhashe, Samir K; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B; Dipasquale, Janet M; Hemashettar, Girish; Yoon, John K; Rasmussen, Robert A; Yang, Feng; Lee, Sandra J; Montefiori, David C; Novembre, Francis J; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R; Robert-Guroff, Marjorie; Johnson, Welkin E; Lieberman, Judy; Ruprecht, Ruth M

2011-08-05

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bone Marrow Stem Cells in Response to Intervertebral Disc-Like Matrix Acidity and Oxygen Concentration: Implications for Cell-based Regenerative Therapy.

PubMed

Naqvi, Syeda M; Buckley, Conor T

2016-05-01

In vitro culture of porcine bone marrow stem cells (BMSCs) in varying pH microenvironments in a three-dimensional hydrogel system. To characterize the response of BMSCs to varying pH environments (blood [pH 7.4], healthy intervertebral disc (IVD) (pH 7.1), mildly degenerated IVD (pH 6.8), and severely degenerated IVD (pH 6.5) in three-dimensional culture under normoxic (20%) and hypoxic (5%) conditions. The IVD is an avascular organ relying on diffusion of essential nutrients through the cartilaginous endplates (CEPs) thereby creating a challenging microenvironment. Within a degenerated IVD, oxygen and glucose concentrations decrease further (<5% oxygen, <5 mmol/L glucose) and matrix acidity (GAG) and collagen accumulation at pH 6.5 with increased proliferation, sustained cell viability and increased sGAG and collagen accumulation in pH 6.8 or higher. These findings suggest that there is a threshold at pH 6.8, below which cells cannot survive and accumulate nucleus pulposus-like matrix components (sGAG and collagen). Translation into a multimodal protocol requires the survival of stem cells and their ability to function normally amidst the harsh microenvironment. This study demonstrates the critical implication of degeneration stage and suggests stratified targeting to identify suitable candidates through measurement of the local pH thereby maximizing

Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc.

PubMed

Mwale, F; Roughley, P; Antoniou, J

2004-12-15

Tissue engineering of intervertebral discs (IVD) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to compare the proteoglycan to collagen ratio (measured as GAG to hydroxyproline ratio) in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals and test the hypothesis that this ratio can be used for in vivo studies to distinguish between a normal NP and hyaline cartilage phenotype. Whole human lumbar spine specimens from fresh cadavers, ranging in age from 12 weeks to 79 years, were used to harvest the IVDs and adjacent endplates. The GAG to hydroxyproline ratio within the NP of young adults is approximately 27:1, whereas the ratio within the hyaline cartilage endplate of the same aged individuals is about 2:1. The production of an extracellular matrix with a high proteoglycan to collagen ratio can be used in vivo to distinguish NP cells from chondrocytes, and could help in identifying a NP-like phenotype in vivo as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc.

Fluorescein diacetate for determination of cell viability in 3D fibroblast-collagen-GAG constructs.

PubMed

Powell, Heather M; Armour, Alexis D; Boyce, Steven T

2011-01-01

Quantification of cell viability and distribution within engineered tissues currently relies on representative histology, phenotypic assays, and destructive assays of viability. To evaluate uniformity of cell density throughout 3D collagen scaffolds prior to in vivo use, a nondestructive, field assessment of cell viability is advantageous. Here, we describe a field measure of cell viability in lyophilized collagen-glycosaminoglycan (C-GAG) scaffolds in vitro using fluorescein diacetate (FdA). Fibroblast-C-GAG constructs are stained 1 day after cellular inoculation using 0.04 mg/ml FdA followed by exposure to 366 nm UV light. Construct fluorescence quantified using Metamorph image analysis is correlated with inoculation density, MTT values, and histology of corresponding biopsies. Construct fluorescence correlates significantly with inoculation density (p  <  0.001) and MTT values (p  <  0.001) of biopsies collected immediately after FdA staining. No toxicity is detected in the constructs, as measured by MTT assay before and after the FdA assay at different time points; normal in vitro histology is demonstrated for the FdA-exposed constructs. In conclusion, measurement of intracellular fluorescence with FdA allows for the early, comprehensive measurement of cellular distributions and viability in engineered tissue.

Effectiveness of Intellectual Distraction on Gagging and Anxiety Management in Children: A Prospective Clinical Study

PubMed Central

Debs, Nahla Nassif; Aboujaoude, Samia

2017-01-01

Objectives: The aim of the present prospective study is to determine the effect of an intellectual colored game (ICG) on the severity of gag reflex (GR) and anxiety in children during dental alginate impression. Materials and Methods: Forty-one children, aging between 5 and 11 years, having a GR varying from normal to moderate had upper alginate impressions. The children's anxiety was evaluated with a facial image scale (FIS) before (T0) and after first failed impression (T1), then, after playing an intellectual colored game (ICG) at T2, while taking an upper alginate impression. Results: 42.9 % of the children had a gag reflex of stage 2 and 31.0 % a facial scale of 3. Initial GR was not significantly associated with the final success of the impression (P =0.260) whereas final impression success was strongly associated with FIS (P <0.001). There was a statistically significant reduction in median GR score from T0 to T2 (P < 0.001) and FIS dropped significantly at T2 with ICG (P < 0.001). Conclusion: This study highlights the clinical performance of the intellectual distraction approach in GR management PMID:29387614

Exosomes carring gag/env of ALV-J possess negative effect on immunocytes.

PubMed

Wang, Guihua; Wang, Zhenzhen; Zhuang, Pingping; Zhao, Xiaomin; Cheng, Ziqiang

2017-11-01

J subgroup avian leukosis virus (ALV-J) is an exogenous retrovirus of avian. A key feature of ALV-J infection is leading to severe immunosuppressive characteristic of diseases. Viral components of retrovirus were reported closely associated with immunosuppression, and several similarities between exosomes and retrovirus preparations have lead to the hypotheses of retrovirus hijacker exosomes pathway. In this study, we purified exosomes from DF-1 cells infected and uninfected by ALV-J. Electron microscopy and mass spectrometry (MS) analysis showed that ALV-J not only increased the production of exosomes from ALV-J infected DF-1 cells (Exo-J) but also stimulated some proteins expression, especially ALV-J components secreted in exosomes. Immunosuppressive domain peptide (ISD) of envelope subunit transmembrane (TM) and gag of ALV-J were secreted in Exo-J. It has been reported that HIV gag was budded from endosome-like domains of the T cell plasma membrane. But env protein was first detected in exosomes from retrovirus infected cells. We found that Exo-J caused negative effects on splenocytes in a dose-dependant manner by flow cytometric analysis. And low dose of Exo-J activated immune activity of splenocytes, while high dose possessed immunosuppressive properties. Interestingly, Exo-J has no significant effects on the immunosuppression induced by ALV-J, and the immunosuppressive effects induced by Exo-J lower than that by ALV-J. Taken together, our data indicated that Exo-J supplied a microenvironment for the replication and transformation of ALV-J. Copyright © 2017. Published by Elsevier Ltd.

Global cerebral ischemia with subsequent respiratory arrest in a cat after repeated use of a spring-loaded mouth gag.

PubMed

Hartman, Emily A; McCarthy, Robert J; Labato, Mary A

2017-01-01

A 10-year-old neutered male domestic shorthair cat was evaluated because of signs of stertorous breathing and reverse sneezing of 8 months' duration. A CT scan performed 1 week before evaluation indicated nasopharyngeal stenosis or collapse. Increased respiratory effort, stertorous breathing, coughing, reverse sneezing, bilateral black ocular discharge and mucoid left nasal discharge were noted. Rhinoscopy suggested possible nasopharyngeal stenosis. Balloon dilation was attempted but unsuccessful. Ventral rhinotomy was performed the following day using a spring-loaded mouth gag to access the surgical site. After rhinotomy, the patient had neurologic signs attributed to global cerebral ischemia that progressed to respiratory arrest, subsequently resulting in euthanasia. While ischemic brain injury has been associated with the use of a spring-loaded mouth gag in cats, to our knowledge this is the first reported instance where use resulted in respiratory arrest culminating in euthanasia.

Glycol chitosan/oxidized hyaluronic acid hydrogels functionalized with cartilage extracellular matrix particles and incorporating BMSCs for cartilage repair.

PubMed

Liu, Chun; Liu, Deshuai; Wang, Yingying; Li, Yun; Li, Tao; Zhou, Zhiyou; Yang, Zhijian; Wang, Jianhua; Zhang, Qiqing

2018-02-05

In this article, we fabricated a bioactive hydrogel composed of glycol chitosan (G-CS) and oxidized hyaluronic acid (OHA) via Schiff base reaction. Cartilage extracellular matrix (ECM) particles with different concentrations were used to functionalize G-CS/OHA (S1) hydrogel. The results demonstrated that S3 (G-CS/OHA/ECM 2% w/v) hydrogel exhibited the most suitable compression strength and provided the optimal environment for proliferation of bone marrow mesenchymal stem cells (BMSCs). To assess the chondroinductivity of ECM in vitro, we compared the chondrogenesis of BMSCs in S1 (G-CS/OHA) and S3 (G-CS/OHA/ECM 2% w/v) hydrogels. The results confirmed that the higher levels of glycosaminoglycans (GAGs) and collagen type II (COL II) were accumulated in S3 hydrogel. In vivo, cartilage defects of rats generated most mature tissue within BMSCs-laden S3 hydrogel (S3/BMSCs group). The tissues were more integrative and contained higher levels of COL II and GAGs compared to the other groups. All these results suggested that the G-CS/OHA hydrogel functionalized with ECM particles is a good candidate biomaterial to be applied in cartilage tissue engineering.

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates

PubMed Central

Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

2005-01-01

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain. PMID:16262906

Shell extracts from the marine bivalve Pecten maximus regulate the synthesis of extracellular matrix in primary cultured human skin fibroblasts.

PubMed

Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

2014-01-01

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the -112/-61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications.

Shell Extracts from the Marine Bivalve Pecten maximus Regulate the Synthesis of Extracellular Matrix in Primary Cultured Human Skin Fibroblasts

PubMed Central

Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

2014-01-01

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the −112/−61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications. PMID:24949635

The capsid-spacer peptide 1 Gag processing intermediate is a dominant-negative inhibitor of HIV-1 maturation.

PubMed

Checkley, Mary Ann; Luttge, Benjamin G; Soheilian, Ferri; Nagashima, Kunio; Freed, Eric O

2010-04-25

The human immunodeficiency virus type 1 (HIV-1) maturation inhibitor bevirimat disrupts virus replication by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) Gag processing intermediate to mature CA. The observation that bevirimat delays but does not completely block CA-SP1 processing suggests that the presence of uncleaved CA-SP1 may disrupt the maturation process in trans. In this study, we validate this hypothesis by using a genetic approach to demonstrate that a non-cleavable CA-SP1 mutant exerts a dominant-negative effect on maturation of wild-type HIV-1. In contrast, a mutant in which cleavage can occur internally within SP1 is significantly less potent as a dominant-negative inhibitor. We also show that bevirimat blocks processing at both the major CA-SP1 cleavage site and the internal site. These data underscore the importance of full CA-SP1 processing for HIV-1 maturation and highlight the therapeutic potential of inhibitors that target this Gag cleavage event. Published by Elsevier Inc.

Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

PubMed Central

Lin, Ying-Chuan; Brik, Ashraf; de Parseval, Aymeric; Tam, Karen; Torbett, Bruce E.; Wong, Chi-Huey; Elder, John H.

2006-01-01

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations. PMID:16873240

Identifying a New Mechanism of HIV Core Formation | Center for Cancer Research

Cancer.gov

During the maturation of human immunodeficiency virus 1 (HIV-1), viral particles transition from a noninfectious form to an infectious one, and this conversion requires the cleavage of the HIV-1 Gag polyprotein. Gag is made up of three structural proteins—matrix (MA), capsid (CA), and nucleocapsid (NC)—connected by linkers. MA anchors Gag in the membrane, CA surrounds the

PTAP motif duplication in the p6 Gag protein confers a replication advantage on HIV-1 subtype C.

PubMed

Sharma, Shilpee; Arunachalam, Prabhu S; Menon, Malini; Ragupathy, Viswanath; Satya, Ravi Vijaya; Jebaraj, Joshua; Ganeshappa Aralaguppe, Shambhu; Rao, Chaitra; Pal, Sreshtha; Saravanan, Shanmugam; Murugavel, Kailapuri G; Balakrishnan, Pachamuthu; Solomon, Suniti; Hewlett, Indira; Ranga, Udaykumar

2018-05-17

HIV-1 subtype C (HIV-1C) may duplicate longer amino acid stretches in the p6 Gag protein, leading to the creation of an additional Pro-Thr/Ser-Ala-Pro (PTAP) motif necessary for viral packaging. However, the biological significance of a duplication of the PTAP motif for HIV-1 replication and pathogenesis has not been experimentally validated. In a longitudinal study of two different clinical cohorts of select HIV-1 seropositive, drug-naive individuals from India, we found that 8 of 50 of these individuals harbored a mixed infection of viral strains discordant for the PTAP duplication. Conventional and next-generation sequencing of six primary viral quasispecies at multiple time points disclosed that in a mixed infection, the viral strains containing the PTAP duplication dominated the infection. The dominance of the double-PTAP viral strains over a genetically similar single-PTAP viral clone was confirmed in viral proliferation and pairwise competition assays. Of note, in the proximity ligation assay, double-PTAP Gag proteins exhibited a significantly enhanced interaction with the host protein tumor susceptibility gene 101 (Tsg101). Moreover, Tsg101 overexpression resulted in a biphasic effect on HIV-1C proliferation - an enhanced effect at low concentration and an inhibitory effect only at higher concentrations - unlike a uniformly inhibitory effect on subtype B strains. In summary, our results indicate that the duplication of the PTAP motif in the p6 Gag protein enhances the replication fitness of HIV-1C by engaging the Tsg101 host protein with a higher affinity. Our results have implications for HIV-1 pathogenesis, especially of HIV-1C. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon, Portugal.

PubMed

Esteves, Aida; Parreira, Ricardo; Piedade, João; Venenno, Teresa; Franco, Margarida; Germano de Sousa, José; Patrício, Luis; Brum, Paula; Costa, António; Canas-Ferreira, Wanda F

2003-06-01

We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique.

Detection of Hemoglobin New York [β113 (G15) Val→Glu, GTG>GAG] in a Thai Woman by Capillary Electrophoresis.

PubMed

Panyasai, Sitthichai; Pornprasert, Sakorn

2016-12-01

Hemoglobin (Hb) New York [β113 (G15) Val→Glu, GTG>GAG] is a very rare β-chain variant found in Thailand. This variant is often missed by routine laboratory testing because Hb New York and Hb A have the identical retention time on high performance liquid chromatography. We reported here for the first time that the detection of Hb New York in a Thai woman by using capillary electrophoresis (CE). A peak of Hb New York located ahead of Hb A at the electrophoretic zone 11 with a level of 42.8 %. The DNA sequencing revealed the GTG>GAG mutation at codon 113 for Hb New York on one allele of β-globin gene. Therefore, the CE has a high efficiency to prevent the misinterpretation of hemoglobin analysis in patients who are heterozygote of this variant.

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

PubMed Central

Henderson, L E; Sowder, R; Copeland, T D; Smythers, G; Oroszlan, S

1984-01-01

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

PubMed Central

Barros, Marilia; Nanda, Hirsh

2016-01-01

ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

Recombinant modified vaccinia virus Ankara–simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer

PubMed Central

Seth, Aruna; Ourmanov, Ilnour; Kuroda, Marcelo J.; Schmitz, Jörn E.; Carroll, Miles W.; Wyatt, Linda S.; Moss, Bernard; Forman, Meryl A.; Hirsch, Vanessa M.; Letvin, Norman L.

1998-01-01

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans. PMID:9707609

Unusual Asymptomatic Fluorodeoxyglucose Avid Pheochromocytoma in a Case of Myxoid Liposarcoma of the Extremity on 18-F Fluorodeoxyglucose Positron Emission Tomography-computed Tomography.

PubMed

Shivdasani, Divya; Singh, Natasha; Pereira, Melvika; Zade, Anand

2017-01-01

Sarcomas are a heterogeneous group of rare tumors and arise either from soft tissue or from bone. Soft-tissue sarcomas (STSs) initially metastasize to the lungs. Metastases to extrapulmonary sites such as liver, brain, and soft tissue distant from primary tumor usually develop later. However, cases with isolated adrenal metastasis without disseminated disease have been reported in literature. We present a case of primary myxoid liposarcoma of the lower limb, in which staging 18 -F fluorodeoxyglucose positron emission tomography-computed tomography (FDG PET-CT) scan detected a suspicious FDG avid adrenal lesion which eventually on resection was diagnosed as asymptomatic pheochromocytoma. Pheochromocytomas have been reported to demonstrate FDG uptake mimicking metastasis. Hence, while interpreting FDG PET-CT scans in the context of STSs, both the extrapulmonary metastatic potential of aggressive histological subtypes of sarcoma and rare possibility of FDG avid coexistent benign tumor should be taken into consideration.

Enhancement of Gag-specific but reduction of Env- and Pol-specific CD8+ T cell responses by simian immunodeficiency virus nonstructural proteins in mice.

PubMed

Zhang, Yinfeng; Sun, Caijun; Feng, Liqiang; Xiao, Lijun; Chen, Ling

2012-04-01

Accessory and regulatory proteins (nonstructural proteins) have received increasing attention as components in novel HIV/SIV vaccine design. However, the complicated interactions between nonstructural proteins and structural proteins remain poorly understood, especially their effects on immunogenicity. In this study, the immunogenicity of structural proteins in the presence and absence of nonstructural proteins was compared. First, a series of recombinant plasmids and adenoviral vectors carrying various SIVmac239 nonstructural and structural genes was constructed. Then mice were primed with DNA plasmids and boosted with corresponding Ad5 vectors of different combinations, and the resulting immune responses were measured. Our results demonstrated that when the individual Gag, Pol, or Env gene products were coimmunized with the whole repertoire of nonstructural proteins, the Gag-specific CD8(+) T response was greatly enhanced, while the Env- and Pol-specific CD8(+) T responses were significantly reduced. The same pattern was not observed in CD4(+) T cell responses. Antibody responses against both the Gag and Env proteins were elicited more effectively when these structural antigens were immunized together with nonstructural antigens. These findings may provide helpful insights into the development of novel HIV/SIV vaccines.

X-ray crystallographic characterization of rhesus macaque MHC Mamu-A*02 complexed with an immunodominant SIV-Gag nonapeptide

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

Feng, Youjun; Graduate School, Chinese Academy of Sciences, Beijing; Qi, Jianxun

2006-01-01

X-ray crystallographic characterization of rhesus macaque MHC Mamu-A*02 complexed with an immunodominant SIV-Gag nonapeptide. Simian immunodeficiency virus (SIV) in the rhesus macaque is regarded as a classic animal model, playing a crucial role in HIV vaccine strategies and therapeutics by characterizing various cytotoxic T-lymphocyte (CTL) responses in macaque monkeys. However, the availability of well documented structural reports focusing on rhesus macaque major histocompatibility complex class I (MHC I) molecules remains extremely limited. Here, a complex of the rhesus macaque MHC I molecule (Mamu-A*02) with human β{sub 2}m and an immunodominant SIV-Gag nonapeptide, GESNLKSLY (GY9), has been crystallized. The crystal diffractsmore » X-rays to 2.7 Å resolution and belongs to space group C2, with unit-cell parameters a = 124.11, b = 110.45, c = 100.06 Å, and contains two molecules in the asymmetric unit. The availability of the structure, which is being solved by molecular replacement, will provide new insights into rhesus macaque MHC I (Mamu-A*02) presenting pathogenic SIV peptides.« less

Early evolution of HLA-associated escape mutations in variable Gag proteins predicts CD4+ decline in HIV-1 subtype C infected women

PubMed Central

Chopera, Denis R.; Ntale, Roman; Ndabambi, Nonkululeko; Garrett, Nigel; Gray, Clive M.; Matten, David; Karim, Quarraisha Abdool; Karim, Salim Abdool; Williamson, Carolyn

2016-01-01

Objective HIV-1 escape from cytotoxic T-lymphocytes (CTL) results in the accumulation of HLA-associated mutations in the viral genome. To understand the contribution of early escape to disease progression, this study investigated the evolution and pathogenic implications of CTL escape in a cohort followed from infection for five years. Methods Viral loads and CD4+ counts were monitored in 78 subtype C infected individuals from onset of infection until CD4+ decline to <350 cells/μl or five years post-infection. The gag gene was sequenced and HLA-associated changes between enrolment and 12 months post-infection were mapped. Results HLA-associated escape mutations were identified in 48 (62%) of the participants and were associated with CD4+ decline to <350 copies/ml (p=0.05). Escape mutations in variable Gag proteins (p17 and p7p6) had a greater impact on disease progression than escape in more conserved regions (p24) (p=0.03). The association between HLA-associated escape mutations and CD4+ decline was independent of protective HLA allele (B*57, B*58:01, B*81) expression. Conclusion The high frequency of escape contributed to rapid disease progression in this cohort. While HLA-adaption in both conserved and variable Gag domains in the first year of infection was detrimental to long term clinical outcome, escape in variable domains had greater impact. PMID:27755110

N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

PubMed Central

Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

2016-01-01

The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

Reaction-diffusion basis of retroviral infectivity

NASA Astrophysics Data System (ADS)

Sadiq, S. Kashif

2016-11-01

Retrovirus particle (virion) infectivity requires diffusion and clustering of multiple transmembrane envelope proteins (Env3) on the virion exterior, yet is triggered by protease-dependent degradation of a partially occluding, membrane-bound Gag polyprotein lattice on the virion interior. The physical mechanism underlying such coupling is unclear and only indirectly accessible via experiment. Modelling stands to provide insight but the required spatio-temporal range far exceeds current accessibility by all-atom or even coarse-grained molecular dynamics simulations. Nor do such approaches account for chemical reactions, while conversely, reaction kinetics approaches handle neither diffusion nor clustering. Here, a recently developed multiscale approach is considered that applies an ultra-coarse-graining scheme to treat entire proteins at near-single particle resolution, but which also couples chemical reactions with diffusion and interactions. A model is developed of Env3 molecules embedded in a truncated Gag lattice composed of membrane-bound matrix proteins linked to capsid subunits, with freely diffusing protease molecules. Simulations suggest that in the presence of Gag but in the absence of lateral lattice-forming interactions, Env3 diffuses comparably to Gag-absent Env3. Initial immobility of Env3 is conferred through lateral caging by matrix trimers vertically coupled to the underlying hexameric capsid layer. Gag cleavage by protease vertically decouples the matrix and capsid layers, induces both matrix and Env3 diffusion, and permits Env3 clustering. Spreading across the entire membrane surface reduces crowding, in turn, enhancing the effect and promoting infectivity. This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'.

Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

NASA Astrophysics Data System (ADS)

Vogel, Sarah; Arnoldini, Simon; Möller, Stephanie; Schnabelrauch, Matthias; Hempel, Ute

2016-11-01

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Enamel Matrix Derivative has No Effect on the Chondrogenic Differentiation of Mesenchymal Stem Cells

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

Groeneveldt, Lisanne C.; Knuth, Callie; Witte-Bouma, Janneke

2014-09-02

Background: Treatment of large bone defects due to trauma, tumor resection, or congenital abnormalities is challenging. Bone tissue engineering using mesenchymal stem cells (MSCs) represents a promising treatment option. However, the quantity and quality of engineered bone tissue are not sufficient to fill large bone defects. The aim of this study was to determine if the addition of enamel matrix derivative (EMD) improves in vitro chondrogenic priming of MSCs to ultimately improve in vivo MSC mediated endochondral bone formation. Methods: MSCs were chondrogenically differentiated in 2.0 × 10{sup 5} cell pellets in medium supplemented with TGFβ3 in the absence ormore » presence of 1, 10, or 100 μg/mL EMD. Samples were analyzed for gene expression of RUNX2, Col II, Col X, and Sox9. Protein and glycoaminoglycan (GAG) production were also investigated via DMB assays, histology, and immunohistochemistry. Osteogenic and adipogenic differentiation capacity were also assessed. Results: The addition of EMD did not negatively affect chondrogenic differentiation of adult human MSCs. EMD did not appear to alter GAG production or expression of chondrogenic genes. Osteogenic and adipogenic differentiation were also unaffected though a trend toward decreased adipogenic gene expression was observed. Conclusion: EMD does not affect chondrogenic differentiation of adult human MSCs. As such the use of EMD in combination with chondrogenically primed MSCs for periodontal bone tissue repair is unlikely to have negative effects on MSC differentiation.« less

HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection.

PubMed

Laher, Faatima; Ranasinghe, Srinika; Porichis, Filippos; Mewalal, Nikoshia; Pretorius, Karyn; Ismail, Nasreen; Buus, Søren; Stryhn, Anette; Carrington, Mary; Walker, Bruce D; Ndung'u, Thumbi; Ndhlovu, Zaza M

2017-04-01

Immune control of viral infections is heavily dependent on helper CD4 + T cell function. However, the understanding of the contribution of HIV-specific CD4 + T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4 + T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4 + T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4 + T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4 + T cells in HIV controllers than progressors ( P = 0.0001), and these expanded Gag-specific CD4 + T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control ( r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4 + T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4 + T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4 + T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV

HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection

PubMed Central

Laher, Faatima; Ranasinghe, Srinika; Porichis, Filippos; Mewalal, Nikoshia; Pretorius, Karyn; Ismail, Nasreen; Buus, Søren; Stryhn, Anette; Carrington, Mary; Walker, Bruce D.; Ndung'u, Thumbi

2017-01-01

ABSTRACT Immune control of viral infections is heavily dependent on helper CD4+ T cell function. However, the understanding of the contribution of HIV-specific CD4+ T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4+ T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4+ T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4+ T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4+ T cells in HIV controllers than progressors (P = 0.0001), and these expanded Gag-specific CD4+ T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r = −0.5, P = 0.02). These data identify an association between HIV-specific CD4+ T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4+ T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4+ T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4+ T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV

The first case of Hb G-Honolulu [α30(B11)Glu→Gln (GAG>CAG); HBA2:c.91G>A] observed in association with Hb S [β6(A3)Glu→Val, GAG>GTG] in a healthy Italian child.

PubMed

Paleari, Renata; Caruso, Donatella; Giavarini, Flavio; Colzani, Carlo; Brunati, Pietro; Mosca, Andrea

2012-01-01

We report the first observation of Hb G-Honolulu [α30(B11)Glu→Gln (GAG>CAG); HBA2:c.91G>A] in a Caucasian family and the first case of this variant to be found in association with Hb S [β6(A3)Glu→Val, GAG>GTG]. The proband was a healthy 4-year-old Italian boy. His chromatographic hemoglobin (Hb) pattern showed an abnormal peak having the typical retention time of Hb S (25.6% ), a second abnormal peak eluted soon after (13.6%) and a third minor peak eluted at the end of the run (6.5%). Identification of Hb variants were performed by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Two abnormal peptides at m/z 765.1 and 922 were found, corresponding to the αT-4 and βT-1 peptides characteristic for Hb G-Honolulu and Hb S, respectively. The third minor abnormal peak presumably corresponded to the hybrid molecule (α(G-Honolulu)/β(S)). The concomitant presence of Hb G-Honolulu and Hb S does not seem to produce any relevant clinical manifestation.

Characterization of the interaction of interleukin-8 with hyaluronan, chondroitin sulfate, dermatan sulfate and their sulfated derivatives by spectroscopy and molecular modeling.

PubMed

Pichert, Annelie; Samsonov, Sergey A; Theisgen, Stephan; Thomas, Lars; Baumann, Lars; Schiller, Jürgen; Beck-Sickinger, Annette G; Huster, Daniel; Pisabarro, M Teresa

2012-01-01

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG-protein systems.

An anticholinergic reverses motor control and corticostriatal LTD deficits in Dyt1 ΔGAG knock-in mice

PubMed Central

Dang, Mai T.; Yokoi, Fumiaki; Cheetham, Chad C.; Lu, Jun; Vo, Viet; Lovinger, David M.; Li, Yuqing

2011-01-01

DYT1 early-onset generalized torsion dystonia is an inherited movement disorder associated with mutations in DYT1 that codes for torsinA protein. The most common mutation seen in this gene is a trinucleotide deletion of GAG. We previously reported a motor control deficit on a beam-walking task in our Dyt1 ΔGAG knock-in heterozygous mice. In this report we show the reversal of this motor deficit with the anticholinergic trihexyphenidyl (THP), a drug commonly used to treat movement problems in dystonia patients. THP also restored the reduced corticostriatal long-term depression (LTD) observed in these mice. Corticostriatal LTD has long been known to be dependent on D2 receptor activation. In this mouse model, striatal D2 receptors were expressed at lower quantities in comparison to wild-type mice. Furthermore, the mice were also partially resistant to FPL64176, an agonist of L-type calcium channels that have been previously reported to cause severe dystonic-like symptoms in wild-type mice. Our findings collectively suggest that altered communication between cholinergic interneurons and medium spiny neurons is responsible for the LTD deficit and that this synaptic plasticity modification may be involved in the striatal motor control abnormalities in our mouse model of DYT1 dystonia. PMID:21995941

Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

PubMed

Steppert, Petra; Burgstaller, Daniel; Klausberger, Miriam; Kramberger, Petra; Tover, Andres; Berger, Eva; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Jungbauer, Alois

2017-02-01

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 10 12 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 10 11 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 10 9 particles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Identification of Proteoglycans and Glycosaminoglycans in Archaeological Human Bones and Teeth

PubMed Central

Coulson-Thomas, Yvette M.; Coulson-Thomas, Vivien J.; Norton, Andrew L.; Gesteira, Tarsis F.; Cavalheiro, Renan P.; Meneghetti, Maria Cecília Z.; Martins, João R.; Dixon, Ronald A.; Nader, Helena B.

2015-01-01

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology. PMID:26107959

In Vitro Evolution of the Human Immunodeficiency Virus Type 1 Gag-Protease Region and Maintenance of Reverse Transcriptase Resistance following Prolonged Drug Exposure†

PubMed Central

La Seta Catamancio, Simona; De Pasquale, Maria Pia; Citterio, Paola; Kurtagic, Semir; Galli, Massimo; Rusconi, Stefano

2001-01-01

We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drug-resistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions. PMID:11230439

Genetic diversity of HIV-1 non-B strains in Sicily: evidence of intersubtype recombinants by sequence analysis of gag, pol, and env genes.

PubMed

Tramuto, Fabio; Bonura, Filippa; Perna, Anna Maria; Mancuso, Salvatrice; Firenze, Alberto; Romano, Nino; Vitale, Francesco

2007-09-01

The molecular epidemiology of HIV-1 strains in Sicily (Italy) was phylogenetically investigated by the analysis of HIV-1 gag, pol, and env gene sequences from 11 HIV-1 non-B strains from 408 HIV-1-seropositive patients observed from September 2001 to August 2006. Sequences suggestive of recombination were further investigated by bootscanning analysis of various fragments. Overall, we identified several second-generation recombinant (SGRs) strains, which contained genetic material of CRF02_AG in at least one gene. Notably, three individuals were found to be infected with subsubtype A3, and one of them showed genetic recombination with subsubtype A4. The current study emphasizes the genetic analysis of gag, pol, and env genes as a powerful tool to trace the spread of complex HIV-1 recombinant forms, and highlight the genetic diversity of HIV-1 non-B strains in Italy.

Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag

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

Ramirez, Jean Marie; Houzet, Laurent; Koller, Richard

2004-12-20

Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associatedmore » with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.« less

Defined surface immobilization of glycosaminoglycan molecules for probing and modulation of cell-material interactions.

PubMed

Wang, Kai; Luo, Ying

2013-07-08

As one important category of biological molecules on the cell surface and in the extracellular matrix (ECM), glycosaminoglycans (GAGs) have been widely studied for biomedical applications. With the understanding that the biological functions of GAGs are driven by the complex dynamics of physiological and pathological processes, methodologies are desired to allow the elucidation of cell-GAG interactions with molecular level precision. In this study, a microtiter plate-based system was devised through a new surface modification strategy involving polydopamine (PDA) and GAG molecules functionalized with hydrazide chemical groups. A small library of GAGs including hyaluronic acid (with different molecular weights), heparin, and chondroitin sulfate was successfully immobilized via defined binding sites onto the microtiter plate surface under facile aqueous conditions. The methodology then allowed parallel studies of the GAG-modified surfaces in a high-throughput format. The results show that immobilized GAGs possess distinct properties to mediate protein adsorption, cell adhesion, and inflammatory responses, with each property showing dependence on the type and molecular weight of specific GAG molecules. The PDA-assisted immobilization of hydrazide-functionalized GAGs allows biomimetic attachment of GAG molecules and retains their bioactivity, providing a new methodology to systematically probe fundamental cell-GAG interactions to modulate the bioactivity and biocompatibility of biomaterials.

Polyfunctional analysis of Gag and Nef specific CD8+ T-cell responses in HIV-1 infected Indian individuals.

PubMed

Mendiratta, Sanjay; Vajpayee, Madhu; Mojumdar, Kamalika; Chauhan, Neeraj K; Sreenivas, Vishnubhatla

2011-02-01

Polyfunctional CD8+ T-cells have been described as most competent in controlling viral replication. We studied the impact of antigen persistence on the polyfunctional immune responses of CD8+ T-lymphocytes to HIV Gag and Nef peptides and polyclonal stimuli in 40 ART naïve HIV infected individuals and analyzed the alterations in T-cell functionality in early and late stages of infection. Significantly elevated level of global response and polyfunctional profile of CD8+ T-cells were observed to polyclonal stimulation, than HIV specific antigens in chronically infected individuals. However no key differences were observed in CD8+ T-cell functional profile in any of the 15 unique subsets for Gag and Nef specific antigens. The subjects in early stage of infection (defined as a gap of 6 months or less between seroconversion and enrolment and with no apparent clinical symptoms) had a higher degree of response functionality (4+ or 3+ different functions simultaneously) than in the late stage infection (defined as time duration since seroconversion greater than 6 months). The data suggest that persistence of antigen during chronic infection leads to functional impairment of HIV specific responses. Copyright © 2010 Elsevier Ltd. All rights reserved.

An anticholinergic reverses motor control and corticostriatal LTD deficits in Dyt1 ΔGAG knock-in mice.

PubMed

Dang, Mai T; Yokoi, Fumiaki; Cheetham, Chad C; Lu, Jun; Vo, Viet; Lovinger, David M; Li, Yuqing

2012-01-15

DYT1 early-onset generalized torsion dystonia is an inherited movement disorder associated with mutations in DYT1 that codes for torsinA protein. The most common mutation seen in this gene is a trinucleotide deletion of GAG. We previously reported a motor control deficit on a beam-walking task in our Dyt1 ΔGAG knock-in heterozygous mice. In this report we show the reversal of this motor deficit with the anticholinergic trihexyphenidyl (THP), a drug commonly used to treat movement problems in dystonia patients. THP also restored the reduced corticostriatal long-term depression (LTD) observed in these mice. Corticostriatal LTD has long been known to be dependent on D2 receptor activation. In this mouse model, striatal D2 receptors were expressed at lower quantities in comparison to wild-type mice. Furthermore, the mice were also partially resistant to FPL64176, an agonist of L-type calcium channels that have been previously reported to cause severe dystonic-like symptoms in wild-type mice. Our findings collectively suggest that altered communication between cholinergic interneurons and medium spiny neurons is responsible for the LTD deficit and that this synaptic plasticity modification may be involved in the striatal motor control abnormalities in our mouse model of DYT1 dystonia. Copyright © 2011 Elsevier B.V. All rights reserved.

Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β

PubMed Central

2013-01-01

Introduction Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen. Methods Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1β (10 ng/ml), carprofen (100 μg/ml), and carprofen (100 μg/ml) + IL-1β (10 ng/ml). Explants were incubated (37°C, 5% CO2) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs). Results Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1β stimulation increased the release of all three MMPs. IL-1β also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by β-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1β-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67%�

Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β.

PubMed

Williams, Adam; Smith, Julia R; Allaway, David; Harris, Pat; Liddell, Susan; Mobasheri, Ali

2013-01-01

Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen. Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1β (10 ng/ml), carprofen (100 μg/ml), and carprofen (100 μg/ml) + IL-1β (10 ng/ml). Explants were incubated (37°C, 5% CO2) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs). Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1β stimulation increased the release of all three MMPs. IL-1β also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by β-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1β-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1�

Relaxin's induction of metalloproteinases is associated with the loss of collagen and glycosaminoglycans in synovial joint fibrocartilaginous explants

PubMed Central

Naqvi, Tabassum; Duong, Trang T; Hashem, Gihan; Shiga, Momotoshi; Zhang, Qin; Kapila, Sunil

2005-01-01

Diseases of specific fibrocartilaginous joints are especially common in women of reproductive age, suggesting that female hormones contribute to their etiopathogenesis. Previously, we showed that relaxin dose-dependently induces matrix metalloproteinase (MMP) expression in isolated joint fibrocartilaginous cells. Here we determined the effects of relaxin with or without β-estradiol on the modulation of MMPs in joint fibrocartilaginous explants, and assessed the contribution of these proteinases to the loss of collagen and glycosaminoglycan (GAG) in this tissue. Fibrocartilaginous discs from temporomandibular joints of female rabbits were cultured in medium alone or in medium containing relaxin (0.1 ng/ml) or β-estradiol (20 ng/ml) or relaxin plus β-estradiol. Additional experiments were done in the presence of the MMP inhibitor GM6001 or its control analog. After 48 hours of culture, the medium was assayed for MMPs and the discs were analyzed for collagen and GAG concentrations. Relaxin and β-estradiol plus relaxin induced the MMPs collagenase-1 and stromelysin-1 in fibrocartilaginous explants – a finding similar to that which we observed in pubic symphysis fibrocartilage, but not in articular cartilage explants. The induction of these proteinases by relaxin or β-estradiol plus relaxin was accompanied by a loss of GAGs and collagen in joint fibrocartilage. None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation. Indeed, fibrocartilaginous explants cultured in the presence of GM6001 showed an inhibition of relaxin-induced and β-estradiol plus relaxin-induced collagenase and stromelysin activities to control baseline levels that were accompanied by the maintenance of collagen or GAG content at control levels. These findings show for the first time that relaxin has degradative effects on non-reproductive synovial joint fibrocartilaginous tissue and

Vulnerability of the Superficial Zone of Immature Articular Cartilage to Compressive Injury

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

Rolauffs, R.; Muehleman, C; Li, J

The zonal composition and functioning of adult articular cartilage causes depth-dependent responses to compressive injury. In immature cartilage, shear and compressive moduli as well as collagen and sulfated glycosaminoglycan (sGAG) content also vary with depth. However, there is little understanding of the depth-dependent damage caused by injury. Since injury to immature knee joints most often causes articular cartilage lesions, this study was undertaken to characterize the zonal dependence of biomechanical, biochemical, and matrix-associated changes caused by compressive injury. Disks from the superficial and deeper zones of bovine calves were biomechanically characterized. Injury to the disks was achieved by applying amore » final strain of 50% compression at 100%/second, followed by biomechanical recharacterization. Tissue compaction upon injury as well as sGAG density, sGAG loss, and biosynthesis were measured. Collagen fiber orientation and matrix damage were assessed using histology, diffraction-enhanced x-ray imaging, and texture analysis. Injured superficial zone disks showed surface disruption, tissue compaction by 20.3 {+-} 4.3% (mean {+-} SEM), and immediate biomechanical impairment that was revealed by a mean {+-} SEM decrease in dynamic stiffness to 7.1 {+-} 3.3% of the value before injury and equilibrium moduli that were below the level of detection. Tissue areas that appeared intact on histology showed clear textural alterations. Injured deeper zone disks showed collagen crimping but remained undamaged and biomechanically intact. Superficial zone disks did not lose sGAG immediately after injury, but lost 17.8 {+-} 1.4% of sGAG after 48 hours; deeper zone disks lost only 2.8 {+-} 0.3% of sGAG content. Biomechanical impairment was associated primarily with structural damage. The soft superficial zone of immature cartilage is vulnerable to compressive injury, causing superficial matrix disruption, extensive compaction, and textural alteration, which

Development and Characterization of Acellular Extracellular Matrix Scaffolds from Porcine Menisci for Use in Cartilage Tissue Engineering

PubMed Central

Chen, Ying-Chen; Chen, Ray-Neng; Jhan, Hua-Jing; Liu, Der-Zen; Ho, Hsiu-O; Mao, Yong; Kohn, Joachim

2015-01-01

Given the growing number of arthritis patients and the limitations of current treatments, there is great urgency to explore cartilage substitutes by tissue engineering. In this study, we developed a novel decellularization method for menisci to prepare acellular extracellular matrix (ECM) scaffolds with minimal adverse effects on the ECM. Among all the acid treatments, formic acid treatment removed most of the cellular contents and preserved the highest ECM contents in the decellularized porcine menisci. Compared with fresh porcine menisci, the content of DNA decreased to 4.10%±0.03%, and there was no significant damage to glycosaminoglycan (GAG) or collagen. Histological staining also confirmed the presence of ECM and the absence of cellularity. In addition, a highly hydrophilic scaffold with three-dimensional interconnected porous structure was fabricated from decellularized menisci tissue. Human chondrocytes showed enhanced cell proliferation and synthesis of chondrocyte ECM including type II collagen and GAG when cultured in this acellular scaffold. Moreover, the scaffold effectively supported chondrogenesis of human bone marrow-derived mesenchymal stem cells. Finally, in vivo implantation was conducted in rats to assess the biocompatibility of the scaffolds. No significant inflammatory response was observed. The acellular ECM scaffold provided a native environment for cells with diverse physiological functions to promote cell proliferation and new tissue formation. This study reported a novel way to prepare decellularized meniscus tissue and demonstrated the potential as scaffolds to support cartilage repair. PMID:25919905

Inhibition of chondroitin sulfate glycosaminoglycans incorporation affected odontoblast differentiation in cultured embryonic mouse molars.

PubMed

Liu, Lipei; Chen, Weiting; Li, Lefeng; Xu, Fangfang; Jiang, Beizhan

2017-12-01

Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without β-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by β-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that β-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.

Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function.

PubMed

Kharytonchyk, Siarhei; Brown, Joshua D; Stilger, Krista; Yasin, Saif; Iyer, Aishwarya S; Collins, John; Summers, Michael F; Telesnitsky, Alice

2018-07-06

The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5' leader Ψ elements plus poorly defined additional features. We previously defined minimal 5' leader sequences competitive with intact Ψ for HIV-1 packaging, and here examined the potential roles of additional downstream elements. The findings confirmed that together, HIV-1 5' leader Ψ sequences plus a nuclear export element are sufficient to specify packaging. However, RNAs trafficked using a heterologous export element did not compete well with RNAs using HIV-1's RRE. Furthermore, some RNA additions to well-packaged minimal vectors rendered them packaging-defective. These defects were rescued by extending gag sequences in their native context. To understand these packaging defects' causes, in vitro dimerization properties of RNAs containing minimal packaging elements were compared to RNAs with sequence extensions that were or were not compatible with packaging. In vitro dimerization was found to correlate with packaging phenotypes, suggesting that HIV-1 evolved to prevent 5' leader residues' base pairing with downstream residues and misfolding of the packaging signal. Our findings explain why gag sequences have been implicated in packaging and show that RRE's packaging contributions appear more specific than nuclear export alone. Paired with recent work showing that sequences upstream of Ψ can dictate RNA folds, the current work explains how genetic context of minimal packaging elements contributes to HIV-1 RNA fate determination. Copyright © 2018 Elsevier Ltd. All rights reserved.

Vitamin D and calcium supplementation for three years in postmenopausal osteoporosis significantly alters bone mineral and organic matrix quality.

PubMed

Paschalis, E P; Gamsjaeger, S; Hassler, N; Fahrleitner-Pammer, A; Dobnig, H; Stepan, J J; Pavo, I; Eriksen, E F; Klaushofer, K

2017-02-01

Prospective, controlled clinical trials in postmenopausal osteoporosis typically compare effects of an active drug with placebo in addition to vitamin D and calcium supplementation in both treatment arms. While clinical benefits are documented, the effect of this supplementation in the placebo arm and in clinical practice on bone material composition properties is unknown. The purpose of the present study was to evaluate these bone quality indices (specifically mineral/matrix, nanoporosity, glycosaminoglycan content, mineral maturity/crystallinity, and pyridinoline content) in patients that either received long-term vitamin D (400-1200IU) and calcium (1.0-1.5g) supplementation, or did not. We have analyzed by Raman microspectroscopy the bone forming trabecular surfaces of iliac crest in pre-treatment samples of a teriparatide study and the endpoint biopsies of the control arm obtained from the HORIZON trial. In general, the mineral/matrix ratio and the glycosaminoglycan (GAG) content was higher while nanoporosity, (a surrogate for tissue water content), the mineral maturity/crystallinity (MMC) and the pyridinoline (Pyd) content was lower in patients without long-term supplementation. Moreover, all indices were significantly dependent on tissue age. In conclusion, vitamin D and calcium supplementation is associated with altered mineral and organic matrix properties. Copyright © 2016 Elsevier Inc. All rights reserved.

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

PubMed Central

Ryden, T A; de Mars, M; Beemon, K

1993-01-01

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive. Images PMID:8386280

Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes.

PubMed

Du, Yushen; Zhang, Tian-Hao; Dai, Lei; Zheng, Xiaojuan; Gorin, Aleksandr M; Oishi, John; Wu, Ting-Ting; Yoshizawa, Janice M; Li, Xinmin; Yang, Otto O; Martinez-Maza, Otoniel; Detels, Roger; Sun, Ren

2017-11-28

Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8 + cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

PubMed Central

Ghiglione, Yanina; Falivene, Juliana; Socias, María Eugenia; Laufer, Natalia; Coloccini, Romina Soledad; Rodriguez, Ana María; Ruiz, María Julia; Pando, María Ángeles; Giavedoni, Luis David; Cahn, Pedro; Sued, Omar; Salomon, Horacio; Gherardi, María Magdalena

2013-01-01

The important role of the CD8+ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8+ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8+ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4+ T-cell set points were observed in PHI subjects with higher HIV-specific CD8+ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8+ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection. PMID:23616666

Non-Glycanated Biglycan and LTBP4: Leveraging the extracellular matrix for Duchenne Muscular Dystrophy therapeutics.

PubMed

Fallon, Justin R; McNally, Elizabeth M

2018-08-01

The extracellular matrix (ECM) plays key roles in normal and diseased skeletal and cardiac muscle. In healthy muscle the ECM is essential for transmitting contractile force, maintaining myofiber integrity and orchestrating cellular signaling. Duchenne Muscular Dystrophy (DMD) is caused by loss of dystrophin, a cytosolic protein that anchors a transmembrane complex and serves as a vital link between the actin cytoskeleton and the basal lamina. Loss of dystrophin leads to membrane fragility and impaired signaling, resulting in myofiber death and cycles of inflammation and regeneration. Fibrosis is also a cardinal feature of DMD. In this review, we will focus on two cases where understanding the normal function and regulation of ECM in muscle has led to the discovery of candidate therapeutics for DMD. Biglycan is a small leucine rich repeat ECM protein present as two glycoforms in muscle that have dramatically different functions. One widely expressed form is biglycan proteoglycan (PG) that bears two chondroitin sulfate GAG chains (typically chondroitin sulfate) and two N-linked carbohydrates. The second glycoform, referred to as 'NG' (non-glycanated) biglycan, lacks the GAG side chains. NG, but not PG biglycan recruits utrophin, an autosomal paralog of dystrophin, and an NOS-containing signaling complex to the muscle cell membrane. Recombinant NG biglycan can be systemically delivered to dystrophic mice where it upregulates utrophin at the membrane and improves muscle health and function. An optimized version of NG biglycan, 'TVN-102', is under development as a candidate therapeutic for DMD. A second matrix-embedded protein being evaluated for therapeutic potential is latent TGFβ binding protein 4 (LTBP4). Identified in a genomic screen for modifiers of muscular dystrophy, LTBP4 binds both TGFβ and myostatin. Genetic studies identified the hinge region of LTBP4 as linked to TGFβ release and contributing to the "hyper-TGFβ" signaling state that promotes fibrosis

Clinicopathologic and radiologic features of extraskeletal myxoid chondrosarcoma: a retrospective study of 40 Chinese cases with literature review.

PubMed

Shao, Rui; Lao, I Weng; Wang, Lei; Yu, Lin; Wang, Jian; Fan, Qinhe

2016-08-01

The aim of this study is to describe the clinicopathologic and radiologic features of 40 cases of extraskeletal myxoid chondrosarcoma (EMC) from China. There were 25 males and 15 females (sex ratio, 1.7:1). Apart from an adolescent, all patients were adults with a median age of 49years. Twenty-four tumors (60%) occurred in the lower limb and limb girdles, especially the thigh, followed by the upper limb and limb girdles (20%) and trunk (10%). Other less commonly involved locations included the head and neck, sacrococcygeal region, and perineum. Tumors ranged in size from 1.5 to 19cm (mean, 7cm). By radiology, they appeared as hypoattenuated or isoattenuated masses on computed tomography with hyperintense signal on T2-weighted magnetic resonance imaging. Intralesional hypointense septa were present in most cases. Of the 40 tumors, 30 belonged to the classic subtype, whereas 9 cases were cellular, and 1 case had a rhabdoid phenotype. Tumor cells showed variable expression of synaptophysin (36%), S-100 protein (29%), epithelial membrane antigen (11%), and neuron-specific enolase (7%). Ki-67 index was remarkably higher in the cellular variant (mean, 30%). EWSR1-related rearrangement was detected in 12 of 14 cases tested by fluorescence in situ hybridization using break-apart probes. The overall 5- and 7-year survival was 71% and 60%, respectively. Awareness of the imaging features may help pathologists in the diagnosis of EMC. Fluorescence in situ hybridization also serves as a useful diagnostic tool for EMC, especially in the distinction from its mimics. Copyright © 2016 Elsevier Inc. All rights reserved.

[Functional analysis of Grp and Iris, the gag and env domesticated errantivirus genes, in the Drosophila melanogaster genome].

PubMed

Makhnovskii, P A; Kuzmin, I V; Nefedova, L N; Kima, A I

2016-01-01

Drosophila melanogaster is the only invertebrate that contains endogenous retroviruses, which are called errantiviruses. Two domesticated genes, Grp and Iris, which originate from errantivirus gag and env, respectively, have been found in the D. melanogaster genome. The functions performed by the genes in Drosophila are still unclear. To identify the functions of domesticated gag and env in the D. melanogaster genome, expression of Iris and Grp was studied in strains differing by the presence or absence of the functional gypsy errantivirus. In addition, the expression levels were measured after injection of gram-positive and gram-negative bacteria, which activate different immune response pathways, and exposure to various abiotic stress factors. The presence of functional D. melanogaster retrovirus gypsy was found to increase the Grp expression level in somatic tissues of the carcass, while exerting no effect on the Iris expression level. Activation of the immune response in D. melanogaster by bacteria Bacillus cereus increased the Grp expression level and did not affect Iris expression. As for the effects of abiotic stress factors (oxidative stress, starvation, and heat and cold stress), the Grp expression level increased in response to starvation in D. melanogaster females, and the Iris expression level was downregulated in heat shock and oxidative stress. Based on the findings, Grp was assumed to play a direct role in the immune response in D. melanogaster; Iris is not involved in immune responses, but and apparently performs a cell function that is inhibited in stress.

Generation of the novel monoclonal antibody against TLS/EWS-CHOP chimeric oncoproteins that is applicable to one of the most sensitive assays for myxoid and round cell liposarcomas.

PubMed

Oikawa, Kosuke; Ishida, Tsuyoshi; Imamura, Tetsuo; Yoshida, Keiichi; Takanashi, Masakatsu; Hattori, Hiroyuki; Ishikawa, Akio; Fujita, Koji; Yamamoto, Kengo; Matsubayashi, Jun; Kuroda, Masahiko; Mukai, Kiyoshi

2006-03-01

The fusion oncoproteins, TLS-CHOP and EWS-CHOP, are characteristic markers for myxoid and round cell liposarcomas (MLS/RCLS). Especially, the peptide sequence of 26 amino acids corresponding to the normally untranslated CHOP exon 2 and parts of exon 3 (5'-UTR) is a unique structure for these chimeric proteins. In this report, we have generated monoclonal antibodies against the unique peptide sequence of TLS/EWS-CHOP oncoproteins. These antibodies reacted with TLS-CHOP fusion protein, but not reacted with normal TLS and CHOP proteins by Western blot analysis. In addition, one of the antibodies also recognized the chimeric oncoprotein in archival paraffin-embedded tissue samples of MLS/RCLS. The oncoprotein was detectable by the antibody even in the paraffin-embedded tissue samples whose mRNAs were too degraded to be detected by a nested reverse transcription-polymerase chain reaction-based assay. Thus, the molecular assay using the novel antibody is expected to be one of the most sensitive diagnostic assays for MLS/RCLS.

Micro-Scale Distribution of CA4+ in Ex Vivo Human Articular Cartilage Detected with Contrast-Enhanced Micro-Computed Tomography Imaging

NASA Astrophysics Data System (ADS)

Karhula, Sakari S.; Finnilä, Mikko A.; Freedman, Jonathan D.; Kauppinen, Sami; Valkealahti, Maarit; Lehenkari, Petri; Pritzker, Kenneth P. H.; Nieminen, Heikki J.; Snyder, Brian D.; Grinstaff, Mark W.; Saarakkala, Simo

2017-08-01

Contrast-enhanced micro-computed tomography (CEµCT) with cationic and anionic contrast agents reveals glycosaminoglycan (GAG) content and distribution in articular cartilage (AC). The advantage of using cationic stains (e.g. CA4+) compared to anionic stains (e.g. Hexabrix®), is that it distributes proportionally with GAGs, while anionic stain distribution in AC is inversely proportional to the GAG content. To date, studies using cationic stains have been conducted with sufficient resolution to study its distributions on the macro-scale, but with insufficient resolution to study its distributions on the micro-scale. Therefore, it is not known whether the cationic contrast agents accumulate in extra/pericellular matrix and if they interact with chondrocytes. The insufficient resolution has also prevented to answer the question whether CA4+ accumulation in chondrons could lead to an erroneous quantification of GAG distribution with low-resolution µCT setups. In this study, we use high-resolution µCT to investigate whether CA4+ accumulates in chondrocytes, and further, to determine whether it affects the low-resolution ex vivo µCT studies of CA4+ stained human AC with varying degree of osteoarthritis. Human osteochondral samples were immersed in three different concentrations of CA4+ (3 mgI/ml, 6mgI/ml, and 24 mgI/ml) and imaged with high-resolution µCT at several timepoints. Different uptake diffusion profiles of CA4+ were observed between the segmented chondrons and the rest of the tissue. While the X-ray -detected CA4+ concentration in chondrons was greater than in the rest of the AC, its contribution to the uptake into the whole tissue was negligible and in line with macro-scale GAG content detected from histology. The efficient uptake of CA4+ into chondrons and surrounding territorial matrix can be explained by the micro-scale distribution of GAG content. CA4+ uptake in chondrons occurred regardless of the progression stage of osteoarthritis in the samples

[Serum glycosaminoglycans in Graves' disease patients].

PubMed

Winsz-Szczotka, Katarzyna B; Olczyk, Krystyna Z; Koźma, Ewa M; Komosińska-Vassev, Katarzyna B; Wisowski, Grzegorz R; Marcisz, Czesław

2006-01-01

The aim of the study was to determine the blood serum sulfated glycosaminoglycans (GAGs) and hyaluronic acid (HA) concentration of Graves' disease patients before treatment and after attainment of the euthyroid state. The study was carried out on the blood serum obtained from 17 patients with newly recognised Graves' disease and from the same patients after attainment of the euthyroid state. Graves' patients had not any clinical symptoms neither of ophthalmopathy nor pretibial myxedema. GAGs were isolated from the blood serum by the multistage extraction and purification using papaine hydrolysis, alkali elimination, as well as cetylpyridium chloride binding. Total amount of GAGs was quantified by the hexuronic acids assay. HA content in obtained GAGs sample was evaluated by the ELISA method. Increased serum concentration of sulfated GAGs in non-treated Graves' disease patients was found. Similarly, serum HA level in untreated patients was significantly elevated. The attainment of euthyroid state was accompanied by the decreased serum sulfated GAGs level and by normalization of serum HA concentration. In conclusion, the results obtained demonstrate that the alterations of GAGs metabolism connected with Graves' disease can lead to systemic changes of the extracellular matrix properties.

Live attenuated measles vaccine expressing HIV-1 Gag virus like particles covered with gp160DELTAV1V2 is strongly immunogenic

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

Guerbois, Mathilde; Moris, Arnaud; Combredet, Chantal

Although a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy inducing both T cells and neutralizing antibodies to native assembled HIV-1 particles expressed by a replicating virus might mimic the advantageous characteristics of live attenuated vaccine. To this aim, we generated a live attenuated recombinant measles vaccine expressing HIV-1 Gag virus-like particles (VLPs) covered with gp160DELTAV1V2 Env protein. The measles-HIV virus replicated efficiently in cell culture and induced the intense budding of HIV particles covered with Env. In mice sensitive to MV infection, this recombinant vaccine stimulated high levels of cellular and humoral immunity tomore » both MV and HIV with neutralizing activity. The measles-HIV virus infected human professional antigen-presenting cells, such as dendritic cells and B cells, and induced efficient presentation of HIV-1 epitopes and subsequent activation of human HIV-1 Gag-specific T cell clones. This candidate vaccine will be next tested in non-human primates. As a pediatric vaccine, it might protect children and adolescents simultaneously from measles and HIV.« less

Dental treatment in patients with severe gag reflex using propofol-remifentanil intravenous sedation

PubMed Central

Shin, Sooil

2017-01-01

Patients with severe gag reflex (SGR) have difficulty getting the treatment they require in local clinics, and many tend to postpone the start of their treatment. To address this problem, dentists have used behavioral techniques and/or pharmacological techniques for treatment. Among the pharmacological methods available, propofol IV sedation is preferred over general anesthesia because it is a simpler procedure. Propofol in combination with remifentanil is characterized by stable sedative effects and quick recovery, leading to a deep sedation. Remifentanil acts to reduce the pain caused by lipid-soluble propofol on injection. The synergistic effects of propofol-remifentanil include reduction in the total amount of drug required to achieve a desired sedation level and anti-emetic effects. In this case report, we outline how the use of propofol-remifentanil IV sedation enabled us to successfully complete a wide range of dental treatments in a patient with SGR. PMID:28879331

Dental treatment in patients with severe gag reflex using propofol-remifentanil intravenous sedation.

PubMed

Shin, Sooil; Kim, Seungoh

2017-03-01

Patients with severe gag reflex (SGR) have difficulty getting the treatment they require in local clinics, and many tend to postpone the start of their treatment. To address this problem, dentists have used behavioral techniques and/or pharmacological techniques for treatment. Among the pharmacological methods available, propofol IV sedation is preferred over general anesthesia because it is a simpler procedure. Propofol in combination with remifentanil is characterized by stable sedative effects and quick recovery, leading to a deep sedation. Remifentanil acts to reduce the pain caused by lipid-soluble propofol on injection. The synergistic effects of propofol-remifentanil include reduction in the total amount of drug required to achieve a desired sedation level and anti-emetic effects. In this case report, we outline how the use of propofol-remifentanil IV sedation enabled us to successfully complete a wide range of dental treatments in a patient with SGR.

Homologous structure-function relationships between native fibrocartilage and tissue engineered from MSC-seeded nanofibrous scaffolds.

PubMed

Nerurkar, Nandan L; Han, Woojin; Mauck, Robert L; Elliott, Dawn M

2011-01-01

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of tissues to replace diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials. Copyright © 2010 Elsevier Ltd. All rights reserved.

HOMOLOGOUS STRUCTURE-FUNCTION RELATIONSHIPS BETWEEN NATIVE FIBROCARTILAGE AND TISSUE ENGINEERED FROM MSC-SEEDED NANOFIBROUS SCAFFOLDS

PubMed Central

Nerurkar, Nandan L.; Han, Woojin; Mauck, Robert L.; Elliott, Dawn M.

2010-01-01

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of replacement tissues for diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials. PMID:20880577

Complex assembly, crystallization and preliminary X-ray crystallographic studies of rhesus macaque MHC Mamu-A*01 complexed with an immunodominant SIV-Gag nonapeptide

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

Chu, Fuliang; Graduate School, Chinese Academy of Sciences, Beijing; Lou, Zhiyong

2005-06-01

Crystallization of the first rhesus macaque MHC class I complex. Simian immunodeficiency virus (SIV) infection in rhesus macaques has been used as the best model for the study of human immunodeficiency virus (HIV) infection in humans, especially in the cytotoxic T-lymphocyte (CTL) response. However, the structure of rhesus macaque (or any other monkey model) major histocompatibility complex class I (MHC I) presenting a specific peptide (the ligand for CTL) has not yet been elucidated. Here, using in vitro refolding, the preparation of the complex of the rhesus macaque MHC I allele (Mamu-A*01) with human β{sub 2}m and an immunodominant peptide,more » CTPYDINQM (Gag-CM9), derived from SIV Gag protein is reported. The complex (45 kDa) was crystallized; the crystal belongs to space group I422, with unit-cell parameters a = b = 183.8, c = 155.2 Å. The crystal contains two molecules in the asymmetric unit and diffracts X-rays to 2.8 Å resolution. The structure is being solved by molecular replacement and this is the first attempt to determined the crystal structure of a peptide–nonhuman primate MHC complex.« less

Consumers, gag rules, and health plans: strategies for a patient-focused market.

PubMed

Etheredge, L; Jones, S B

1997-05-01

The "gag rule" controversy has become a symbol of new tensions and changing relationships among patients, physicians, and health plans. This paper offers a consumer-focused analysis of these fundamental issues from the point of view of a patient with a chronic illness. It starts with a case study of a specific individual and then considers the systemic incentives and other factors that lead to conflicts among patients, physicians, and health plans. This consumer focus invites the reader to consider managed care with the following question in mind: "What would you want for yourself it you were the patient?" The paper suggests that many private-sector initiatives, as well as government actions, could contribute to a better health care market. Among the reform strategies discussed are (a) professional responsibility and private-sector standards, (b) consumer assistance, and (c) government regulation. All of us, including persons with chronic illness, need a consumer-focused health system. So, too, do physicians and health plans that want to provide excellent care for all of their patients.

Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein*

PubMed Central

Vlach, Jiri; Samal, Alexandra B.; Saad, Jamil S.

2014-01-01

Subcellular distribution of calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM co-localizes and interacts with the HIV-1 Gag protein in the cytosol of infected cells. Although it has been shown that binding of Gag to CaM is mediated by the matrix (MA) domain, the structural details of this interaction are not known. We have recently shown that binding of CaM to MA induces a conformational change that triggers myristate exposure, and that the CaM-binding domain of MA is confined to a region spanning residues 8–43 (MA-(8–43)). Here, we present the NMR structure of CaM bound to MA-(8–43). Our data revealed that MA-(8–43), which contains a novel CaM-binding motif, binds to CaM in an antiparallel mode with the N-terminal helix (α1) anchored to the CaM C-terminal lobe, and the C-terminal helix (α2) of MA-(8–43) bound to the N-terminal lobe of CaM. The CaM protein preserves a semiextended conformation. Binding of MA-(8–43) to CaM is mediated by numerous hydrophobic interactions and stabilized by favorable electrostatic contacts. Our structural data are consistent with the findings that CaM induces unfolding of the MA protein to have access to helices α1 and α2. It is noteworthy that several MA residues involved in CaM binding have been previously implicated in membrane binding, envelope incorporation, and particle production. The present findings may ultimately help in identification of the functional role of CaM in HIV-1 replication. PMID:24500712

Analyzing the effects of mechanical and osmotic loading on glycosaminoglycan synthesis rate in cartilaginous tissues.

PubMed

Gao, Xin; Zhu, Qiaoqiao; Gu, Weiyong

2015-02-26

The glycosaminoglycan (GAG) plays an important role in cartilaginous tissues to support and transmit mechanical loads. Many extracellular biophysical stimuli could affect GAG synthesis by cells. It has been hypothesized that the change of cell volume is a primary mechanism for cells to perceive the stimuli. Experimental studies have shown that the maximum synthesis rate of GAG is achieved at an optimal cell volume, larger or smaller than this level the GAG synthesis rate decreases. Based on the hypothesis and experimental findings in the literature, we proposed a mathematical model to quantitatively describe the cell volume dependent GAG synthesis rate in the cartilaginous tissues. Using this model, we investigated the effects of osmotic loading and mechanical loading on GAG synthesis rate. It is found our proposed mathematical model is able to well describe the change of GAG synthesis rate in isolated cells or in cartilage with variations of the osmotic loading or mechanical loading. This model is important for evaluating the GAG synthesis activity within cartilaginous tissues as well as understanding the role of mechanical loading in tissue growth or degeneration. It is also important for designing a bioreactor system with proper extracellular environment or mechanical loading for growing tissue at the maximum synthesis rate of the extracellular matrix. Copyright © 2015 Elsevier Ltd. All rights reserved.

Neoadjuvant radiation in primary extremity liposarcoma: correlation of MRI features with histopathology.

PubMed

Wortman, Jeremy R; Tirumani, Sree Harsha; Tirumani, Harika; Shinagare, Atul B; Jagannathan, Jyothi P; Hornick, Jason L; Ramaiya, Nikhil H

2016-05-01

To evaluate MRI features of response of primary extremity liposarcoma (LPS) to neoadjuvant radiation therapy (RT) with histopathologic correlation. In this IRB-approved study including 125 patients with extremity LPS treated with neoadjuvant RT from 2000 to 2013, MRI of the primary tumour in 18 patients (5 pleomorphic LPS, 13 myxoid LPS) before and after RT were reviewed by two radiologists by consensus. Histopathology of the surgical specimens was reviewed by a pathologist with expertise in sarcomas. In the pleomorphic LPS cohort, 3/5 tumours increased in size; 3/5 decreased in enhancing component; and 3/5 increased in peritumoral oedema, intratumoral haemorrhage, and necrosis. In the myxoid LPS cohort, 12/13 tumours decreased in size, 8/13 decreased in enhancing component, and 5/13 increased in internal fat following RT. Histopathology showed ≥50% residual tumour in 1/5 pleomorphic LPS and 2/13 myxoid LPS. Hyalinization/necrosis of ≥75% was noted in 4/5 pleomorphic LPS and 11/13 myxoid LPS. Cytodifferentiation was noted in 1/5 pleomorphic and 9/13 myxoid LPS. While pleomorphic LPS showed an increase in size, peritumoral oedema, intratumoral haemorrhage, and necrosis on MRI following neoadjuvant RT, myxoid LPS showed a decrease in size and enhancement with an increase in internal fat. • Pleomorphic LPS commonly increase in size and necrosis on MRI following RT. • Myxoid LPS commonly decrease in size and enhancement on MRI following RT. • Myxoid LPS often increase in fatty component on MRI following RT.

Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner.

PubMed

Hahn, Friedrich; Schmalen, Adrian; Setz, Christian; Friedrich, Melanie; Schlößer, Stefan; Kölle, Julia; Spranger, Robert; Rauch, Pia; Fraedrich, Kirsten; Reif, Tatjana; Karius-Fischer, Julia; Balasubramanyam, Ashok; Henklein, Petra; Fossen, Torgils; Schubert, Ulrich

2017-01-01

There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.

HIV-1 Gag p17 presented as virus-like particles on the E2 scaffold from Geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of IFN{gamma} production by CD4+ T cells

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

Caivano, Antonella; Doria-Rose, Nicole A.; Dept. of Molecular and Cell Biology, University of Washington, Seattle, WA 98124-6108

2010-11-25

We have constructed stable virus-like particles displaying the HIV-1 Gag(p17) protein as an N-terminal fusion with an engineered protein domain from the Geobacillus stearothermophilus pyruvate dehydrogenase subunit E2. Mice immunized with the Gag(p17)-E2 60-mer scaffold particles mounted a strong and sustained antibody response. Antibodies directed to Gag(p17) were boosted significantly with additional immunizations, while anti-E2 responses reached a plateau. The isotype of the induced antibodies was biased towards IgG1, and the E2-primed CD4+ T cells did not secrete IFN{gamma}. Using transgenic mouse model systems, we demonstrated that CD8+ T cells primed with E2 particles were able to exert lytic activitymore » and produce IFN{gamma}. These results show that the E2 scaffold represents a powerful vaccine delivery system for whole antigenic proteins or polyepitope engineered proteins, evoking antibody production and antigen specific CTL activity even in the absence of IFN{gamma}-producing CD4+ T cells.« less

Chondroitin-4-sulfation negatively regulates axonal guidance and growth

PubMed Central

Wang, Hang; Katagiri, Yasuhiro; McCann, Thomas E.; Unsworth, Edward; Goldsmith, Paul; Yu, Zu-Xi; Tan, Fei; Santiago, Lizzie; Mills, Edward M.; Wang, Yu; Symes, Aviva J.; Geller, Herbert M.

2008-01-01

Summary Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition, and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate (CS) mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated CS GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings provide the evidence showing that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function. PMID:18768934

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

PubMed

Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

Noninvasive Assessment of Glycosaminoglycan Production in Injectable Tissue-Engineered Cartilage Constructs Using Magnetic Resonance Imaging

PubMed Central

Ramaswamy, Sharan; Uluer, Mehmet C.; Leen, Stephanie; Bajaj, Preeti; Fishbein, Kenneth W.

2008-01-01

Abstract The glycosaminoglycan (GAG) content of engineered cartilage is a determinant of biochemical and mechanical quality. The ability to measure the degree to which GAG content is maintained or increases in an implant is therefore of importance in cartilage repair procedures. The gadolinium exclusion magnetic resonance imaging (MRI) method for estimating matrix fixed charge density (FCD) is ideally suited to this. One promising approach to cartilage repair is use of seeded injectable hydrogels. Accordingly, we assess the reliability of measuring GAG content in such a system ex vivo using MRI. Samples of the photo-polymerizable hydrogel, poly(ethylene oxide) diacrylate, were seeded with bovine chondrocytes (∼2.4 million cells/sample). The FCD of the constructs was determined using MRI after 9, 16, 29, 36, 43, and 50 days of incubation. Values were correlated with the results of biochemical determination of GAG from the same samples. FCD and GAG were found to be statistically significantly correlated (R2 = 0.91, p <0.01). We conclude that MRI-derived FCD measurements of FCD in injectable hydrogels reflect tissue GAG content and that this methodology therefore has potential for in vivo monitoring of such constructs. PMID:18620483

Products resulting from cleavage of the interglobular domain of aggrecan in samples of synovial fluid collected from dogs with early- and late-stage osteoarthritis.

PubMed

Innes, John F; Little, Chris B; Hughes, Clare E; Caterson, Bruce

2005-10-01

To investigate interglobular domain (IGD) cleavage of aggrecan in dogs with naturally developing osteoarthritis (OA). Samples of synovial fluid (SF) obtained from 3 cubital (elbow) joints and 3 stifle joints of 4 clinically normal dogs, 24 elbow joints of 12 dogs with early-stage OA, 8 stifle joints of 5 dogs with early-stage OA, and 10 stifle joints of 9 dogs with late-stage OA. Fractions of SF were assayed for total glycosaminoglycan (GAG) content and also subjected to Western blot analysis by use of monoclonal antibodies against neoepitopes generated by cleavage of the IGD of the aggrecan protein core by matrix metalloproteinase (MMP; BC-14) and aggrecanase (BC-3). Total GAG content of SF from joints of clinically normal dogs did not differ from that of dogs with early-stage OA. The GAG content of SF from joints of dogs with late-stage OA was significantly lower, compared with GAG content for other SF samples. Aggrecanase-generated fragments were detected in SF from all groups but not in all samples. Matrix metalloproteinase-generated fragments were not detected in any SF samples. In early-stage OA, high-molecular-weight aggrecanase-generated aggrecan catabolites were evident. GAG content of SF obtained from dogs with late-stage OA is significantly decreased, suggesting proteoglycan depletion of cartilage. Aggrecanases, but not MMPs, are the major proteolytic enzymes responsible for IGD cleavage of aggrecan in canine joints. Analyses of SF samples to detect aggrecanase-generated catabolites may provide an early biomarker for discriminating early- and late-stage OA in dogs.

Conditioned Medium Derived from Notochordal Cell-Rich Nucleus Pulposus Tissue Stimulates Matrix Production by Canine Nucleus Pulposus Cells and Bone Marrow-Derived Stromal Cells

PubMed Central

de Vries, Stefan A.H.; Potier, Esther; van Doeselaar, Marina; Meij, Björn P.; Tryfonidou, Marianna A.

2015-01-01

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×106 cells/mL) and NPCs+BMSCs (6×106 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10 ng/mL transforming growth factor-β1 (TGF-β1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this

Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow.

PubMed

Staples, Gregory O; Naimy, Hicham; Yin, Hongfeng; Kileen, Kevin; Kraiczek, Karsten; Costello, Catherine E; Zaia, Joseph

2010-01-15

Heparan sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. High-performance liquid chromatography (HPLC)-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of postcolumn makeup flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding the oligosaccharide profile, degree of sulfation, and nonreducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.

Simultaneous analysis of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan disaccharides by glycoblotting-assisted sample preparation followed by single-step zwitter-ionic-hydrophilic interaction chromatography.

PubMed

Takegawa, Yasuhiro; Araki, Kayo; Fujitani, Naoki; Furukawa, Jun-ichi; Sugiyama, Hiroaki; Sakai, Hideaki; Shinohara, Yasuro

2011-12-15

Glycosaminoglycans (GAGs) play important roles in cell adhesion and growth, maintenance of extracellular matrix (ECM) integrity, and signal transduction. To fully understand the biological functions of GAGs, there is a growing need for sensitive, rapid, and quantitative analysis of GAGs. The present work describes a novel analytical technique that enables high throughput cellular/tissue glycosaminoglycomics for all three families of uronic acid-containing GAGs, hyaluronan (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), and heparan sulfate (HS). A one-pot purification and labeling procedure for GAG Δ-disaccharides was established by chemo-selective ligation of disaccharides onto high density hydrazide beads (glycoblotting) and subsequent labeling by fluorescence. The 17 most common disaccharides (eight comprising HS, eight CS/DS, and one comprising HA) could be separated with a single chromatography for the first time by employing a zwitter-ionic type of hydrophilic-interaction chromatography column. These novel analytical techniques were able to precisely characterize the glycosaminoglycome in various cell types including embryonal carcinoma cells and ocular epithelial tissues (cornea, conjunctiva, and limbus).

Responses to altered oxygen tension are distinct between human stem cells of high and low chondrogenic capacity.

PubMed

Anderson, Devon E; Markway, Brandon D; Bond, Derek; McCarthy, Helen E; Johnstone, Brian

2016-10-20

Lowering oxygen from atmospheric level (hyperoxia) to the physiological level (physioxia) of articular cartilage promotes mesenchymal stem cell (MSC) chondrogenesis. However, the literature is equivocal regarding the benefits of physioxic culture on preventing hypertrophy of MSC-derived chondrocytes. Articular cartilage progenitors (ACPs) undergo chondrogenic differentiation with reduced hypertrophy marker expression in hyperoxia but have not been studied in physioxia. This study sought to delineate the effects of physioxic culture on both cell types undergoing chondrogenesis. MSCs were isolated from human bone marrow aspirates and ACP clones were isolated from healthy human cartilage. Cells were differentiated in pellet culture in physioxia (2 % oxygen) or hyperoxia (20 % oxygen) over 14 days. Chondrogenesis was characterized by biochemical assays and gene and protein expression analysis. MSC preparations and ACP clones of high intrinsic chondrogenicity (termed high-GAG) produced abundant matrix in hyperoxia and physioxia. Poorly chondrogenic cells (low-GAG) demonstrated a significant fold-change matrix increase in physioxia. Both high-GAG and low-GAG groups of MSCs and ACPs significantly upregulated chondrogenic genes; however, only high-GAG groups had a concomitant decrease in hypertrophy-related genes. High-GAG MSCs upregulated many common hypoxia-responsive genes in physioxia while low-GAG cells downregulated most of these genes. In physioxia, high-GAG MSCs and ACPs produced comparable type II collagen but less type I collagen than those in hyperoxia. Type X collagen was detectable in some ACP pellets in hyperoxia but reduced or absent in physioxia. In contrast, type X collagen was detectable in all MSC preparations in hyperoxia and physioxia. MSC preparations and ACP clones had a wide range of chondrogenicity between donors. Physioxia significantly enhanced the chondrogenic potential of both ACPs and MSCs compared with hyperoxia, but the magnitude of

Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells

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

Wang, Tsung-Pao; Pan, Yun-Ru; Fu, Chien-Yu

2010-10-15

UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellularmore » spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.« less

Brain ageing changes proteoglycan sulfation, rendering perineuronal nets more inhibitory.

PubMed

Foscarin, Simona; Raha-Chowdhury, Ruma; Fawcett, James W; Kwok, Jessica C F

2017-06-28

Chondroitin sulfate (CS) proteoglycans in perineuronal nets (PNNs) from the central nervous system (CNS) are involved in the control of plasticity and memory. Removing PNNs reactivates plasticity and restores memory in models of Alzheimer's disease and ageing. Their actions depend on the glycosaminoglycan (GAG) chains of CS proteoglycans, which are mainly sulfated in the 4 (C4S) or 6 (C6S) positions. While C4S is inhibitory, C6S is more permissive to axon growth, regeneration and plasticity. C6S decreases during critical period closure. We asked whether there is a late change in CS-GAG sulfation associated with memory loss in aged rats. Immunohistochemistry revealed a progressive increase in C4S and decrease in C6S from 3 to 18 months. GAGs extracted from brain PNNs showed a large reduction in C6S at 12 and 18 months, increasing the C4S/C6S ratio. There was no significant change in mRNA levels of the chondroitin sulfotransferases. PNN GAGs were more inhibitory to axon growth than those from the diffuse extracellular matrix. The 18-month PNN GAGs were more inhibitory than 3-month PNN GAGs. We suggest that the change in PNN GAG sulfation in aged brains renders the PNNs more inhibitory, which lead to a decrease in plasticity and adversely affect memory.

Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein.

PubMed

Serrière, Jennifer; Robert, Xavier; Perez, Magali; Gouet, Patrice; Guillon, Christophe

2013-06-24

Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins

The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

PubMed Central

Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

In vitro culture increases mechanical stability of human tissue engineered cartilage constructs by prevention of microscale scaffold buckling.

PubMed

Middendorf, Jill M; Shortkroff, Sonya; Dugopolski, Caroline; Kennedy, Stephen; Siemiatkoski, Joseph; Bartell, Lena R; Cohen, Itai; Bonassar, Lawrence J

2017-11-07

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling. Copyright © 2017 Elsevier Ltd. All rights reserved.

High prevalence of natural polymorphisms in Gag (CA-SP1) associated with reduced response to Bevirimat, an HIV-1 maturation inhibitor.

PubMed

Seclén, Eduardo; González, María Del Mar; Corral, Angélica; de Mendoza, Carmen; Soriano, Vincent; Poveda, Eva

2010-01-28

Mutations H358Y, L363F/M, A364V and A366T/V confer in-vitro resistance to bevirimat. Moreover, polymorphisms at the Glutamine-Valine-Threonine (QVT) motif (369-371) have been associated with reduced bevirimat activity in vivo. The rate of these changes was assessed in 389 HIV+ patients naïve for bevirimat. QVT polymorphisms were frequent (47%), especially in non-B subtypes (93%). Conversely, only four patients (1%) harbored major bevirimat resistance mutations. Finally, specific gag changes were associated with protease inhibitor resistance mutations in subtype B viruses.

Identifying a New Mechanism of HIV Core Formation | Center for Cancer Research

Cancer.gov

During the maturation of human immunodeficiency virus 1 (HIV-1), viral particles transition from a noninfectious form to an infectious one, and this conversion requires the cleavage of the HIV-1 Gag polyprotein. Gag is made up of three structural proteins—matrix (MA), capsid (CA), and nucleocapsid (NC)—connected by linkers. MA anchors Gag in the membrane, CA surrounds the HIV-1 core, and NC packages the viral RNA within the core. Current models of the development of HIV-1 suggest that when CA is cleaved from Gag it dissociates from the membrane and moves into the virus interior before nucleating, in a concentration-dependent manner, into the core, which is the last step in virus maturation. The core is thought to grow from its narrow end stopping only when it reaches the opposite side of the virus membrane. Since blocking the formation of infectious viral particles is an important therapeutic strategy, it is critical to understand the detailed mechanism of core maturation.

Diffusion-weighted imaging for the cellularity assessment and matrix characterization of soft tissue tumour.

PubMed

Robba, Tiziana; Chianca, Vito; Albano, Domenico; Clementi, Valeria; Piana, Raimondo; Linari, Alessandra; Comandone, Alessandro; Regis, Guido; Stratta, Maurizio; Faletti, Carlo; Borrè, Alda

2017-11-01

To evaluate whether apparent diffusion coefficient (ADC) of diffusion-weighted imaging (DWI) is able to investigate the histological features of soft tissue tumours. We reviewed MRIs of soft tissue tumours performed from 2012 to 2015 to calculate the average ADCs. We included 46 patients (27 male; mean age: 57 years, range 12-85 years) with histologically proven soft tissue tumours (10 benign, 2 intermediate 34 malignant) grouped into eight tumour type classes. An experienced pathologist assigned a semi-quantitative cellularity score (very high, high, medium and low) and tumour grading. The t test, ANOVA and linear regression were used to correlate ADC with clinicopathological data. Approximate receiver operating characteristic curves were created to predict possible uses of ADC to differentiate benign from malignant tumours. There was a significant difference (p < 0.01) in ADCs between these three groups excluding myxoid sarcomas. A significant difference was also evident between the tumour type classes (p < 0.001), grade II and III myxoid lesions (p < 0.05), tumour grading classes (p < 0.001) and cellularity scores classes (p < 0.001), with the lowest ADCs in the very high cellularity. While the linear regression analysis showed a significant relationship between ADC and tumour cellularity (r = 0.590, p ≤ 0.05) and grading (r = 0.437, p ≤ 0.05), no significant relationship was found with age, gender, tumour size and histological subtype. An optimal cut-off ADC value of 1.45 × 10 -3 mm 2 /s with 76.8% accuracy was found to differentiate benign from malignant tumours. DWI may offer adjunctive information about soft tissue tumours, but its clinical role is still to be defined.

Identification of potential biophysical and molecular signalling mechanisms underlying hyaluronic acid enhancement of cartilage formation

PubMed Central

Responte, Donald J.; Natoli, Roman M.; Athanasiou, Kyriacos A.

2012-01-01

This study determined the effects of exogenous hyaluronic acid (HA) on the biomechanical and biochemical properties of self-assembled bovine chondrocytes, and investigated biophysical and genetic mechanisms underlying these effects. The effects of HA commencement time, concentration, application duration and molecular weight were examined using histology, biomechanics and biochemistry. Additionally, the effects of HA application on sulphated glycosaminoglycan (GAG) retention were assessed. To investigate the influence of HA on gene expression, microarray analysis was conducted. HA treatment of developing neocartilage increased compressive stiffness onefold and increased sulphated GAG content by 35 per cent. These effects were dependent on HA molecular weight, concentration and application commencement time. Additionally, applying HA increased sulphated GAG retention within self-assembled neotissue. HA administration also upregulated 503 genes, including multiple genes associated with TGF-β1 signalling. Increased sulphated GAG retention indicated that HA could enhance compressive stiffness by increasing the osmotic pressure that negatively charged GAGs create. The gene expression data demonstrate that HA treatment differentially regulates genes related to TGF-β1 signalling, revealing a potential mechanism for altering matrix composition. These results illustrate the potential use of HA to improve cartilage regeneration efforts and better understand cartilage development. PMID:22809846

Hb S [β6(A3)Glu→Val, GAG>GTG] and β-globin gene cluster haplotype distribution in Mauritania.

PubMed

Veten, Fatimetou M; Abdelhamid, Isselmou O; Meiloud, Ghlana M; Ghaber, Sidi M; Salem, Mohamed L; Abbes, Salem; Houmeida, Ahmed O

2012-01-01

Of 1050 Mauritanian blood donors screened from the two main racial groups, i.e., the Moors and Black Africans, 60 were found to carry Hb S [β6(A3)Glu→Val, GAG>GTG], giving a global frequency of 5.71%. The prevalence observed in the Black African Mauritanians (10.69%) is almost five times that found in the Moor group (2.25%). Four of the five main β(S) haplotypes were detected in this study: Senegal (77.8%), Benin (8.8%), Arab-Indian (5.5%) and Bantu (4.4%). These data showed that Hb S is a serious public health problem in Mauritania. They also confirm the ethnic heterogeneity of the Mauritanian population.

Plasmacytoid dendritic cell and functional HIV Gag p55-specific T cells before treatment interruption can inform set-point plasma HIV viral load after treatment interruption in chronically suppressed HIV-1(+) patients.

PubMed

Papasavvas, Emmanouil; Foulkes, Andrea; Yin, Xiangfan; Joseph, Jocelin; Ross, Brian; Azzoni, Livio; Kostman, Jay R; Mounzer, Karam; Shull, Jane; Montaner, Luis J

2015-07-01

The identification of immune correlates of HIV control is important for the design of immunotherapies that could support cure or antiretroviral therapy (ART) intensification-related strategies. ART interruptions may facilitate this task through exposure of an ART partially reconstituted immune system to endogenous virus. We investigated the relationship between set-point plasma HIV viral load (VL) during an ART interruption and innate/adaptive parameters before or after interruption. Dendritic cell (DC), natural killer (NK) cell and HIV Gag p55-specific T-cell functional responses were measured in paired cryopreserved peripheral blood mononuclear cells obtained at the beginning (on ART) and at set-point of an open-ended interruption from 31 ART-suppressed chronically HIV-1(+) patients. Spearman correlation and linear regression modeling were used. Frequencies of plasmacytoid DC (pDC), and HIV Gag p55-specific CD3(+)  CD4(-)  perforin(+)  IFN-γ(+) cells at the beginning of interruption associated negatively with set-point plasma VL. Inclusion of both variables with interaction into a model resulted in the best fit (adjusted R(2)  = 0·6874). Frequencies of pDC or HIV Gag p55-specific CD3(+)  CD4(-)  CSFE(lo)  CD107a(+) cells at set-point associated negatively with set-point plasma VL. The dual contribution of pDC and anti-HIV T-cell responses to viral control, supported by our models, suggests that these variables may serve as immune correlates of viral control and could be integrated in cure or ART-intensification strategies. © 2015 John Wiley & Sons Ltd.

Extracellular-Matrix-Based and Arg-Gly-Asp–Modified Photopolymerizing Hydrogels for Cartilage Tissue Engineering

PubMed Central

Kim, Hwan D.; Heo, Jiseung; Hwang, Yongsung; Kwak, Seon-Yeong; Park, Ok Kyu; Kim, Hyunbum; Varghese, Shyni

2015-01-01

Articular cartilage damage is a persistent and increasing problem with the aging population. Strategies to achieve complete repair or functional restoration remain a challenge. Photopolymerizing-based hydrogels have long received an attention in the cartilage tissue engineering, due to their unique bioactivities, flexible method of synthesis, range of constituents, and desirable physical characteristics. In the present study, we have introduced unique bioactivity within the photopolymerizing-based hydrogels by copolymerizing polyethylene glycol (PEG) macromers with methacrylated extracellular matrix (ECM) molecules (hyaluronic acid and chondroitin sulfate [CS]) and integrin binding peptides (RGD peptide). Results indicate that cellular morphology, as observed by the actin cytoskeleton structures, was strongly dependent on the type of ECM component as well as the presence of integrin binding moieties. Further, CS-based hydrogel with integrin binding RGD moieties increased the lubricin (or known as superficial zone protein [SZP]) gene expression of the encapsulated chondrocytes. Additionally, CS-based hydrogel displayed cell-responsive degradation and resulted in increased DNA, GAG, and collagen accumulation compared with other hydrogels. This study demonstrates that integrin-mediated interactions within CS microenvironment provide an optimal hydrogel scaffold for cartilage tissue engineering application. PMID:25266634

Prediction of glycosaminoglycan synthesis in intervertebral disc under mechanical loading.

PubMed

Gao, Xin; Zhu, Qiaoqiao; Gu, Weiyong

2016-09-06

The loss of glycosaminoglycan (GAG) content is a major biochemical change during intervertebral disc (IVD) degeneration. Abnormal mechanical loading is one of the major factors causing disc degeneration. In this study, a multiscale mathematical model was developed to quantify the effect of mechanical loading on GAG synthesis. This model was based on a recently developed cell volume dependent GAG synthesis theory that predicts the variation of GAG synthesis rate of a cell under the influence of mechanical stimuli, and the biphasic theory that describes the deformation of IVD under mechanical loading. The GAG synthesis (at the cell level) was coupled with the mechanical loading (at the tissue level) via a cell-matrix unit approach which established a relationship between the variation of cell dilatation and the local tissue dilatation. This multiscale mathematical model was used to predict the effect of static load (creep load) on GAG synthesis in bovine tail discs. The predicted results are in the range of experimental results. This model was also used to investigate the effect of static (0.2MPa) and diurnal loads (0.1/0.3MPa and 0.15/0.25MPa in 12/12 hours shift with an average of 0.2MPa over a cycle) on GAG synthesis. It was found that static load and diurnal loads have different effects on GAG synthesis in a diurnal cycle, and the diurnal load effects depend on the amplitude of the load. The model is important to understand the effect of mechanical loading at the tissue level on GAG synthesis at the cellular level, as well as to optimize the mechanical loading in growing engineered tissue. Copyright © 2016 Elsevier Ltd. All rights reserved.

Co-culture with infrapatellar fat pad differentially stimulates proteoglycan synthesis and accumulation in cartilage and meniscus tissues.

PubMed

Nishimuta, James F; Bendernagel, Monica F; Levenston, Marc E

2017-09-01

Although osteoarthritis is widely viewed as a disease of the whole joint, relatively few studies have focused on interactions among joint tissues in joint homeostasis and degeneration. In particular, few studies have examined the effects of the infrapatellar fat pad (IFP) on cartilaginous tissues. The aim of this study was to test the hypothesis that co-culture with healthy IFP would induce degradation of cartilage and meniscus tissues. Bovine articular cartilage, meniscus, and IFP were cultured isolated or as cartilage-fat or meniscus-fat co-cultures for up to 14 days. Conditioned media were assayed for sulfated glycosaminoglycan (sGAG) content, nitrite content, and matrix metalloproteinase (MMP) activity, and explants were assayed for sGAG and DNA contents. Co-cultures exhibited increased cumulative sGAG release and sGAG release rates for both cartilage and meniscus, and the cartilage (but not meniscus) exhibited a substantial synergistic effect of co-culture (sGAG release in co-culture was significantly greater than the summed release from isolated cartilage and fat). Fat co-culture did not significantly alter the sGAG content of either cartilage or meniscus explants, indicating that IFP co-culture stimulated net sGAG production by cartilage. Nitrite release was increased relative to isolated tissue controls in co-cultured meniscus, but not the cartilage, with no synergistic effect of co-culture. Interestingly, MMP-2 production was decreased by co-culture for both cartilage and meniscus. This study demonstrates that healthy IFP may modulate joint homeostasis by stimulating sGAG production in cartilage. Counter to our hypothesis, healthy IFP did not promote degradation of either cartilage or meniscus tissues.

Docking glycosaminoglycans to proteins: analysis of solvent inclusion

NASA Astrophysics Data System (ADS)

Samsonov, Sergey A.; Teyra, Joan; Pisabarro, M. Teresa

2011-05-01

Glycosaminoglycans (GAGs) are anionic polysaccharides, which participate in key processes in the extracellular matrix by interactions with protein targets. Due to their charged nature, accurate consideration of electrostatic and water-mediated interactions is indispensable for understanding GAGs binding properties. However, solvent is often overlooked in molecular recognition studies. Here we analyze the abundance of solvent in GAG-protein interfaces and investigate the challenges of adding explicit solvent in GAG-protein docking experiments. We observe PDB GAG-protein interfaces being significantly more hydrated than protein-protein interfaces. Furthermore, by applying molecular dynamics approaches we estimate that about half of GAG-protein interactions are water-mediated. With a dataset of eleven GAG-protein complexes we analyze how solvent inclusion affects Autodock 3, eHiTs, MOE and FlexX docking. We develop an approach to de novo place explicit solvent into the binding site prior to docking, which uses the GRID program to predict positions of waters and to locate possible areas of solvent displacement upon ligand binding. To investigate how solvent placement affects docking performance, we compare these results with those obtained by taking into account information about the solvent position in the crystal structure. In general, we observe that inclusion of solvent improves the results obtained with these methods. Our data show that Autodock 3 performs best, though it experiences difficulties to quantitatively reproduce experimental data on specificity of heparin/heparan sulfate disaccharides binding to IL-8. Our work highlights the current challenges of introducing solvent in protein-GAGs recognition studies, which is crucial for exploiting the full potential of these molecules for rational engineering.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

PubMed

Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

2016-06-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. © 2016 Kalloush et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

PubMed Central

Kalloush, Rawan M.; Vivet-Boudou, Valérie; Ali, Lizna M.; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A.

2016-01-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2′hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. PMID:27095024

Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues.

PubMed

Peng, Tao; Huang, Bingzhen; Sun, Yao; Lu, Yongbo; Bonewald, Lynda; Chen, Shuo; Butler, William T; Feng, Jerry Q; D'Souza, Rena N; Qin, Chunlin

2009-01-01

Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH(2)- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH(2) termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp(181) (corresponding to Asp(197) of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH(2) terminus of Asp(197) of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp(197) is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp(197) with Ala(197) by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH(2) terminus of Asp(197) is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH(2)-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser(74) in rat DMP1 (Ser(89) in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser(89), we substituted Ser(89) by Gly(89). Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser(89) in mouse DMP1. Copyright 2008 S. Karger AG, Basel.

Quantifying integrated SIV-DNA by repetitive-sampling Alu-gag PCR.

PubMed

Mavigner, Maud; Lee, S Thera; Habib, Jakob; Robinson, Cameron; Silvestri, Guido; O'Doherty, Una; Chahroudi, Ann

2016-10-05

Although antiretroviral therapy (ART) effectively suppresses HIV-1 replication, it does not eradicate the virus and ART interruption consistently results in rebound of viraemia, demonstrating the persistence of a long-lived viral reservoir. Several approaches aimed at reducing virus persistence are being developed, and accurate measurements of the latent reservoir (LR) are necessary to assess the effectiveness of anti-latency interventions. We sought to measure the LR in SIV/SHIV-infected rhesus macaques (RMs) by quantifying integrated SIV-DNA. We optimised a repetitive sampling Alu-gag PCR to quantify integrated SIV-DNA ex vivo in ART-naïve and ART-experienced SIV/SHIV-infected RMs. In ART-naïve RMs, we found the median level of integrated SIV-DNA to be 1660 copies and 866 copies per million PBMC during untreated acute and chronic SHIV infection, respectively. Integrated and total SIV-DNA levels were positively correlated with one another. In ART-treated RMs, integrated SIV-DNA was readily detected in lymph nodes and spleen and levels of total (3319 copies/million cells) and integrated (3160 copies/million cells) SIV-DNA were similar after a median of 404 days of ART. In peripheral blood CD4+ T cells from ART-treated RMs, levels of total (3319 copies/million cells) and integrated (2742 copies/million cells) SIV-DNA were not significantly different and were positively correlated. The assay described here is validated and can be used in interventional studies testing HIV/SIV cure strategies in RMs. Measurement of integrated SIV-DNA in ART-treated RMs, along with other reservoir analyses, gives an estimate of the size of the LR.

Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

PubMed

Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

2017-12-01

The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media.

PubMed

Lynch, Alisson; Meyers, Ann E; Williamson, Anna-Lise; Rybicki, Edward P

2012-09-18

HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4°C, -20°C and -70°C) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at -70°C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at -70°C for 12 months is most effective in retaining VLP stability.

Frequent and Variable Cytotoxic-T-Lymphocyte Escape-Associated Fitness Costs in the Human Immunodeficiency Virus Type 1 Subtype B Gag Proteins

PubMed Central

Boutwell, Christian L.; Carlson, Jonathan M.; Lin, Tien-Ho; Seese, Aaron; Power, Karen A.; Peng, Jian; Tang, Yanhua; Brumme, Zabrina L.; Heckerman, David; Schneidewind, Arne

2013-01-01

Cytotoxic-T-lymphocyte (CTL) escape mutations undermine the durability of effective human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cell responses. The rate of CTL escape from a given response is largely governed by the net of all escape-associated viral fitness costs and benefits. The observation that CTL escape mutations can carry an associated fitness cost in terms of reduced virus replication capacity (RC) suggests a fitness cost-benefit trade-off that could delay CTL escape and thereby prolong CD8 response effectiveness. However, our understanding of this potential fitness trade-off is limited by the small number of CTL escape mutations for which a fitness cost has been quantified. Here, we quantified the fitness cost of the 29 most common HIV-1B Gag CTL escape mutations using an in vitro RC assay. The majority (20/29) of mutations reduced RC by more than the benchmark M184V antiretroviral drug resistance mutation, with impacts ranging from 8% to 69%. Notably, the reduction in RC was significantly greater for CTL escape mutations associated with protective HLA class I alleles than for those associated with nonprotective alleles. To speed the future evaluation of CTL escape costs, we also developed an in silico approach for inferring the relative impact of a mutation on RC based on its computed impact on protein thermodynamic stability. These data illustrate that the magnitude of CTL escape-associated fitness costs, and thus the barrier to CTL escape, varies widely even in the conserved Gag proteins and suggest that differential escape costs may contribute to the relative efficacy of CD8 responses. PMID:23365420

Matrix completion by deep matrix factorization.

PubMed

Fan, Jicong; Cheng, Jieyu

2018-02-01

Conventional methods of matrix completion are linear methods that are not effective in handling data of nonlinear structures. Recently a few researchers attempted to incorporate nonlinear techniques into matrix completion but there still exists considerable limitations. In this paper, a novel method called deep matrix factorization (DMF) is proposed for nonlinear matrix completion. Different from conventional matrix completion methods that are based on linear latent variable models, DMF is on the basis of a nonlinear latent variable model. DMF is formulated as a deep-structure neural network, in which the inputs are the low-dimensional unknown latent variables and the outputs are the partially observed variables. In DMF, the inputs and the parameters of the multilayer neural network are simultaneously optimized to minimize the reconstruction errors for the observed entries. Then the missing entries can be readily recovered by propagating the latent variables to the output layer. DMF is compared with state-of-the-art methods of linear and nonlinear matrix completion in the tasks of toy matrix completion, image inpainting and collaborative filtering. The experimental results verify that DMF is able to provide higher matrix completion accuracy than existing methods do and DMF is applicable to large matrices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Macroscopic and histological investigation of guanaco footpads (Lama guanicoe, Müller 1776).

PubMed

König, Horst Erich; Skewes, Oscar; Helmreich, Magda; Böck, Peter

2015-03-01

The surface of guanaco footpads is characterized by hairless skin with up to 4-mm-thick stratum corneum that protects from abrasion. The horny layer is pliable and elastic, and ensures firm contact with irregular ground. It is padded with a particular structure of the subcutaneous layer, the digital cushion. The flat cushions of each of the two digits are of elongated ovate shape, each about 45-mm long, up to 20-mm wide, and 8-mm thick. The cushions are lined by a 1-2-mm-thick capsule that resembles a tunica albuginea. The capsule consists of coarse collagen fibers, with elastic fibers absent. The cushion capsule and dermis approach each other, and fuse along a line that runs parallel to the longitudinal axes of cushion and digit. Loose connective tissue rich in elastic fibers and acidic glycosaminoglycans separates dermis and cushion capsule lateral to the narrow interconnecting zone. The cushion capsule encloses cloudy yellowish, gelatinous material. Microscopy shows bundles of elastic fibers in abundant mucinous matrix. Tightly gathered elastic bundles adjoin the inner surface of the capsule. Rough cords of elastic fibers branch out from there and traverse to the opposite side. The cushion is pressed flat, and elastic fibers are stretched when bearing weight. With relief of load, elastic fibers contract and reset the cushion's shape. Contractile cells are absent. A resistant capsule and easily malleable mucinous contents establish the functioning as a gel pad. Mucinous connective tissue between elastic fiber bundles contains abundant basophilic matrix. Hyaluronan, chondroitin sulfate, and dermatan sulfate are main matrix constituents. Spindle-shaped or stellate fibroblasts contain vimentin, S100 protein, and neuron specific enolase. Moprhology, staining characteristics and synthesis activities of these cells meet the criteria to be classified as myxoid cells. The connective tissue in guanaco digital cushions represents myxoid tissue. © 2014 Wiley Periodicals

A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.

PubMed

Lee, Sook-Kyung; Cheng, Nancy; Hull-Ryde, Emily; Potempa, Marc; Schiffer, Celia A; Janzen, William; Swanstrom, Ronald

2013-07-23

The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.

Controlled release of GAG-binding enhanced transduction (GET) peptides for sustained and highly efficient intracellular delivery.

PubMed

Abu-Awwad, Hosam Al-Deen M; Thiagarajan, Lalitha; Dixon, James E

2017-07-15

Controlled release systems for therapeutic molecules are vital to allow the sustained local delivery of their activities which direct cell behaviour and enable novel regenerative strategies. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor signalling but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding domains which promote cell targeting, and cell penetrating peptides (CPPs) which allow cell entry. Herein we demonstrate that GET system can be used in controlled release systems to mediate sustained intracellular transduction over one week. We assessed the stability and activity of GET peptides in poly(dl-lactic acid-co-glycolic acid) (PLGA) microparticles (MPs) prepared using a S/O/W double emulsion method. Efficient encapsulation (∼65%) and tailored protein release profiles could be achieved, however intracellular transduction was significantly inhibited post-release. To retain GET peptide activity we optimized a strategy of co-encapsulation of l-Histidine, which may form a complex with the PLGA degradation products under acidic conditions. Simulations of the polymer microclimate showed that hydrolytic acidic PLGA degradation products directly inhibited GET peptide transduction activity, and use of l-Histidine significantly enhanced released protein delivery. The ability to control the intracellular transduction of functional proteins into cells will facilitate new localized delivery methods and allow approaches to direct cellular behaviour for many regenerative medicine applications. The goal for regenerative medicine is to restore functional biological tissue by controlling and augmenting cellular behaviour. Either Transcription (TFs) or growth factors (GFs) can be presented to cells in spatio-temporal gradients for

Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulfate-glycosaminoglycan depleted cartilage.

PubMed

Siebelt, Michiel; Groen, Harald C; Koelewijn, Stuart J; de Blois, Erik; Sandker, Marjan; Waarsing, Jan H; Müller, Cristina; van Osch, Gerjo J V M; de Jong, Marion; Weinans, Harrie

2014-01-29

Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration. sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness. All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation. Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage damage, but also resulted in pronounced

Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulfate-glycosaminoglycan depleted cartilage

PubMed Central

2014-01-01

Introduction Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration. Methods sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness. Results All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation. Conclusions Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage

Electrostatic, elastic and hydration-dependent interactions in dermis influencing volume exclusion and macromolecular transport.

PubMed

Øien, Alf H; Wiig, Helge

2016-07-07

Interstitial exclusion refers to the limitation of space available for plasma proteins and other macromolecules based on collagen and negatively charged glycosaminoglycans (GAGs) in the interstitial space. It is of particular importance to interstitial fluid and plasma volume regulation. Here we present a novel mechanical and mathematical model of the dynamic interactions of structural elements within the interstitium of the dermis at the microscopic level that may explain volume exclusion of charged and neutral macroparticles. At this level, the interstitium is considered to consist of elements called extracellular matrix (ECM) cells, again containing two main interacting structural components on a fluid background including anions and cations setting up osmotic forces: one smaller GAG component, having an intrinsic expansive electric force, and one bigger collagen component, having an intrinsic elastic force. Because of size differences, the GAG component interacts with a fraction of the collagen component only at normal hydration. This fraction, however, increases with rising hydration as a consequence of the modeled form of the interaction force between the GAGs and collagen. Collagen is locally displaced at variable degrees as hydration changes. Two models of GAGs are considered, having largely different geometries which demands different, but related, forms of GAG-collagen interaction forces. The effects of variable fixed charges on GAGs and of GAG density in tissue are evaluated taking into account observed volume exclusion properties of charged macromolecules as a function of tissue hydration. The presented models may improve our biophysical understanding of acting forces influencing tissue fluid dynamics. Such knowledge is significant when evaluating the transport of electrically charged and neutral macromolecules into and through the interstitium, and therefore to drug uptake and the therapeutic effects of macromolecular agents. Copyright © 2016 Elsevier

Deformation simulation of cells seeded on a collagen-GAG scaffold in a flow perfusion bioreactor using a sequential 3D CFD-elastostatics model.

PubMed

Jungreuthmayer, C; Jaasma, M J; Al-Munajjed, A A; Zanghellini, J; Kelly, D J; O'Brien, F J

2009-05-01

Tissue-engineered bone shows promise in meeting the huge demand for bone grafts caused by up to 4 million bone replacement procedures per year, worldwide. State-of-the-art bone tissue engineering strategies use flow perfusion bioreactors to apply biophysical stimuli to cells seeded on scaffolds and to grow tissue suitable for implantation into the patient's body. The aim of this study was to quantify the deformation of cells seeded on a collagen-GAG scaffold which was perfused by culture medium inside a flow perfusion bioreactor. Using a microCT scan of an unseeded collagen-GAG scaffold, a sequential 3D CFD-deformation model was developed. The wall shear stress and the hydrostatic wall pressure acting on the cells were computed through the use of a CFD simulation and fed into a linear elastostatics model in order to calculate the deformation of the cells. The model used numerically seeded cells of two common morphologies where cells are either attached flatly on the scaffold wall or bridging two struts of the scaffold. Our study showed that the displacement of the cells is primarily determined by the cell morphology. Although cells of both attachment profiles were subjected to the same mechanical load, cells bridging two struts experienced a deformation up to 500 times higher than cells only attached to one strut. As the scaffold's pore size determines both the mechanical load and the type of attachment, the design of an optimal scaffold must take into account the interplay of these two features and requires a design process that optimizes both parameters at the same time.

Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels.

PubMed

Bian, Liming; Zhai, David Y; Zhang, Emily C; Mauck, Robert L; Burdick, Jason A

2012-04-01

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair, and there is growing evidence that mechanical signals play a critical role in the regulation of stem cell chondrogenesis and in cartilage development. In this study we investigated the effect of dynamic compressive loading on chondrogenesis, the production and distribution of cartilage specific matrix, and the hypertrophic differentiation of human MSCs encapsulated in hyaluronic acid (HA) hydrogels during long term culture. After 70 days of culture, dynamic compressive loading increased the mechanical properties, as well as the glycosaminoglycan (GAG) and collagen contents of HA hydrogel constructs in a seeding density dependent manner. The impact of loading on HA hydrogel construct properties was delayed when applied to lower density (20 million MSCs/ml) compared to higher seeding density (60 million MSCs/ml) constructs. Furthermore, loading promoted a more uniform spatial distribution of cartilage matrix in HA hydrogels with both seeding densities, leading to significantly improved mechanical properties as compared to free swelling constructs. Using a previously developed in vitro hypertrophy model, dynamic compressive loading was also shown to significantly reduce the expression of hypertrophic markers by human MSCs and to suppress the degree of calcification in MSC-seeded HA hydrogels. Findings from this study highlight the importance of mechanical loading in stem cell based therapy for cartilage repair in improving neocartilage properties and in potentially maintaining the cartilage phenotype.

Expression of p24 gag protein of bovine leukemia virus in insect cells and its use in immunodetection of the disease.

PubMed

Larsen, Alejandra; Gonzalez, Ester Teresa; Serena, María Soledad; Echeverría, María Gabriela; Mortola, Eduardo

2013-06-01

Bovine leukemia is a common retroviral infection of cattle. The disease is characterized by a strong immunological response to several viral proteins, but the antibodies against p24 and gp51 are predominant. In this study, a recombinant baculovirus containing the gag gene p24 was constructed and the protein, used as antigen, analyzed by western blot and an indirect in-house rp24-ELISA test. This allowed detecting the presence of antibodies for bovine leukemia virus in a panel of cattle sera. The authentication of the protein expands its potential use for different medical applications, from improved diagnosis of the disease to source of antigens to be included in a subunit vaccine.

Surface plasmon resonance-based molecular detection of Hb S [beta6(A3)Glu-->Val, GAG-->GTG] at the gene level.

PubMed

Atalay, Erol O; Ustel, Emre; Yildiz, Sanem; Atalay, Ayfer

2006-01-01

The surface plasmon resonance (SPR) approach, being a relatively novel biophysical method, is used to detect many different targets by biomolecular interaction. The SPR system uses optical and evanescent wave phenomenon. This approach does not need any labels, such as enzymes or isotopes, and the monitored interactions are in real time. In DNA-DNA interaction, the SPR approach is Tm-independent. Here we report our preliminary results for the molecular detection of the Hb S (GAG -->GTG) mutation at codon 6 of the human beta-globin gene. Our preliminary results show that the SPR approach could be applied as an inexpensive and fast routine test system for the molecular diagnosis of abnormal hemoglobins (Hbs), especially in premarital screening programs.

Mapping the Differential Distribution of Glycosaminoglycans in the Adult Human Retina, Choroid, and Sclera

PubMed Central

Clark, Simon J.; Keenan, Tiarnan D. L.; Fielder, Helen L.; Collinson, Lisa J.; Holley, Rebecca J.; Merry, Catherine L. R.; van Kuppevelt, Toin H.; Day, Anthony J.; Bishop, Paul N.

2011-01-01

Purpose. To map the distribution of different classes of glycosaminoglycans (GAGs) in the healthy human retina, choroid, and sclera. Methods. Frozen tissue sections were made from adult human donor eyes. The GAG chains of proteoglycans (PGs) were detected with antibodies directed against various GAG structures (either directly or after pretreatment with GAG-degrading enzymes); hyaluronan (HA) was detected using biotinylated recombinant G1-domain of human versican. The primary detection reagents were identified with FITC-labeled probes and analyzed by fluorescence microscopy. Results. Heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and HA were present throughout the retina and choroid, but keratan sulfate (KS) was detected only in the sclera. HS labeling was particularly strong in basement membrane–containing structures, the nerve fiber layer (NFL), and retinal pigment epithelium (RPE)—for example, intense staining was seen with an antibody that binds strongly to sequences containing 3-O-sulfation in the internal limiting membrane (ILM) and in the basement membrane of blood vessels. Unsulfated CS was seen throughout the retina, particularly in the ILM and interphotoreceptor matrix (IPM) with 6-O-sulfated CS also prominent in the IPM. There was labeling for DS throughout the retina and choroid, especially in the NFL, ganglion cell layer, and blood vessels. Conclusions. The detection of GAG chains with specific probes and fluorescence microscopy provides for the first time a detailed analysis of their compartmentalization in the human retina, by both GAG chain type and sulfation pattern. This reference map provides a basis for understanding the functional regulation of GAG-binding proteins in health and disease processes. PMID:21746802

Human immunodeficiency virus type 1 resistance to the small molecule maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid is conferred by a variety of single amino acid substitutions at the CA-SP1 cleavage site in Gag.

PubMed

Zhou, Jing; Chen, Chin Ho; Aiken, Christopher

2006-12-01

The compound 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.

Co-culture of chondrons and mesenchymal stromal cells reduces the loss of collagen VI and improves extracellular matrix production.

PubMed

Owida, H A; De Las Heras Ruiz, T; Dhillon, A; Yang, Y; Kuiper, N J

2017-12-01

Adult articular chondrocytes are surrounded by a pericellular matrix (PCM) to form a chondron. The PCM is rich in hyaluronan, proteoglycans, and collagen II, and it is the exclusive location of collagen VI in articular cartilage. Collagen VI anchors the chondrocyte to the PCM. It has been suggested that co-culture of chondrons with mesenchymal stromal cells (MSCs) might enhance extracellular matrix (ECM) production. This co-culture study investigates whether MSCs help to preserve the PCM and increase ECM production. Primary bovine chondrons or chondrocytes or rat MSCs were cultured alone to establish a baseline level for ECM production. A xenogeneic co-culture monolayer model using rat MSCs (20, 50, and 80%) was established. PCM maintenance and ECM production were assessed by biochemical assays, immunofluorescence, and histological staining. Co-culture of MSCs with chondrons enhanced ECM matrix production, as compared to chondrocyte or chondron only cultures. The ratio 50:50 co-culture of MSCs and chondrons resulted in the highest increase in GAG production (18.5 ± 0.54 pg/cell at day 1 and 11 ± 0.38 pg/cell at day 7 in 50:50 co-culture versus 16.8 ± 0.61 pg/cell at day 1 and 10 ± 0.45 pg/cell at day 7 in chondron monoculture). The co-culture of MSCs with chondrons appeared to decelerate the loss of the PCM as determined by collagen VI expression, whilst the expression of high-temperature requirement serine protease A1 (HtrA1) demonstrated an inverse relationship to that of the collagen VI. Together, this implies that MSCs directly or indirectly inhibited HtrA1 activity and the co-culture of MSCs with chondrons enhanced ECM synthesis and the preservation of the PCM.

Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic Machinery for Biomedical Applications.

PubMed

Chua, Jie Shi; Kuberan, Balagurunathan

2017-11-21

Glycosaminoglycans (GAGs) are polysaccharides ubiquitously found on cell surfaces and in the extracellular matrix (ECM). They regulate numerous cellular signaling events involved in many developmental and pathophysiological processes. GAGs are composed of complex sequences of repeating disaccharide units, each of which can carry many different modifications. The tremendous structural variations account for their ability to bind many proteins and thus, for their numerous functions. Although the sequence of GAG biosynthetic events and the enzymes involved mostly were deduced a decade ago, the emergence of tissue or cell specific GAGs from a nontemplate driven process remains an enigma. Current knowledge favors the hypothesis that macromolecular assemblies of GAG biosynthetic enzymes termed "GAGOSOMEs" coordinate polymerization and fine structural modifications in the Golgi apparatus. Distinct GAG structures arise from the differential channeling of substrates through the Golgi apparatus to various GAGOSOMEs. As GAGs perform multiple regulatory roles, it is of great interest to develop molecular strategies to selectively interfere with GAG biosynthesis for therapeutic applications. In this Account, we assess our present knowledge on GAG biosynthesis, the manipulation of GAG biosynthesis using synthetic xylosides, and the unrealized potential of these xylosides in various biomedical applications. Synthetic xylosides are small molecules consisting of a xylose attached to an aglycone group, and they compete with endogenous proteins for precursors and biosynthetic enzymes to assemble GAGs. This competition reduces endogenous proteoglycan-bound GAGs while increasing xyloside-bound free GAGs, mostly chondroitin sulfate (CS) and less heparan sulfate (HS), resulting in a variety of biological consequences. To date, hundreds of xylosides have been published and the importance of the aglycone group in determining the structure of the primed GAG chains is well established

Role of a GAG Hinge in the Nucleotide-induced Conformational Change Governing Nucleotide Specificity by T7 DNA Polymerase*

PubMed Central

Jin, Zhinan; Johnson, Kenneth A.

2011-01-01

A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches. PMID:20978284

Effects of hydration on steric and electric charge-induced interstitial volume exclusion--a model.

PubMed

Øien, Alf H; Justad, Sigrid R; Tenstad, Olav; Wiig, Helge

2013-09-03

The presence of collagen and charged macromolecules like glycosaminoglycans (GAGs) in the interstitial space limits the space available for plasma proteins and other macromolecules. This phenomenon, known as interstitial exclusion, is of importance for interstitial fluid volume regulation. Physical/mathematical models are presented for calculating the exclusion of electrically charged and neutral macromolecules that equilibrate in the interstitium under various degrees of hydration. Here, a central hypothesis is that the swelling of highly electrically charged GAGs with increased hydration shields parts of the neutral collagen of the interstitial matrix from interacting with electrically charged macromolecules, such that exclusion of charged macromolecules exhibits change due to steric and charge effects. GAGs are also thought to allow relatively small neutral, but also charged macromolecules neutralized by a very high ionic strength, diffuse into the interior of GAGs, whereas larger macromolecules may not. Thus, in the model, relatively small electrically charged macromolecules, such as human serum albumin, and larger neutral macromolecules such as IgG, will have quite similar total volume exclusion properties in the interstitium. Our results are in agreement with ex vivo and in vivo experiments, and suggest that the charge of GAGs or macromolecular drugs may be targeted to increase the tissue uptake of macromolecular therapeutic agents. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Large proteoglycan complexes and disturbed collagen architecture in the corneal extracellular matrix of mucopolysaccharidosis type VII (Sly syndrome).

PubMed

Young, Robert D; Liskova, Petra; Pinali, Christian; Palka, Barbara P; Palos, Michalis; Jirsova, Katerina; Hrdlickova, Enkela; Tesarova, Marketa; Elleder, Milan; Zeman, Jiri; Meek, Keith M; Knupp, Carlo; Quantock, Andrew J

2011-08-24

Deficiencies in enzymes involved in proteoglycan (PG) turnover underlie a number of rare mucopolysaccharidoses (MPS), investigations of which can considerably aid understanding of the roles of PGs in corneal matrix biology. Here, the authors analyze novel pathologic changes in MPS VII (Sly syndrome) to determine the nature of PG-collagen associations in stromal ultrastructure. Transmission electron microscopy and electron tomography were used to investigate PG-collagen architectures and interactions in a cornea obtained at keratoplasty from a 22-year-old man with MPS VII, which was caused by a compound heterozygous mutation in the GUSB gene. Transmission electron microscopy showed atypical morphology of the epithelial basement membrane and Bowman's layer in MPS VII. Keratocytes were packed with cytoplasmic vacuoles containing abnormal glycosaminoglycan (GAG) material, and collagen fibrils were thinner than in normal cornea and varied considerably throughout anterior (14-32 nm), mid (13-42 nm), and posterior (17-39 nm) regions of the MPS VII stroma. PGs viewed in three dimensions were striking in appearance in that they were significantly larger than PGs in normal cornea and formed highly extended linkages with multiple collagen fibrils. Cellular changes in the MPS VII cornea resemble those in other MPS. However, the wide range of collagen fibril diameters throughout the stroma and the extensive matrix presence of supranormal-sized PG structures appear to be unique features of this disorder. The findings suggest that the accumulation of stromal chondroitin-, dermatan-, and heparan-sulfate glycosaminoglycans in the absence of β-glucuronidase-mediated degradation can modulate collagen fibrillogenesis.

Design and Fabrication of Anatomical Bioreactor Systems Containing Alginate Scaffolds for Cartilage Tissue Engineering

PubMed Central

Gharravi, Anneh Mohammad; Orazizadeh, Mahmoud; Ansari-Asl, Karim; Banoni, Salem; Izadi, Sina; Hashemitabar, Mahmoud

2012-01-01

The aim of the present study was to develop a tissue-engineering approach through alginate gel molding to mimic cartilage tissue in a three-dimensional culture system. The perfusion biomimetic bioreactor was designed to mimic natural joint. The shear stresses exerting on the bioreactor chamber were calculated by Computational Fluid Dynamic (CFD). Several alginate/bovine chondrocyte constructs were prepared, and were cultured in the bioreactor. Histochemical and immunohistochemical staining methods for the presence of glycosaminoglycan(GAG), overall matrix production and type II collagen protein were performed, respectively. The dynamic mechanical device applied a linear mechanical displacement of 2 mm to 10 mm. The CFD modeling indicated peak velocity and maximum wall shear stress were 1.706×10−3 m/s and 0.02407 dyne/cm 2, respectively. Histochemical and immunohistochemical analysis revealed evidence of cartilage-like tissue with lacunas similar to those of natural cartilage and the production of sulfated GAG of matrix by the chondrons, metachromatic territorial matrix-surrounded cells and accumulation of type II collagen around the cells. The present study indicated that when chondrocytes were seeded in alginate hydrogel and cultured in biomimetic cell culture system, cells survived well and secreted newly synthesized matrix led to improvement of chondrogenesis. PMID:23408660

Gag rule is formally suspended as Title X renewal advances in House.

PubMed

1993-02-09

In February 1993, the US House of Representatives reintroduced a bill (HR 670) which was similar to an earlier bill introduced during the Bush Administration in late 1992 to override a Supreme Court decision upholding Reagan's executive order and vetoed by President Bush. This executive order restricted abortion counseling at federally funded clinics. Bill HR 670 clarified congressional intent of Title X. It also called for increased funding for Title X programs. The bill cleared the full Energy and Commerce Committee within 2 days. Family planning groups and the Clinton Administration strongly supported this bill. Acting Assistant Secretary for Health opposed a proposed restrictive parental notification amendment, however, which the full committee had defeated (18-25). The bill should have reached the Senate shortly after the winter recess. Also in February, the Clinton Administration suspended the Title X gag rule regulations implemented during the waning days of the Reagan Administration and upheld during the waning days of the Reagan Administration and upheld during the Bush Administration. The Clinton Administration did so by issuing an interim rule which returned oversight of federally supported family planning clinics to previous regulations and policies, e.g., US Department of Health and Human Services (DHHS) 1981 Program Guidelines for Family Planning Projects. It also issued a proposed rule which could become law after a 60-day comment period. Therefore the new rulings required federally supported clinics to provide nondirective counseling and abortion referrals upon request for women with unwanted pregnancies. DHHS' Office for Population Affairs continued to administer the Title X program.

Possible genetic defects in regulation of glycosaminoglycans in patients with diabetic nephropathy

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

Deckert, T.; Horowitz, I.M.; Kofoed-Enevoldsen, A.

1991-06-01

The hypothesis of genetic defects in glycosaminoglycan (GAG) regulation among patients with insulin-dependent diabetes mellitus (IDDM) and nephropathy was assessed by studies in tissue cultures of fibroblasts obtained from 7 patients with normal urinary albumin excretion, 11 patients with diabetic nephropathy, and 6 nondiabetic control subjects. The incorporation of (2H) glucosamine and (35S) sulfate into hyaluronic acid (HA), chondroitin sulfate and dermatan sulfate (CS + DS), and heparan sulfate (HS) was measured in cells, matrix, and medium and related to micrograms of tissue protein. Large interindividual variations were seen in all three groups, and the incorporation of (3H) glucosamine intomore » HA, CS + DS, and HS and (35S) sulfate into CS + DS and HS were not significantly different between the three groups. However, the fractional incorporation of (3H)glucosamine into HS was significantly reduced in diabetic patients with nephropathy compared with control subjects. This was the case not only when related to the total amount of GAGs (P = 0.014) but also when related to HA (P = 0.014). No significant difference was seen between control subjects and normoalbuminuric diabetic patients. The degree of N-sulfation of HS was not significantly different between the experimental groups. The results suggest that patients with diabetic nephropathy may suffer from deficiencies of coordinate regulation in the biosynthesis of GAG in fibroblasts, which may lead to a reduced density of HS in the extracellular matrix. If these changes reflect alterations in the biosynthesis of GAG from endothelial, myomedial, and mesangial cells, this observation may be relevant for the pathogenesis of severe diabetic complications.« less

Electrostatic interactions and binding orientation of HIV-1 matrix studied by neutron reflectivity.

PubMed

Nanda, Hirsh; Datta, Siddhartha A K; Heinrich, Frank; Lösche, Mathias; Rein, Alan; Krueger, Susan; Curtis, Joseph E

2010-10-20

The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1 maturation inhibitor bevirimat.

PubMed

Adamson, Catherine S; Sakalian, Michael; Salzwedel, Karl; Freed, Eric O

2010-04-20

The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient's failure to respond correlated with baseline polymorphisms at SP1 residues 6-8. In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance, and SP1-V7M and T8Delta mutations conferred intermediate levels of BVM resistance. Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.

Matrix product operators, matrix product states, and ab initio density matrix renormalization group algorithms

NASA Astrophysics Data System (ADS)

Chan, Garnet Kin-Lic; Keselman, Anna; Nakatani, Naoki; Li, Zhendong; White, Steven R.

2016-07-01

Current descriptions of the ab initio density matrix renormalization group (DMRG) algorithm use two superficially different languages: an older language of the renormalization group and renormalized operators, and a more recent language of matrix product states and matrix product operators. The same algorithm can appear dramatically different when written in the two different vocabularies. In this work, we carefully describe the translation between the two languages in several contexts. First, we describe how to efficiently implement the ab initio DMRG sweep using a matrix product operator based code, and the equivalence to the original renormalized operator implementation. Next we describe how to implement the general matrix product operator/matrix product state algebra within a pure renormalized operator-based DMRG code. Finally, we discuss two improvements of the ab initio DMRG sweep algorithm motivated by matrix product operator language: Hamiltonian compression, and a sum over operators representation that allows for perfect computational parallelism. The connections and correspondences described here serve to link the future developments with the past and are important in the efficient implementation of continuing advances in ab initio DMRG and related algorithms.

Matrix product operators, matrix product states, and ab initio density matrix renormalization group algorithms.

PubMed

Chan, Garnet Kin-Lic; Keselman, Anna; Nakatani, Naoki; Li, Zhendong; White, Steven R

2016-07-07

Current descriptions of the ab initio density matrix renormalization group (DMRG) algorithm use two superficially different languages: an older language of the renormalization group and renormalized operators, and a more recent language of matrix product states and matrix product operators. The same algorithm can appear dramatically different when written in the two different vocabularies. In this work, we carefully describe the translation between the two languages in several contexts. First, we describe how to efficiently implement the ab initio DMRG sweep using a matrix product operator based code, and the equivalence to the original renormalized operator implementation. Next we describe how to implement the general matrix product operator/matrix product state algebra within a pure renormalized operator-based DMRG code. Finally, we discuss two improvements of the ab initio DMRG sweep algorithm motivated by matrix product operator language: Hamiltonian compression, and a sum over operators representation that allows for perfect computational parallelism. The connections and correspondences described here serve to link the future developments with the past and are important in the efficient implementation of continuing advances in ab initio DMRG and related algorithms.

Membrane culture and reduced oxygen tension enhances cartilage matrix formation from equine cord blood mesenchymal stromal cells in vitro.

PubMed

Co, C; Vickaryous, M K; Koch, T G

2014-03-01

Ongoing research is aimed at increasing cartilage tissue yield and quality from multipotent mesenchymal stromal cells (MSC) for the purpose of treating cartilage damage in horses. Low oxygen culture has been shown to enhance chondrogenesis, and novel membrane culture has been proposed to increase tissue yield and homogeneity. The objective of this study was to evaluate and compare the effect of reduced oxygen and membrane culture during in vitro chondrogenesis of equine cord blood (CB) MSC. CB-MSC (n = 5 foals) were expanded at 21% oxygen prior to 3-week differentiation in membrane or pellet culture at 5% and 21% oxygen. Assessment included histological examination (H&E, toluidine Blue, immunohistochemistry (IHC) for collagen type I and II), protein quantification by hydroxyproline assay and dimethylmethylene assay, and mRNA analysis for collagen IA1, collagen IIA1, collagen XA1, HIF1α and Sox9. Among treatment groups, 5% membrane culture produced neocartilage most closely resembling hyaline cartilage. Membrane culture resulted in increased wet mass, homogenous matrix morphology and an increase in total collagen content, while 5% oxygen culture resulted in higher GAG and type II collagen content. No significant differences were observed for mRNA analysis. Membrane culture at 5% oxygen produces a comparatively larger amount of higher quality neocartilage. Matrix homogeneity is attributed to a uniform diffusion gradient and reduced surface tension. Membrane culture holds promise for scale-up for therapeutic purposes, for cellular preconditioning prior to cytotherapeutic applications, and for modeling system for gas-dependent chondrogenic differentiation studies. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Characterization of mesenchymal stem cells and fibrochondrocytes in three-dimensional co-culture: analysis of cell shape, matrix production, and mechanical performance.

PubMed

McCorry, Mary Clare; Puetzer, Jennifer L; Bonassar, Lawrence J

2016-03-12

Bone marrow mesenchymal stem cells (MSCs) have shown positive therapeutic effects for meniscus regeneration and repair. Preliminary in vitro work has indicated positive results for MSC applications for meniscus tissue engineering; however, more information is needed on how to direct MSC behavior. The objective of this study was to examine the effect of MSC co-culture with primary meniscal fibrochondrocytes (FCCs) in a three-dimensional collagen scaffold in fibrochondrogenic media. Co-culture of MSCs and FCCs was hypothesized to facilitate the transition of MSCs to a FCC cell phenotype as measured by matrix secretion and morphology. MSCs and FCCs were isolated from bovine bone marrow and meniscus, respectively. Cells were seeded in a 20 mg/mL high-density type I collagen gel at MSC:FCC ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs were cultured for up to 2 weeks and then analyzed for cell morphology, glycosaminoglycan content, collagen content, and production of collagen type I, II, and X. Cells were homogeneously mixed throughout the scaffold and cells had limited direct cell-cell contact. After 2 weeks in culture, MSCs transitioned from a spindle-like morphology toward a rounded phenotype, while FCCs remained rounded throughout culture. Although MSC shape changed with culture, the overall size was significantly larger than FCCs throughout culture. While 75:25 and 100:0 (MSC mono-culture) culture groups produced significantly more glycosaminoglycan (GAG)/DNA than FCCs in mono-culture, GAG retention was highest in 50:50 co-cultures. Similarly, the aggregate modulus was highest in 100:0 and 50:50 co-cultures. All samples contained both collagen types I and II after 2 weeks, and collagen type X expression was evident only in MSC mono-culture gels. MSCs shift to a FCC morphology in both mono- and co-culture. Co-culture reduced hypertrophy by MSCs, indicated by collagen type X. This study shows that MSC phenotype can be influenced by indirect homogeneous

Colorimetric molecular diagnosis of the HIV gag gene using DNAzyme and a complementary DNA-extended primer.

PubMed

Kim, Seong U; Batule, Bhagwan S; Mun, Hyoyoung; Byun, Ju-Young; Shim, Won-Bo; Kim, Min-Gon

2018-02-07

We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H 2 O 2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection

PubMed Central

Asher, Tedi E.; Wilson, Nancy A.; Nason, Martha C.; Brenchley, Jason M.; Metzler, Ian S.; Venturi, Vanessa; Gostick, Emma; Chattopadhyay, Pratip K.; Roederer, Mario; Davenport, Miles P.; Watkins, David I.; Douek, Daniel C.

2009-01-01

Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8+ T cell populations in Mamu-A*01+ rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR β-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8+ T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181–189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8+ T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection. PMID:19349463

Corneal Sulfated Glycosaminoglycans and Their Effects on Trigeminal Nerve Growth Cone Behavior In Vitro: Roles for ECM in Cornea Innervation

PubMed Central

Schwend, Tyler; Deaton, Ryan J.; Zhang, Yuntao; Caterson, Bruce; Conrad, Gary W.

2012-01-01

Purpose. Sensory trigeminal nerve growth cones innervate the cornea in a highly coordinated fashion. The purpose of this study was to determine if extracellular matrix glycosaminoglycans (ECM–GAGs), including keratan sulfate (KS), dermatan sulfate (DS), and chondroitin sulfate A (CSA) and C (CSC), polymerized in developing eyefronts, may provide guidance cues to nerves during cornea innervation. Methods. Immunostaining using antineuron-specific-β-tubulin and monoclonal antibodies for KS, DS, and CSA/C was performed on eyefronts from embryonic day (E) 9 to E14 and staining visualized by confocal microscopy. Effects of purified GAGs on trigeminal nerve growth cone behavior were tested using in vitro neuronal explant cultures. Results. At E9 to E10, nerves exiting the pericorneal nerve ring grew as tight fascicles, advancing straight toward the corneal stroma. In contrast, upon entering the stroma, nerves bifurcated repeatedly as they extended anteriorly toward the epithelium. KS was localized in the path of trigeminal nerves, whereas DS and CSA/C–rich areas were avoided by growth cones. When E10 trigeminal neurons were cultured on different substrates comprised of purified GAG molecules, their neurite growth cone behavior varied depending on GAG type, concentration, and mode of presentation (immobilized versus soluble). High concentrations of immobilized KS, DS, and CSA/C inhibited neurite growth to varying degrees. Neurites traversing lower, permissive concentrations of immobilized DS and CSA/C displayed increased fasciculation and decreased branching, whereas KS caused decreased fasciculation and increased branching. Enzymatic digestion of sulfated GAGs canceled their effects on trigeminal neurons. Conclusions. Data herein suggest that GAGs may direct the movement of trigeminal nerve growth cones innervating the cornea. PMID:23132805

Ceramic matrix composite article and process of fabricating a ceramic matrix composite article

DOEpatents

Cairo, Ronald Robert; DiMascio, Paul Stephen; Parolini, Jason Robert

2016-01-12

A ceramic matrix composite article and a process of fabricating a ceramic matrix composite are disclosed. The ceramic matrix composite article includes a matrix distribution pattern formed by a manifold and ceramic matrix composite plies laid up on the matrix distribution pattern, includes the manifold, or a combination thereof. The manifold includes one or more matrix distribution channels operably connected to a delivery interface, the delivery interface configured for providing matrix material to one or more of the ceramic matrix composite plies. The process includes providing the manifold, forming the matrix distribution pattern by transporting the matrix material through the manifold, and contacting the ceramic matrix composite plies with the matrix material.

Recombinant yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 gag induces SIV-specific CD8+ T-cell responses in rhesus macaques.

PubMed

Bonaldo, Myrna C; Martins, Mauricio A; Rudersdorf, Richard; Mudd, Philip A; Sacha, Jonah B; Piaskowski, Shari M; Costa Neves, Patrícia C; Veloso de Santana, Marlon G; Vojnov, Lara; Capuano, Saverio; Rakasz, Eva G; Wilson, Nancy A; Fulkerson, John; Sadoff, Jerald C; Watkins, David I; Galler, Ricardo

2010-04-01

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8(+) T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8(+) T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8(+) T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4(+) T cells.

Matrix management in hospitals: testing theories of matrix structure and development.

PubMed

Burns, L R

1989-09-01

A study of 315 hospitals with matrix management programs was used to test several hypotheses concerning matrix management advanced by earlier theorists. The study verifies that matrix management involves several distinctive elements that can be scaled to form increasingly complex types of lateral coordinative devices. The scalability of these elements is evident only cross-sectionally. The results show that matrix complexity is not an outcome of program age, nor does matrix complexity at the time of implementation appear to influence program survival. Matrix complexity, finally, is not determined by the organization's task diversity and uncertainty. The results suggest several modifications in prevailing theories of matrix organization.

Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo.

PubMed

Yang, Yuanheng; Lin, Hang; Shen, He; Wang, Bing; Lei, Guanghua; Tuan, Rocky S

2018-03-15

Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for

Synthesis of glycosaminoglycans by undifferentiated and differentiated HT29 human colonic cancer cells.

PubMed

Simon-Assmann, P; Bouziges, F; Daviaud, D; Haffen, K; Kedinger, M

1987-08-15

Among the extracellular matrix components which have been suggested to be involved in developmental and neoplastic changes are glycosaminoglycans (GAGs). To try to correlate their amount and nature with the process of enterocytic differentiation, we studied glycosaminoglycan synthesis of human colonic adenocarcinoma cells (HT29 cell line) by [3H]glucosamine and [35S]sulfate incorporation. Enterocytic differentiation of the cells obtained in a sugar-free medium (for review, see A. Zweibaum et al. In: Handbook of Physiology. Intestinal Transport of the Gastrointestinal System, in press, 1987) resulted in a marked increase in total incorporation of labeled precursors (20-fold for [3H]glucosamine, 4.5-fold for [35S]sulfate) as well as in uronic acid content (5-fold); most of the synthesized GAGs were found associated with the cell pellet. Chromatographic and electrophoretic analysis of the labeled GAGs revealed that undifferentiated cells synthesized and secreted hyaluronic acid, heparan sulfate, and one class of chondroitin sulfate. Differentiation of HT29 cells because associated with the synthesis of an additional class of chondroitin sulfate (CS4) concomitant to a decrease in heparan sulfate which is no longer found secreted in the medium. Furthermore, the charge density of this latter GAG component varied as assessed by a shift of its affinity on ion-exchange chromatography.

AKT capture by feline leukemia virus.

PubMed

Kawamura, Maki; Umehara, Daigo; Odahara, Yuka; Miyake, Ariko; Ngo, Minh Ha; Ohsato, Yoshiharu; Hisasue, Masaharu; Nakaya, Masa-Aki; Watanabe, Shinya; Nishigaki, Kazuo

2017-04-01

Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called "oncogene capture". The captured cellular genes, referred to as "v-onc" genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated "FeLV-AKT", that has captured feline c-AKT1 in feline lymphoma. FeLV-AKT contains a gag-AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

Chondroprotective effect of zinc oxide nanoparticles in conjunction with hypoxia on bovine cartilage-matrix synthesis.

PubMed

Mirza, Eraj Humayun; Pan-Pan, Chong; Wan Ibrahim, Wan Mohd Azhar Bin; Djordjevic, Ivan; Pingguan-Murphy, Belinda

2015-11-01

Articular cartilage is a tissue specifically adapted to a specific niche with a low oxygen tension (hypoxia), and the presence of such conditions is a key factor in regulating growth and survival of chondrocytes. Zinc deficiency has been linked to cartilage-related disease, and presence of Zinc is known to provide antibacterial benefits, which makes its inclusion attractive in an in vitro system to reduce infection. Inclusion of 1% zinc oxide nanoparticles (ZnONP) in poly octanediol citrate (POC) polymer cultured in hypoxia has not been well determined. In this study we investigated the effects of ZnONP on chondrocyte proliferation and matrix synthesis cultured under normoxia (21% O2 ) and hypoxia (5% O2 ). We report an upregulation of chondrocyte proliferation and sulfated glycosaminoglycan (S-GAG) in hypoxic culture. Results demonstrate a synergistic effect of oxygen concentration and 1% ZnONP in up-regulation of anabolic gene expression (Type II collagen and aggrecan), and a down regulation of catabolic (MMP-13) gene expression. Furthermore, production of transcription factor hypoxia-inducible factor 1A (HIF-1A) in response to hypoxic condition to regulate chondrocyte survival under hypoxia is not affected by the presence of 1% ZnONP. Presence of 1% ZnONP appears to act to preserve homeostasis of cartilage in its hypoxic environment. © 2015 Wiley Periodicals, Inc.

BASIC Matrix Operations.

ERIC Educational Resources Information Center

Digital Equipment Corp., Maynard, MA.

The curriculum materials and computer programs in this booklet introduce the idea of a matrix. They go on to discuss matrix operations of addition, subtraction, multiplication by a scalar, and matrix multiplication. The last section covers several contemporary applications of matrix multiplication, including problems of communication…

The Mexico City Policy: a "gag rule" that violates free speech and democratic values.

PubMed

Cohen, S A

1998-04-01

With the support of the Republican leadership of the US House of Representatives, Representative Chris Smith of New Jersey continues to attempt to limit provision of US family planning (FP) funding to foreign nongovernmental organizations (NGOs) who agree to enforce absolute prohibitions on engaging in abortion-related activities. Despite the fact that the House is withholding funds to meet US obligations to the UN and to support the International Monetary Fund, US President Clinton refuses to give in to pressure to enforce what amounts to a "gag rule." The Secretary of State explains that Clinton has no intention of punishing organizations engaging in free-speech protected democratic activities in foreign countries. Smith has offered a self-styled "compromise" that would allow Clinton to waive disqualification for a foreign NGO offering abortion services with its own funds in compliance with the laws of its country. This waiver would penalize the international FP program by $44 million and would not apply to the broad prohibitions against abortion lobbying (including lobbying for changes in laws, sponsoring conferences and workshops on "alleged" defects in abortion laws, and drafting and distributing materials or public statements on "alleged defects"). Smith's emphasis on the "lobbying" ban has widened the issue from a debate on abortion to a debate on democracy and free speech. Opposition to Smith's proposal is, thus, rising in many quarters. Smith also endorses withholding US contributions to the UN Population Fund because the fund has resumed work in China.

Ceramic matrix and resin matrix composites: A comparison

NASA Technical Reports Server (NTRS)

Hurwitz, Frances I.

1987-01-01

The underlying theory of continuous fiber reinforcement of ceramic matrix and resin matrix composites, their fabrication, microstructure, physical and mechanical properties are contrasted. The growing use of organometallic polymers as precursors to ceramic matrices is discussed as a means of providing low temperature processing capability without the fiber degradation encountered with more conventional ceramic processing techniques. Examples of ceramic matrix composites derived from particulate-filled, high char yield polymers and silsesquioxane precursors are provided.

Ceramic matrix and resin matrix composites - A comparison

NASA Technical Reports Server (NTRS)

Hurwitz, Frances I.

1987-01-01

The underlying theory of continuous fiber reinforcement of ceramic matrix and resin matrix composites, their fabrication, microstructure, physical and mechanical properties are contrasted. The growing use of organometallic polymers as precursors to ceramic matrices is discussed as a means of providing low temperature processing capability without the fiber degradation encountered with more conventional ceramic processing techniques. Examples of ceramic matrix composites derived from particulate-filled, high char yield polymers and silsesquioxane precursors are provided.

Results of sub-analysis of a phase 2 study on trabectedin treatment for extraskeletal myxoid chondrosarcoma and mesenchymal chondrosarcoma.

PubMed

Morioka, Hideo; Takahashi, Shunji; Araki, Nobuhito; Sugiura, Hideshi; Ueda, Takafumi; Takahashi, Mitsuru; Yonemoto, Tsukasa; Hiraga, Hiroaki; Hiruma, Toru; Kunisada, Toshiyuki; Matsumine, Akihiko; Susa, Michiro; Nakayama, Robert; Nishimoto, Kazumasa; Kikuta, Kazutaka; Horiuchi, Keisuke; Kawai, Akira

2016-07-14

Trabectedin is reported to be particularly effective against translocation-related sarcoma. Recently, a randomized phase 2 study in patients with translocation-related sarcomas unresponsive or intolerable to standard chemotherapy was conducted, which showed clinical benefit of trabectedin compared with best supportive care (BSC). Extraskeletal myxoid chondrosarcoma (EMCS) and Mesenchymal chondrosarcoma (MCS) are very rare malignant soft tissue sarcomas, and are associated with translocations resulting in fusion genes. In addition, the previous in vivo data showed that trabectedin affect tumor necrosis and reduction in vascularization in a xenograft model of a human high-grade chondrosarcoma. The aim of the present analysis was to clarify the efficacy of trabectedin for EMCS and MCS subjects in the randomized phase 2 study. Five subjects with EMCS and MCS received trabectedin treatment in the randomized phase 2 study. Three MCS subjects were allocated to the BSC group. Objective response and progression-free survival (PFS) were assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 by central radiology imaging review. The median follow-up time of the randomized phase 2 study was 22.7 months, and one subject with MCS was still receiving trabectedin treatment at the final data cutoff. The median PFS was 12.5 months (95 % CI: 7.4-not reached) in the trabectedin group, while 1.0 months (95 % CI: 0.3-1.0 months) in MCS subjects of the BSC group. The six-month progression-free rate was 100 % in the trabectedin group. One subject with MCS showed partial response, and the others in the trabectedin group showed stable disease. Overall survival of EMCS and MCS subjects was 26.4 months (range, 10.4-26.4 months) in the trabectedin group. At the final data cutoff, two of five subjects were still alive. This sub-analysis shows that trabectedin is effective for patients with EMCS and MCS compared with BSC. The efficacy results were

Hybrid matrix fiber composites

DOEpatents

Deteresa, Steven J.; Lyon, Richard E.; Groves, Scott E.

2003-07-15

Hybrid matrix fiber composites having enhanced compressive performance as well as enhanced stiffness, toughness and durability suitable for compression-critical applications. The methods for producing the fiber composites using matrix hybridization. The hybrid matrix fiber composites include two chemically or physically bonded matrix materials, whereas the first matrix materials are used to impregnate multi-filament fibers formed into ribbons and the second matrix material is placed around and between the fiber ribbons that are impregnated with the first matrix material and both matrix materials are cured and solidified.

Biofilm Matrix Proteins.

PubMed

Fong, Jiunn N C; Yildiz, Fitnat H

2015-04-01

Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins, and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this article, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation.

Incidence of the Hb E [β26(B8)Glu→Lys, GAG>AAG] variant in Totos, one of the smallest primitive tribes in the world.

PubMed

Bhattacharyya, Deboshree; Mukhopadhyay, Ashis; Chakraborty, Abhijit; Dasgupta, Swati; Mukhopadhyay, Soma; Pal, Nabamita; Basak, Jayasri

2013-01-01

Toto is one of the smallest tribes in the world. This primitive sub Himalayan, endogamous tribe lives in a small, isolated village called Totopara in the Jalpaiguri district of West Bengal in India. The tribal communities of West Bengal are vulnerable to various genetic disorders such as β-thalassemia (β-thal). We have studied 443 Totos to define their Hb E [β26(B8)Glu→Lys, GAG>AAG] status. Awareness and screening camps have been organized in various parts of Totopara during the last 2 years. We collected 3 mL peripheral blood from each individual aseptically on which to use the naked eye single tube red cell osmotic fragility test (NESTROFT); complete hemogram and high performance liquid chromatography (HPLC) were done to detect their carrier status. The Hb E variant had been found to be prevalent among the Totos. To confirm the codon 26 (GAG>AAG) mutation in the β-globin gene, amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed. Restriction fragment length polymorphism (RFLP)-PCR was carried out with 44 Hb E alleles to construct the haplotype(s) of the Totos. Our extensive studies have revealed that 49.21% of Totos are Hb E heterozygotes and 19.19% Totos are Hb E homozygotes. The most prevalent haplotype linked with the codon 26 mutation in the Totos is [+ - - - - -] (HincII 5'ϵ, HindIII (G)γ, HindIII (A)γ, HincII 5'ψβ, HincII 3'ψβ and HinfI 3'β). Consanguineous marriages have resulted in a significant increase of the percentages of heterozygotes and homozygotes of Hb E in the Totos. Genetic counseling is essential and important to prevent the spread of this mutation and hence to save them from having any kind of clinically significant hemoglobinopathy in the future.

Matrix diffusion coefficients in volcanic rocks at the Nevada test site: influence of matrix porosity, matrix permeability, and fracture coating minerals.

PubMed

Reimus, Paul W; Callahan, Timothy J; Ware, S Doug; Haga, Marc J; Counce, Dale A

2007-08-15

Diffusion cell experiments were conducted to measure nonsorbing solute matrix diffusion coefficients in forty-seven different volcanic rock matrix samples from eight different locations (with multiple depth intervals represented at several locations) at the Nevada Test Site. The solutes used in the experiments included bromide, iodide, pentafluorobenzoate (PFBA), and tritiated water ((3)HHO). The porosity and saturated permeability of most of the diffusion cell samples were measured to evaluate the correlation of these two variables with tracer matrix diffusion coefficients divided by the free-water diffusion coefficient (D(m)/D*). To investigate the influence of fracture coating minerals on matrix diffusion, ten of the diffusion cells represented paired samples from the same depth interval in which one sample contained a fracture surface with mineral coatings and the other sample consisted of only pure matrix. The log of (D(m)/D*) was found to be positively correlated with both the matrix porosity and the log of matrix permeability. A multiple linear regression analysis indicated that both parameters contributed significantly to the regression at the 95% confidence level. However, the log of the matrix diffusion coefficient was more highly-correlated with the log of matrix permeability than with matrix porosity, which suggests that matrix diffusion coefficients, like matrix permeabilities, have a greater dependence on the interconnectedness of matrix porosity than on the matrix porosity itself. The regression equation for the volcanic rocks was found to provide satisfactory predictions of log(D(m)/D*) for other types of rocks with similar ranges of matrix porosity and permeability as the volcanic rocks, but it did a poorer job predicting log(D(m)/D*) for rocks with lower porosities and/or permeabilities. The presence of mineral coatings on fracture walls did not appear to have a significant effect on matrix diffusion in the ten paired diffusion cell experiments.

Matrix diffusion coefficients in volcanic rocks at the Nevada test site: Influence of matrix porosity, matrix permeability, and fracture coating minerals

NASA Astrophysics Data System (ADS)

Reimus, Paul W.; Callahan, Timothy J.; Ware, S. Doug; Haga, Marc J.; Counce, Dale A.

2007-08-01

Diffusion cell experiments were conducted to measure nonsorbing solute matrix diffusion coefficients in forty-seven different volcanic rock matrix samples from eight different locations (with multiple depth intervals represented at several locations) at the Nevada Test Site. The solutes used in the experiments included bromide, iodide, pentafluorobenzoate (PFBA), and tritiated water ( 3HHO). The porosity and saturated permeability of most of the diffusion cell samples were measured to evaluate the correlation of these two variables with tracer matrix diffusion coefficients divided by the free-water diffusion coefficient ( Dm/ D*). To investigate the influence of fracture coating minerals on matrix diffusion, ten of the diffusion cells represented paired samples from the same depth interval in which one sample contained a fracture surface with mineral coatings and the other sample consisted of only pure matrix. The log of ( Dm/ D*) was found to be positively correlated with both the matrix porosity and the log of matrix permeability. A multiple linear regression analysis indicated that both parameters contributed significantly to the regression at the 95% confidence level. However, the log of the matrix diffusion coefficient was more highly-correlated with the log of matrix permeability than with matrix porosity, which suggests that matrix diffusion coefficients, like matrix permeabilities, have a greater dependence on the interconnectedness of matrix porosity than on the matrix porosity itself. The regression equation for the volcanic rocks was found to provide satisfactory predictions of log( Dm/ D*) for other types of rocks with similar ranges of matrix porosity and permeability as the volcanic rocks, but it did a poorer job predicting log( Dm/ D*) for rocks with lower porosities and/or permeabilities. The presence of mineral coatings on fracture walls did not appear to have a significant effect on matrix diffusion in the ten paired diffusion cell experiments.

Effect of proteoglycans at interfaces as related to location, architecture, and mechanical cues

DOE PAGES

Kurylo, Michael P.; Grandfield, Kathryn; Marshall, Grayson W.; ...

2015-12-03

Covalently bound functional GAGs orchestrate tissue mechanics through time-dependent characteristics. The role of specific glycosaminoglycans (GAGs) at the ligament-cementum and cementum-dentin interfaces within a human periodontal complex were examined. Matrix swelling and resistance to compression under health and modeled diseased states was investigated. The presence of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs at the ligament-cementum and cementum-dentin interfaces in human molars (N = 5) was illustrated by using enzymes, atomic force microscopy (AFM), and AFM-based nanoindentation. Furthermore, the change in physical characteristics of modeled diseased states through sequential digestion of keratin sulfate (KS) and chondroitin sulfate (CS) GAGsmore » was investigated. One-way ANOVA tests with P < 0.05 were performed to determine significant differences between groups. Additionally, the presence of mineral within the seemingly hygroscopic interfaces was investigated using transmission electron microscopy. Immunohistochemistry (N = 3) indicated presence of biglycan and fibromodulin small leucine rich proteoglycans at the interfaces. Digestion of matrices with enzymes confirmed the presence of KS and CS GAGs at the interfaces by illustrating a change in ti ssue architecture and mechanics. A significant increase in height (nm), decrease in elastic modulus (GPa), and tissue deformation rate (nm/s) of the PDL-C attachment site (215 ± 63-424 ± 94 nm; 1.5 ± 0.7-0.4 ± 0.2 GPa; 21 ± 7-48 ± 22 nm/s), and cementum-dentin interface (122 ± 69-360 ± 159 nm; 2.9 ± 1.3-0.7 ± 0.3 GPa; 18 ± 4-30 ± 6 nm/s) was observed. The sequential removal of GAGs indicated loss in intricate structural hierarchy of hygroscopic interfaces. From a mechanics perspective, GAGs provide tissue recovery/resilience. Our results provide insights into the role of GAGs toward conserved tooth movement in the socket in response to mechanical loads, and modulation of potentially

Glycosaminoglycan-functionalized poly-lactide-co-glycolide nanoparticles: synthesis, characterization, cytocompatibility, and cellular uptake

PubMed Central

Lamichhane, Surya P; Arya, Neha; Ojha, Nirdesh; Kohler, Esther; Shastri, V Prasad

2015-01-01

The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP)-based delivery systems remains a significant challenge. This is compounded by the fact that the tumor is highly dynamic and complex environment composed of a plurality of cell types and extracellular matrix. Since glycosaminoglycan (GAG) production is altered in many diseases (or pathologies), NPs bearing GAG moieties on the surface may confer some unique advantages in interrogating the tumor microenvironment. In order to explore this premise, in the study reported here poly-lactide-co-glycolide (PLGA) NPs in the range of 100–150 nm bearing various proteoglycans were synthesized by a single-step nanoprecipitation and characterized. The surface functionalization of the NPs with GAG moieties was verified using zeta potential measurements and X-ray photoelectron spectroscopy. To establish these GAG-bearing NPs as carriers of therapeutics, cellular toxicity assays were undertaken in lung epithelial adenocarcinoma (A549) cells, human pulmonary microvascular endothelial cells (HPMEC), and renal proximal tubular epithelial cells. In general NPs were well tolerated over a wide concentration range (100–600 μg/mL) by all cell types and were taken up to appreciable extents without any adverse cell response in A549 cells and HPMEC. Further, GAG-functionalized PLGA NPs were taken up to different extents in A459 cells and HPMEC. In both cell systems, the uptake of heparin-modified NPs was diminished by 50%–65% in comparison to that of unmodified PLGA. Interestingly, the uptake of chondroitin sulfate NPs was the highest in both cell systems with 40%–60% higher uptake when compared with that of PLGA, and this represented an almost twofold difference over heparin-modified NPs. These findings suggest that GAG modification can be explored as means of changing the uptake behavior of PLGA NPs and these NP systems have potential in cancer therapy. PMID:25632234

Brief announcement: Hypergraph parititioning for parallel sparse matrix-matrix multiplication

DOE PAGES

Ballard, Grey; Druinsky, Alex; Knight, Nicholas; ...

2015-01-01

The performance of parallel algorithms for sparse matrix-matrix multiplication is typically determined by the amount of interprocessor communication performed, which in turn depends on the nonzero structure of the input matrices. In this paper, we characterize the communication cost of a sparse matrix-matrix multiplication algorithm in terms of the size of a cut of an associated hypergraph that encodes the computation for a given input nonzero structure. Obtaining an optimal algorithm corresponds to solving a hypergraph partitioning problem. Furthermore, our hypergraph model generalizes several existing models for sparse matrix-vector multiplication, and we can leverage hypergraph partitioners developed for that computationmore » to improve application-specific algorithms for multiplying sparse matrices.« less

Nanocrystal doped matrixes

DOEpatents

Parce, J. Wallace; Bernatis, Paul; Dubrow, Robert; Freeman, William P.; Gamoras, Joel; Kan, Shihai; Meisel, Andreas; Qian, Baixin; Whiteford, Jeffery A.; Ziebarth, Jonathan

2010-01-12

Matrixes doped with semiconductor nanocrystals are provided. In certain embodiments, the semiconductor nanocrystals have a size and composition such that they absorb or emit light at particular wavelengths. The nanocrystals can comprise ligands that allow for mixing with various matrix materials, including polymers, such that a minimal portion of light is scattered by the matrixes. The matrixes of the present invention can also be utilized in refractive index matching applications. In other embodiments, semiconductor nanocrystals are embedded within matrixes to form a nanocrystal density gradient, thereby creating an effective refractive index gradient. The matrixes of the present invention can also be used as filters and antireflective coatings on optical devices and as down-converting layers. Processes for producing matrixes comprising semiconductor nanocrystals are also provided. Nanostructures having high quantum efficiency, small size, and/or a narrow size distribution are also described, as are methods of producing indium phosphide nanostructures and core-shell nanostructures with Group II-VI shells.

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

PubMed

Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

2015-03-01

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. Copyright © 2014 Elsevier B.V. All rights reserved.

Altered mechano-chemical environment in hip articular cartilage: effect of obesity.

PubMed

Travascio, Francesco; Eltoukhy, Moataz; Cami, Sonila; Asfour, Shihab

2014-10-01

The production of extracellular matrix (ECM) components of articular cartilage is regulated, among other factors, by an intercellular signaling mechanism mediated by the interaction of cell surface receptors (CSR) with insulin-like growth factor-1 (IGF-1). In ECM, the presence of binding proteins (IGFBP) hinders IGF-1 delivery to CSR. It has been reported that levels of IGF-1 and IGFBP in obese population are, respectively, lower and higher than those found in normal population. In this study, an experimental-numerical approach was adopted to quantify the effect of this metabolic alteration found in obese population on the homeostasis of femoral hip cartilage. A new computational model, based on the mechano-electrochemical mixture theory, was developed to describe competitive binding kinetics of IGF-1 with IGFBP and CSR, and associated glycosaminoglycan (GAG) biosynthesis. Moreover, a gait analysis was carried out on obese and normal subjects to experimentally characterize mechanical loads on hip cartilage during walking. This information was deployed into the model to account for effects of physiologically relevant tissue deformation on GAG production in ECM. Numerical simulations were performed to compare GAG biosynthesis in femoral hip cartilage of normal and obese subjects. Results indicated that the lower ratio of IGF-1 to IGFBP found in obese population reduces cartilage GAG concentration up to 18 % when compared to normal population. Moreover, moderate physical activity, such as walking, has a modest beneficial effect on GAG production. The findings of this study suggest that IGF-1/IGFBP metabolic unbalance should be accounted for when considering the association of obesity with hip osteoarthritis.

Chondromyxoid Fibroma of the Ethmoid Sinus

DTIC Science & Technology

2011-06-01

and its perceptible recurrence rate. It is for these reasons that CMF should be distinguished from a myxoid variant of chondrosarcoma (which usually...cartilage of the type seen in chondroma or chondrosarcoma and is a focal finding in less than 20% of CMF cases [5]. Mitotic figures are sparse, accounting...CMF from chondrosarcoma [5]. The histologic differential diagnosis of CMF includes, most importantly, myxoid chondrosarcoma and chondro- blastoma [7

Double heterozygosity for Hb New York [beta 113 GTG-->GAG; VAL-->GLU] and beta degrees-thalassemia mutations manifests as a thalassemia trait.

PubMed

Lee, Anselm C W; Ma, Edmond S K; Chan, Amy Y Y; Szeto, S C; Chan, L C

2008-01-01

An extended family with three individuals affected by two different forms of double heterozygosity for beta-thalassemia and Hb New York is reported. Double heterozygosity of Hb New York [beta 113 GTG-->GAG; VAL-->GLU] and beta degrees codon 17 was detected in a fetus following prenatal screening for thalassemia. The father and a paternal aunt were also found to be heterozygous for Hb New York and beta degrees IVSII-654. Both adults had clinical and hematological features consistent with beta-thalassemia trait. The affected child was followed up after birth and manifested the typical course of a thalassemia trait, with no signs of organomegaly or overt hemolysis. Observations strongly suggest that double heterozygosity of Hb New York and beta degrees thalassemia has mild, if any, clinical symptoms, and is not an indication of therapeutic abortion when detected antenatally.

Matrix Interdiction Problem

NASA Astrophysics Data System (ADS)

Kasiviswanathan, Shiva Prasad; Pan, Feng

In the matrix interdiction problem, a real-valued matrix and an integer k is given. The objective is to remove a set of k matrix columns that minimizes in the residual matrix the sum of the row values, where the value of a row is defined to be the largest entry in that row. This combinatorial problem is closely related to bipartite network interdiction problem that can be applied to minimize the probability that an adversary can successfully smuggle weapons. After introducing the matrix interdiction problem, we study the computational complexity of this problem. We show that the matrix interdiction problem is NP-hard and that there exists a constant γ such that it is even NP-hard to approximate this problem within an n γ additive factor. We also present an algorithm for this problem that achieves an (n - k) multiplicative approximation ratio.

Cellular/intramuscular myxoma and grade I myxofibrosarcoma are characterized by distinct genetic alterations and specific composition of their extracellular matrix

PubMed Central

Willems, Stefan M; Mohseny, Alex B; Balog, Crina; Sewrajsing, Raj; Briaire-de Bruijn, Inge H; Knijnenburg, Jeroen; Cleton-Jansen, Anne-Marie; Sciot, Raf; Fletcher, Christopher D M; Deelder, André M; Szuhai, Karoly; Hensbergen, Paul J; Hogendoorn, Pancras C W

2009-01-01

Cellular myxoma and grade I myxofibrosarcoma are mesenchymal tumours that are characterized by their abundant myxoid extracellular matrix (ECM). Despite their histological overlap, they differ clinically. Diagnosis is therefore difficult though important. We investigated their (cyto) genetics and ECM. GNAS1-activating mutations have been described in intramuscular myxoma, and lead to downstream activation of cFos. KRAS and TP53 mutations are commonly involved in sarcomagenesis whereby KRAS subsequently activates c-Fos. A well-documented series of intramuscular myxoma (three typical cases and seven cases of the more challenging cellular variant) and grade I myxofibrosarcoma (n= 10) cases were karyotyped, analyzed for GNAS1, KRAS and TP53 mutations and downstream activation of c-Fos mRNA and protein expression. ECM was studied by liquid chromatography mass spectrometry and expression of proteins identified was validated by immunohistochemistry and qPCR. Grade I myxofibrosarcoma showed variable, non-specific cyto-genetic aberrations in 83,5% of cases (n= 6) whereas karyotypes of intramuscular myxoma were all normal (n= 7). GNAS1-activating mutations were exclusively found in 50% of intramuscular myxoma. Both tumour types showed over-expression of c-Fos mRNA and protein. No mutations in KRAS codon 12/13 or in TP53 were detected. Liquid chromatography mass spectrometry revealed structural proteins (collagen types I, VI, XII, XIV and decorin) in grade I myxofibrosarcoma lacking in intramuscular myxoma. This was confirmed by immunohistochemistry and qPCR. Intramuscular/cellular myxoma and grade I myxofibrosarcoma show different molecular genetic aberrations and different composition of their ECM that probably contribute to their diverse clinical behaviour. GNAS1 mutation analysis can be helpful to distinguish intramuscular myxoma from grade I myxofibrosarcoma in selected cases. PMID:19320777

Alterations of overused supraspinatus tendon: a possible role of glycosaminoglycans and HARP/pleiotrophin in early tendon pathology.

PubMed

Attia, Mohamed; Scott, Alexander; Duchesnay, Arlette; Carpentier, Gilles; Soslowsky, Louis J; Huynh, Minh Bao; Van Kuppevelt, Toin H; Gossard, Camille; Courty, José; Tassoni, Marie-Claude; Martelly, Isabelle

2012-01-01

Supraspinatus tendon overuse injuries lead to significant pain and disability in athletes and workers. Despite the prevalence and high social cost of these injuries, the early pathological events are not well known. We analyzed the potential relation between glycosaminoglycan (GAG) composition and phenotypic cellular alteration using a rat model of rotator cuff overuse. Total sulfated GAGs increased after 4 weeks of overuse and remained elevated up to 16 weeks. GAG accumulation was preceded by up-regulation of decorin, versican, and aggrecan proteoglycans (PGs) mRNAs and proteins and biglycan PG mRNA after 2 weeks. At 2 weeks, collagen 1 transcript decreased whereas mRNAs for collagen 2, collagen 3, collagen 6, and the transcription factor Sox9 were increased. Protein levels of heparin affine regulatory peptide (HARP)/pleiotrophin, a cytokine known to regulate developmental chondrocyte formation, were enhanced especially at 4 weeks, without up-regulation of HARP/pleiotrophin mRNA. Further results suggest that the increased GAGs present in early lesions may sequester HARP/pleiotrophin, which could contribute to a loss of tenocyte's phenotype. All these modifications are characteristic of a shift towards the chondrocyte phenotype. Identification of these early changes in the extra-cellular matrix may help to prevent the progression of the pathology to more disabling, degenerative alterations. Copyright © 2011 Orthopaedic Research Society.

Effectiveness of metal matrix and ceramic matrix composites as orbital debris shield materials

NASA Technical Reports Server (NTRS)

Mcgill, Preston B.; Mount, Angela R.

1992-01-01

The effectiveness of two metal matrix composites and one ceramic matrix material in defeating hypervelocity impacts at about 3.8 km/s are evaluated to determine the potential of these composites as spacecraft shield materials. The metal matrix composites investigated consist of SiC particles (70 percent by volume) in an aluminum matrix and Al2O3 particles (50 percent by volume) in an Al matrix. The ceramic composite consists of ZrB2 platelets in a ZrC matrix. Both the metal matrix and ceramic matrix composites are found to perform as well or better than 6061-T6 aluminum, which is presently used in the Whipple type bumper shield of Space Station Freedom. Test results indicate that the composites tested may have applications as micrometeoroid/orbital debris shield materials.

Chondroitin sulfates and their binding molecules in the central nervous system.

PubMed

Djerbal, L; Lortat-Jacob, H; Kwok, Jcf

2017-06-01

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in the central nervous system (CNS) matrix. Its sulfation and epimerization patterns give rise to different forms of CS, which enables it to interact specifically and with a significant affinity with various signalling molecules in the matrix including growth factors, receptors and guidance molecules. These interactions control numerous biological and pathological processes, during development and in adulthood. In this review, we describe the specific interactions of different families of proteins involved in various physiological and cognitive mechanisms with CSs in CNS matrix. A better understanding of these interactions could promote a development of inhibitors to treat neurodegenerative diseases.

Modification of natural matrix lac-bagasse for matrix composite films

NASA Astrophysics Data System (ADS)

Nurhayati, Nanik Dwi; Widjaya, Karna; Triyono

2016-02-01

Material technology continues to be developed in order to a material that is more efficient with composite technology is a combination of two or more materials to obtain the desired material properties. The objective of this research was to modification and characterize the natural matrix lac-bagasse as composite films. The first step, natural matrix lac was changed from solid to liquid using an ethanol as a solvent so the matrix homogenly. Natural matrix lac was modified by adding citric acid with concentration variation. Secondly, the bagasse delignification using acid hydrolysis method. The composite films natural matrix lac-bagasse were prepared with optimum modified the addition citric acid 5% (v/v) and delignification bagasse optimum at 1,5% (v/v) in hot press at 80°C 6 Kg/cm-1. Thirdly, composite films without and with modification were characterized functional group analysis using FTIR spectrophotometer and mechanical properties using Universal Testing Machine. The result of research showed natural matrix lac can be modified by reaction with citric acid. FTIR spectra showed without and with modification had functional groups wide absorption 3448 cm-1 group -OH, C=O ester strong on 1712 cm-1 and the methylene group -CH2 on absorption 1465 cm-1. The mechanical properties showed tensile strength 0,55 MPa and elongation at break of 0,95 %. So that composite films natural matrix lac can be made with reinforcement bagasse for material application.

Modeling for Matrix Multicracking Evolution of Cross-ply Ceramic-Matrix Composites Using Energy Balance Approach

NASA Astrophysics Data System (ADS)

Longbiao, Li

2015-12-01

The matrix multicracking evolution of cross-ply ceramic-matrix composites (CMCs) has been investigated using energy balance approach. The multicracking of cross-ply CMCs was classified into five modes, i.e., (1) mode 1: transverse multicracking; (2) mode 2: transverse multicracking and matrix multicracking with perfect fiber/matrix interface bonding; (3) mode 3: transverse multicracking and matrix multicracking with fiber/matrix interface debonding; (4) mode 4: matrix multicracking with perfect fiber/matrix interface bonding; and (5) mode 5: matrix multicracking with fiber/matrix interface debonding. The stress distributions of four cracking modes, i.e., mode 1, mode 2, mode 3 and mode 5, are analysed using shear-lag model. The matrix multicracking evolution of mode 1, mode 2, mode 3 and mode 5, has been determined using energy balance approach. The effects of ply thickness and fiber volume fraction on matrix multicracking evolution of cross-ply CMCs have been investigated.

Matrix superpotentials

NASA Astrophysics Data System (ADS)

Nikitin, Anatoly G.; Karadzhov, Yuri

2011-07-01

We present a collection of matrix-valued shape invariant potentials which give rise to new exactly solvable problems of SUSY quantum mechanics. It includes all irreducible matrix superpotentials of the generic form W=kQ+\\frac{1}{k} R+P, where k is a variable parameter, Q is the unit matrix multiplied by a real-valued function of independent variable x, and P and R are the Hermitian matrices depending on x. In particular, we recover the Pron'ko-Stroganov 'matrix Coulomb potential' and all known scalar shape invariant potentials of SUSY quantum mechanics. In addition, five new shape invariant potentials are presented. Three of them admit a dual shape invariance, i.e. the related Hamiltonians can be factorized using two non-equivalent superpotentials. We find discrete spectrum and eigenvectors for the corresponding Schrödinger equations and prove that these eigenvectors are normalizable.

Matrix differentiation formulas

NASA Technical Reports Server (NTRS)

Usikov, D. A.; Tkhabisimov, D. K.

1983-01-01

A compact differentiation technique (without using indexes) is developed for scalar functions that depend on complex matrix arguments which are combined by operations of complex conjugation, transposition, addition, multiplication, matrix inversion and taking the direct product. The differentiation apparatus is developed in order to simplify the solution of extremum problems of scalar functions of matrix arguments.

Decreasing the metastatic potential in cancers--targeting the heparan sulfate proteoglycans.

PubMed

Fjeldstad, K; Kolset, S O

2005-09-01

The heterogeneity of proteoglycans (PG)s contributes to their functional diversity. Many functions depend on their ability to bind and modulate the activity of components of the extracellular matrix (ECM). The ability of PGs to interact with other molecules, such as growth factors, is largely determined by the fine structure of the glycosaminoglycan (GAG) chains. Tumorigenesis is associated with changes in the PG synthesis. Heparan sulfate (HS) PGs are involved in several aspects of cancer biology including tumor progression, angiogenesis, and metastasis. PGs can have both tumor promoting and tumor suppressing activities depending on the protein core, the GAG attached, molecules they associate with, localization, the tumor subtype, stages, and degree of tumor differentiation. Perlecan is an angiogenic factor involved in tumor invasiveness. The C-terminal domain V of perlecan, named endorepellin, has however been shown to inhibit angiogenesis. Another angiogenic factor is endostatin, the COOH-terminal domain of the part-time PG collagen XVIII. Glypicans and syndecans may promote local cancer cell growth in some cancer tissues, but inhibit tissue invasion and metastasis in others. The GAG hyaluronan (HA) promotes cancer growth by providing a loose matrix for migrating tumor cells and mediates adhesion of cancer cells. HSPG degrading enzymes like heparanase, heparitinase, and other enzymes such as hyaluronidase and MMP are also important in tumor metastasis. Several different treatment strategies that target PGs have been developed. They have the potential to be effective in reducing tumor growth and inhibit the formation of metastases. PGs are also valuable tumor markers in several cancers.

Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

PubMed

Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

2013-10-01

Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12).

PubMed

Ulbricht, David; Oertwig, Kathrin; Arnsburg, Kristin; Saalbach, Anja; Pippel, Jan; Sträter, Norbert; Heiker, John T

2017-01-20

Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (K D = 21 ± 2 nm) and that binding takes place at a basic patch on top of β-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central β-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

An In Vitro Comparison of the Incorporation, Growth, and Chondrogenic Potential of Human Bone Marrow versus Adipose Tissue Mesenchymal Stem Cells in Clinically Relevant Cell Scaffolds Used for Cartilage Repair

PubMed Central

Kohli, Nupur; Wright, Karina T.; Sammons, Rachel L.; Jeys, Lee; Snow, Martyn

2015-01-01

Aim To compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair. Methods Human BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively. Results A greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro-Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source. Conclusion Chondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue. PMID:26425263

Thermal and mechanical behavior of metal matrix and ceramic matrix composites

NASA Technical Reports Server (NTRS)

Kennedy, John M. (Editor); Moeller, Helen H. (Editor); Johnson, W. S. (Editor)

1990-01-01

The present conference discusses local stresses in metal-matrix composites (MMCs) subjected to thermal and mechanical loads, the computational simulation of high-temperature MMCs' cyclic behavior, an analysis of a ceramic-matrix composite (CMC) flexure specimen, and a plasticity analysis of fibrous composite laminates under thermomechanical loads. Also discussed are a comparison of methods for determining the fiber-matrix interface frictional stresses of CMCs, the monotonic and cyclic behavior of an SiC/calcium aluminosilicate CMC, the mechanical and thermal properties of an SiC particle-reinforced Al alloy MMC, the temperature-dependent tensile and shear response of a graphite-reinforced 6061 Al-alloy MMC, the fiber/matrix interface bonding strength of MMCs, and fatigue crack growth in an Al2O3 short fiber-reinforced Al-2Mg matrix MMC.

The symmetries of the system matrix and propagator matrix for anisotropic media and of the system matrix forperiodically layered media

NASA Astrophysics Data System (ADS)

Xu, Guo-Ming; Ni, Si-Dao

1998-11-01

The `auxiliary' symmetry properties of the system matrix (symmetry with respect to the trailing diagonal) for a general anisotropic dissipative medium and the special form for a monoclinic medium are revealed by rearranging the motion-stress vector. The propagator matrix of a single-layer general anisotropic dissipative medium is also shown to have auxiliary symmetry. For the multilayered case, a relatively simple matrix method is utilized to obtain the inverse of the propagator matrix. Further, Woodhouse's inverse of the propagator matrix for a transversely isotropic medium is extended in a clearer form to handle the monoclinic symmetric medium. The properties of a periodic layer system are studied through its system matrix Aly , which is computed from the propagator matrix P. The matrix Aly is then compared with Aeq , the system matrix for the long-wavelength equivalent medium of the periodic isotropic layers. Then we can find how the periodic layered medium departs from its long-wavelength equivalent medium when the wavelength decreases. In our numerical example, the results show that, when λ/D decreases to 6-8, the components of the two matrices will depart from each other. The component ratio of these two matrices increases to its maximum (more than 15 in our numerical test) when λ/D is reduced to 2.3, and then oscillates with λ/D when it is further reduced. The eigenvalues of the system matrix Aly show that the velocities of P and S waves decrease when λ/D is reduced from 6-8 and reach their minimum values when λ/D is reduced to 2.3 and then oscillate afterwards. We compute the time shifts between the peaks of the transmitted waves and the incident waves. The resulting velocity curves show a similar variation to those computed from the eigenvalues of the system matrix Aly , but on a smaller scale. This can be explained by the spectrum width of the incident waves.

Chondrites: The Compaction of Fine Matrix and Matrix-like Chondrule Rims

NASA Astrophysics Data System (ADS)

Wasson, J. T.

1995-09-01

Primitive chondritic meteorites mainly consist of chondrules, sulfide+/-metal, and fine-grained matrix. The most unequilibrated chondrites preserve in their phase compositions and, to a lesser degree, their textures, many details about processes that occurred in the solar nebula. On the other hand, much of the textural evidence records processes that occurred in or on the parent body. I suggest that the low-porosity of chondrule matrix and matrix-like rims reflects compaction processes that occurred in asteroid-size bodies, and that neither matrix lumps nor compact matrix-like rims on chondrules could have achieved their observed low porosities in the solar nebula. Recent theoretical studies by Donn and Meakin (1) and Chokshi et al. (2) have concluded that grain-grain sticking in the solar nebula mainly produces fluffy structures having very high porosities (probably >>50%). If these structures grow large enough, they can provide an aerogel-like matrix that can trap chondrules as well as metal and sulfide grains, and thus form suitable precursors of chondritic meteorites. However, the strength of any such structure formed in the solar nebula must be a trivial fraction of that required to survive passage through the Earth's atmosphere in order to fall as a meteorite. The best evidence of accretionary structures appears to be that reported by Metzler et al. (3). They made SEM images of entire thin sections of CM chondrites, and showed that, in the best preserved chondrites, rims are present on all entitities--on chondrules, chondrule fragments, refractory inclusions, etc. A study by Krot and Wasson (4) shows a more complex situation in ordinary chondrites. Although matrix is common, a sizable fraction of chondrules are not surrounded by matrix-like rims. As summarized by Rubin and Krot (1995), there are reports of small textural and compositional differences between matrix lumps and mean matrix-like chondrule rims, but there is so much overlap in properties between

Multi-threaded Sparse Matrix-Matrix Multiplication for Many-Core and GPU Architectures.

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

Deveci, Mehmet; Rajamanickam, Sivasankaran; Trott, Christian Robert

Sparse Matrix-Matrix multiplication is a key kernel that has applications in several domains such as scienti c computing and graph analysis. Several algorithms have been studied in the past for this foundational kernel. In this paper, we develop parallel algorithms for sparse matrix-matrix multiplication with a focus on performance portability across different high performance computing architectures. The performance of these algorithms depend on the data structures used in them. We compare different types of accumulators in these algorithms and demonstrate the performance difference between these data structures. Furthermore, we develop a meta-algorithm, kkSpGEMM, to choose the right algorithm and datamore » structure based on the characteristics of the problem. We show performance comparisons on three architectures and demonstrate the need for the community to develop two phase sparse matrix-matrix multiplication implementations for efficient reuse of the data structures involved.« less

Automatic switching matrix

DOEpatents

Schlecht, Martin F.; Kassakian, John G.; Caloggero, Anthony J.; Rhodes, Bruce; Otten, David; Rasmussen, Neil

1982-01-01

An automatic switching matrix that includes an apertured matrix board containing a matrix of wires that can be interconnected at each aperture. Each aperture has associated therewith a conductive pin which, when fully inserted into the associated aperture, effects electrical connection between the wires within that particular aperture. Means is provided for automatically inserting the pins in a determined pattern and for removing all the pins to permit other interconnecting patterns.

Mid-infrared matrix assisted laser desorption ionization with a water/glycerol matrix

NASA Astrophysics Data System (ADS)

Caldwell, Kathleen L.; Murray, Kermit K.

1998-05-01

Matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained using a water and glycerol matrix with a tunable mid-infrared optical parametric oscillator. The matrix consists of a 1:1 mixture of water and glycerol deposited on a thin layer of nitrocellulose and cooled to -30°C. When exposed to vacuum, most of the water evaporates, leaving a matrix of glycerol with residual water. The peptide bradykinin and the protein bovine insulin were used to test this new matrix. Mass spectra were obtained for bradykinin between 2.76 and 3.1 μm with the maximum analyte signal at 2.8 μm. Mass resolution in excess of 2000 for bradykinin and 500 for insulin was obtained with delayed ion extraction and a linear time of flight mass spectrometer. The addition of nitrocellulose to the matrix resulted in exceptionally durable samples: more than 10,000 laser shots which produced analyte signal could be obtained from a single sample spot.

Engineering a collagen matrix that replicates the biological properties of native extracellular matrix.

PubMed

Nam, Kwangwoo; Sakai, Yuuki; Funamoto, Seiichi; Kimura, Tsuyoshi; Kishida, Akio

2011-01-01

In this study, we aimed to replicate the function of native tissues that can be used in tissue engineering and regenerative medicine. The key to such replication is the preparation of an artificial collagen matrix that possesses a structure resembling that of the extracellular matrix. We, therefore, prepared a collagen matrix by fibrillogenesis in a NaCl/Na(2)HPO(4) aqueous solution using a dialysis cassette and investigated its biological behavior in vitro and in vivo. The in vitro cell adhesion and proliferation did not show any significant differences. The degradation rate in the living body could be controlled according to the preparation condition, where the collagen matrix with high water content (F-collagen matrix, >98%) showed fast degradation and collagen matrix with lower water content (T-collagen matrix, >80%) showed no degradation for 8 weeks. The degradation did not affect the inflammatory response at all and relatively faster wound healing response was observed. Comparing this result with that of collagen gel and decellularized cornea, it can be concluded that the structural factor is very important and no cell abnormal behavior would be observed for quaternary structured collagen matrix.

Atypical myxoma.

PubMed

Hwang, J J; Lien, W P; Kuan, P; Hung, C R; How, S W

1991-08-01

We report the case of a 38-year-old woman with a large thin-walled cystic mass (6 x 5 x 4.5 cm) filled with arterial blood in the right atrium. The cystic mass with blood content was clearly delineated by transesophageal cross-sectional echocardiography and magnetic resonance imaging of the heart. At operation, a silver-whitish, smooth surfaced cystic mass was found attached to the free wall of the right atrium between the superior vena cava and the right atrial appendage with a broad base. Microscopically, the wall of the cyst was composed of stellate mesenchymal cells embedded within a myxoid matrix which was proved by alcian blue stain. To our knowledge, this type of cardiac myxoma has not been previously reported.

Canine low-grade intra-orbital myxosarcoma: case report.

PubMed

Campos, Cecília B; Nunes, Fernanda C; Gamba, Conrado O; Damasceno, Karine A; Souza, Cristina M; Campos, Liliane C; Cassali, Geovanni D

2015-05-01

The aim of this study was to evaluate important clinical, morphological, histopathological, histochemical, and immunohistochemical characteristics in order to establish the diagnosis and prognosis of a low-grade intra-orbital myxosarcoma. A mongrel dog presented a 2-year history of a neoplastic mass behind the right eye. The neoplasm presented a mesenchymal spindle and stellate cell proliferation with an abundant myxoid matrix, moderate anisocariosis, and a low mitotic index. It stained positive for vimentin, moderately positive for periodic acid-Schiff, and negative for Gomori trichrome stain and α-smooth muscle actin. One year following surgical excision, the patient remains disease free. The histological findings established a diagnosis of a rare canine intra-orbital low-grade myxosarcoma. © 2014 American College of Veterinary Ophthalmologists.

Vaccination with Gag, Vif, and Nef gene fragments affords partial control of viral replication after mucosal challenge with SIVmac239.

PubMed

Martins, Mauricio A; Wilson, Nancy A; Piaskowski, Shari M; Weisgrau, Kim L; Furlott, Jessica R; Bonaldo, Myrna C; Veloso de Santana, Marlon G; Rudersdorf, Richard A; Rakasz, Eva G; Keating, Karen D; Chiuchiolo, Maria J; Piatak, Michael; Allison, David B; Parks, Christopher L; Galler, Ricardo; Lifson, Jeffrey D; Watkins, David I

2014-07-01

Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with "minigenes" encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01(+) vaccinees mounted CD8(+) T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication. Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by

Carbonate fuel cell matrix

DOEpatents

Farooque, Mohammad; Yuh, Chao-Yi

1996-01-01

A carbonate fuel cell matrix comprising support particles and crack attenuator particles which are made platelet in shape to increase the resistance of the matrix to through cracking. Also disclosed is a matrix having porous crack attenuator particles and a matrix whose crack attenuator particles have a thermal coefficient of expansion which is significantly different from that of the support particles, and a method of making platelet-shaped crack attenuator particles.

Method of forming a ceramic matrix composite and a ceramic matrix component

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

de Diego, Peter; Zhang, James

A method of forming a ceramic matrix composite component includes providing a formed ceramic member having a cavity, filling at least a portion of the cavity with a ceramic foam. The ceramic foam is deposited on a barrier layer covering at least one internal passage of the cavity. The method includes processing the formed ceramic member and ceramic foam to obtain a ceramic matrix composite component. Also provided is a method of forming a ceramic matrix composite blade and a ceramic matrix composite component.

Carbonate fuel cell matrix

DOEpatents

Farooque, M.; Yuh, C.Y.

1996-12-03

A carbonate fuel cell matrix is described comprising support particles and crack attenuator particles which are made platelet in shape to increase the resistance of the matrix to through cracking. Also disclosed is a matrix having porous crack attenuator particles and a matrix whose crack attenuator particles have a thermal coefficient of expansion which is significantly different from that of the support particles, and a method of making platelet-shaped crack attenuator particles. 8 figs.

Altered lumbar spine structure, biochemistry and biomechanical properties in a canine model of mucopolysaccharidosis type VII

PubMed Central

Smith, Lachlan J; Martin, John T; Szczesny, Spencer E; Ponder, Katherine P; Haskins, Mark E; Elliott, Dawn M

2010-01-01

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disorder characterized by a deficiency in β-glucuronidase activity, leading to systemic accumulation of poorly degraded glycosaminoglycans (GAG). Along with other morbidities, MPS VII is associated with paediatric spinal deformity. The objective of this study was to examine potential associations between abnormal lumbar spine matrix structure and composition in MPS VII, and spine segment and tissue-level mechanical properties, using a naturally occurring canine model with a similar clinical phenotype to the human form of the disorder. Segments from juvenile MPS VII and unaffected dogs were allocated to: radiography, gross morphology, histology, biochemistry, and mechanical testing. MPS VII spines had radiolucent lesions in the vertebral body epiphyses. Histologically, this corresponded to a GAG-rich cartilaginous region in place of bone, and elevated GAG staining was seen in the annulus fibrosus. Biochemically, MPS VII samples had elevated GAG in the outer annulus fibrosus and epiphyses, low calcium in the epiphyses, and high water content in all regions except the nucleus pulposus. MPS VII spine segments had higher range of motion and lower stiffness than controls. Endplate indentation stiffness and failure loads were significantly lower in MPS VII samples, while annulus fibrosus tensile mechanical properties were normal. Vertebral body lesions in MPS VII spines suggest a failure to convert cartilage to bone during development. Low stiffness in these regions likely contributes to mechanical weakness in motion segments and is a potential factor in the progression of spinal deformity. PMID:19918911

Structural change of human hair induced by mercury exposure.

PubMed

Xing, Xueqing; Du, Rong; Li, Yufeng; Li, Bai; Cai, Quan; Mo, Guang; Gong, Yu; Chen, Zhongjun; Wu, Zhonghua

2013-10-01

Mercury is one of the most hazardous pollutants in the environment. In this paper, the structural change of human hair induced by mercury exposure was studied. Human hair samples were, respectively, collected from the normal Beijing area and the Hg-contaminated Wanshan area of the Guizhou Province, China. Inductively coupled plasma mass spectroscopy was used to detect the element contents. A small angle X-ray scattering technique was used to probe the structural change. Three reflections with 8.8, 6.7, and 4.5 nm spacing were compared between the normal and the Hg-contaminated hair samples. The results confirm that the 4.5 nm reflection is from the ordered fibrillar structure of glycosaminoglycan (GAG) in proteoglycan (PG) that composes the matrix around the intermediate filaments. The increase of Ca content makes the regular oriented fibrillar structure of GAG transform to a random oriented one, broadening the angular extent of the reflection with 4.5 nm spacing. However, overdose Hg makes the core proteins where the ordered fibrils of GAG are attached become coiled, which destroys the ordered arrangements of fibrillar GAG in PG, resulting in the disappearance of the reflections with 4.5 nm spacing. The disappearance of the 4.5 nm reflection can be used as a bioindicator of overdose Hg contamination to the human body. A supercoiled-coil model of hair nanoscale structure and a possible mechanism of mercury effect in human hair are proposed in this paper.

Single-stage cell-based cartilage regeneration using a combination of chondrons and mesenchymal stromal cells: comparison with microfracture.

PubMed

Bekkers, Joris E J; Tsuchida, Anika I; van Rijen, Mattie H P; Vonk, Lucienne A; Dhert, Wouter J A; Creemers, Laura B; Saris, Daniel B F

2013-09-01

Autologous chondrocyte implantation (ACI) is traditionally a 2-step procedure used to repair focal articular cartilage lesions. With use of a combination of chondrons (chondrocytes in their own territorial matrix) and mesenchymal stromal cells (MSCs), ACI could be innovated and performed in a single step, as sufficient cells would be available to fill the defect within a 1-step surgical procedure. Chondrons have been shown to have higher regenerative capacities than chondrocytes without such a pericellular matrix. To evaluate cartilage formation by a combination of chondrons and MSCs in vitro and in both small and large animal models. Controlled laboratory study. Chondrons and MSCs were cultured at different ratios in vitro containing 0%, 5%, 10%, 20%, 50%, or 100% chondrons (n = 3); embedded in injectable fibrin glue (Beriplast); and implanted subcutaneously in nude mice (n = 10; ratios of 0%, 5%, 10%, and 20% chondrons). Also, in a 1-step procedure, a combination of chondrons and MSCs was implanted in a freshly created focal articular cartilage lesion (10% chondrons) in goats (n = 8) and compared with microfracture. The effect of both treatments, after 6-month follow-up, was evaluated using biochemical glycosaminoglycan (GAG) and GAG/DNA analysis and scored using validated scoring systems for macroscopic and microscopic defect repairs. The addition of MSCs to chondron cultures enhanced cartilage-specific matrix production as reflected by a higher GAG production (P < .03), both in absolute levels and normalized to DNA content, compared with chondrocyte and 100% chondron cultures. Similar results were observed after 4 weeks of subcutaneous implantation in nude mice. Treatment of freshly created cartilage defects in goats using a combination of chondrons and MSCs in Beriplast resulted in better microscopic, macroscopic, and biochemical cartilage regeneration (P ≤ .02) compared with microfracture treatment. The combination of chondrons and MSCs increased cartilage

D-MATRIX: A web tool for constructing weight matrix of conserved DNA motifs

PubMed Central

Sen, Naresh; Mishra, Manoj; Khan, Feroz; Meena, Abha; Sharma, Ashok

2009-01-01

Despite considerable efforts to date, DNA motif prediction in whole genome remains a challenge for researchers. Currently the genome wide motif prediction tools required either direct pattern sequence (for single motif) or weight matrix (for multiple motifs). Although there are known motif pattern databases and tools for genome level prediction but no tool for weight matrix construction. Considering this, we developed a D-MATRIX tool which predicts the different types of weight matrix based on user defined aligned motif sequence set and motif width. For retrieval of known motif sequences user can access the commonly used databases such as TFD, RegulonDB, DBTBS, Transfac. D­MATRIX program uses a simple statistical approach for weight matrix construction, which can be converted into different file formats according to user requirement. It provides the possibility to identify the conserved motifs in the co­regulated genes or whole genome. As example, we successfully constructed the weight matrix of LexA transcription factor binding site with the help of known sos­box cis­regulatory elements in Deinococcus radiodurans genome. The algorithm is implemented in C-Sharp and wrapped in ASP.Net to maintain a user friendly web interface. D­MATRIX tool is accessible through the CIMAP domain network. Availability http://203.190.147.116/dmatrix/ PMID:19759861

D-MATRIX: a web tool for constructing weight matrix of conserved DNA motifs.

PubMed

Sen, Naresh; Mishra, Manoj; Khan, Feroz; Meena, Abha; Sharma, Ashok

2009-07-27

Despite considerable efforts to date, DNA motif prediction in whole genome remains a challenge for researchers. Currently the genome wide motif prediction tools required either direct pattern sequence (for single motif) or weight matrix (for multiple motifs). Although there are known motif pattern databases and tools for genome level prediction but no tool for weight matrix construction. Considering this, we developed a D-MATRIX tool which predicts the different types of weight matrix based on user defined aligned motif sequence set and motif width. For retrieval of known motif sequences user can access the commonly used databases such as TFD, RegulonDB, DBTBS, Transfac. D-MATRIX program uses a simple statistical approach for weight matrix construction, which can be converted into different file formats according to user requirement. It provides the possibility to identify the conserved motifs in the co-regulated genes or whole genome. As example, we successfully constructed the weight matrix of LexA transcription factor binding site with the help of known sos-box cis-regulatory elements in Deinococcus radiodurans genome. The algorithm is implemented in C-Sharp and wrapped in ASP.Net to maintain a user friendly web interface. D-MATRIX tool is accessible through the CIMAP domain network. http://203.190.147.116/dmatrix/

The Exopolysaccharide Matrix

PubMed Central

Koo, H.; Falsetta, M.L.; Klein, M.I.

2013-01-01

Many infectious diseases in humans are caused or exacerbated by biofilms. Dental caries is a prime example of a biofilm-dependent disease, resulting from interactions of microorganisms, host factors, and diet (sugars), which modulate the dynamic formation of biofilms on tooth surfaces. All biofilms have a microbial-derived extracellular matrix as an essential constituent. The exopolysaccharides formed through interactions between sucrose- (and starch-) and Streptococcus mutans-derived exoenzymes present in the pellicle and on microbial surfaces (including non-mutans) provide binding sites for cariogenic and other organisms. The polymers formed in situ enmesh the microorganisms while forming a matrix facilitating the assembly of three-dimensional (3D) multicellular structures that encompass a series of microenvironments and are firmly attached to teeth. The metabolic activity of microbes embedded in this exopolysaccharide-rich and diffusion-limiting matrix leads to acidification of the milieu and, eventually, acid-dissolution of enamel. Here, we discuss recent advances concerning spatio-temporal development of the exopolysaccharide matrix and its essential role in the pathogenesis of dental caries. We focus on how the matrix serves as a 3D scaffold for biofilm assembly while creating spatial heterogeneities and low-pH microenvironments/niches. Further understanding on how the matrix modulates microbial activity and virulence expression could lead to new approaches to control cariogenic biofilms. PMID:24045647

MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

PubMed Central

Janes, Holly; Frahm, Nicole; DeCamp, Allan; Rolland, Morgane; Gabriel, Erin; Wolfson, Julian; Hertz, Tomer; Kallas, Esper; Goepfert, Paul; Friedrich, David P.; Corey, Lawrence; Mullins, James I.; McElrath, M. Juliana; Gilbert, Peter

2012-01-01

Background The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. Methods Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. Findings Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0·04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0·06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0·04; Pol p = 0·13; Gag p = 0·89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p>0·50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4·7 vs 5·1) but the difference was not significant (p = 0·27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0·30). Interpretation Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression. PMID

Auger analysis of a fiber/matrix interface in a ceramic matrix composite

NASA Technical Reports Server (NTRS)

Honecy, Frank S.; Pepper, Stephen V.

1988-01-01

Auger electron spectroscopy (AES) depth profiling was used to characterize the fiber/matrix interface of an SiC fiber, reaction bonded Si3N4 matrix composite. Depth profiles of the as received double coated fiber revealed concentration oscillations which disappeared after annealing the fiber in the environment used to fabricate the composite. After the composite was fractured, the Auger depth profiles showed that failure occurred in neither the Beta-SiC fiber body nor in the Si3N4 matrix but, concurrently, at the fiber coating/matrix interface and within the fiber coating itself.

A colinear backscattering Mueller matrix microscope for reflection Muller matrix imaging

NASA Astrophysics Data System (ADS)

Chen, Zhenhua; Yao, Yue; Zhu, Yuanhuan; Ma, Hui

2018-02-01

In a recent attempt, we developed a colinear backscattering Mueller matrix microscope by adding polarization state generator (PSG) and polarization state analyzer (PSA) into the illumination and detection optical paths of a commercial metallurgical microscope. It is found that specific efforts have to be made to reduce the artifacts due to the intrinsic residual polarizations of the optical system, particularly the dichroism due to the 45 degrees beam splitter. In this paper, we present a new calibration method based on numerical reconstruction of the instrument matrix to remove the artifacts introduced by beam splitter. Preliminary tests using a mirror as a standard sample show that the maximum Muller matrix element error of the colinear backscattering Muller matrix microscope can be reduced to a few percent.

Multi-threaded Sparse Matrix Sparse Matrix Multiplication for Many-Core and GPU Architectures.

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

Deveci, Mehmet; Trott, Christian Robert; Rajamanickam, Sivasankaran

Sparse Matrix-Matrix multiplication is a key kernel that has applications in several domains such as scientific computing and graph analysis. Several algorithms have been studied in the past for this foundational kernel. In this paper, we develop parallel algorithms for sparse matrix- matrix multiplication with a focus on performance portability across different high performance computing architectures. The performance of these algorithms depend on the data structures used in them. We compare different types of accumulators in these algorithms and demonstrate the performance difference between these data structures. Furthermore, we develop a meta-algorithm, kkSpGEMM, to choose the right algorithm and datamore » structure based on the characteristics of the problem. We show performance comparisons on three architectures and demonstrate the need for the community to develop two phase sparse matrix-matrix multiplication implementations for efficient reuse of the data structures involved.« less

Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

PubMed

Carpentier, Mathieu; Allain, Fabrice; Slomianny, Marie-Christine; Durieux, Sandrine; Vanpouille, Christophe; Haendler, Bernard; Spik, Geneviève

2002-04-23

Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

[Porous matrix and primary-cell culture: a shared concept for skin and cornea tissue engineering].

PubMed

Auxenfans, C; Builles, N; Andre, V; Lequeux, C; Fievet, A; Rose, S; Braye, F-M; Fradette, J; Janin-Manificat, H; Nataf, S; Burillon, C; Damour, O

2009-06-01

Skin and cornea both feature an epithelium firmly anchored to its underlying connective compartment: dermis for skin and stroma for cornea. A breakthrough in tissue engineering occurred in 1975 when skin stem cells were successfully amplified in culture by Rheinwald and Green. Since 1981, they are used in the clinical arena as cultured epidermal autografts for the treatment of patients with extensive burns. A similar technique has been later adapted to the amplification of limbal-epithelial cells. The basal layer of the limbal epithelium is located in a transitional zone between the cornea and the conjunctiva and contains the stem cell population of the corneal epithelium called limbal-stem cells (LSC). These cells maintain the proper renewal of the corneal epithelium by generating transit-amplifying cells that migrate from the basal layer of the limbus towards the basal layer of the cornea. Tissue-engineering protocols enable the reconstruction of three-dimensional (3D) complex tissues comprising both an epithelium and its underlying connective tissue. Our in vitro reconstruction model is based on the combined use of cells and of a natural collagen-based biodegradable polymer to produce the connective-tissue compartment. This porous substrate acts as a scaffold for fibroblasts, thereby, producing a living dermal/stromal equivalent, which once epithelialized results into a reconstructed skin/hemicornea. This paper presents the reconstruction of surface epithelia for the treatment of pathological conditions of skin and cornea and the development of 3D tissue-engineered substitutes based on a collagen-GAG-chitosan matrix for the regeneration of skin and cornea.

Allele related mutation specific-polymerase chain reaction for rapid diagnosis of Hb New York (beta 113 (G15) Val-->Glu, beta(CD113 GTG-->GAG)).

PubMed

Viprakasit, Vip; Tachavanich, Kalaya; Suwantol, Lerlugsn; Pung-Amritt, Parichat; Chinchang, Worawut; Tanphaichitr, Voravarn S

2002-08-01

Hemoglobin New York (beta 113 (G15) Val-->Glu), a beta-globin variant, was first reported in a Chinese family living in New York. Subsequently, this abnormal hemoglobin was reported in many Chinese descendants from several groups and it was also known as Hb Kaohsiung. The subtle change in alpha1beta1 contact region apart from the heme group connecting area by Val-->Glu substitution has minor changes in both the electrophoretic mobility and stability making this hemoglobin variant difficult to distinguish from Hb A using routine hemoglobin analysis. The authors described a case of heterozygosity of Hb New York diagnosed by a molecular technique and revealed a mutation in beta(CD113 GTG-->GAG). A novel Allele Related Mutation Specific-Polymerase Chain Reaction (ARMS-PCR) for rapid diagnosis of this mutation has been proposed.

Differential regulation of immature articular cartilage compressive moduli and Poisson's ratios by in vitro stimulation with IGF-1 and TGF-beta1.

PubMed

Williams, Gregory M; Dills, Kristin J; Flores, Christian R; Stender, Michael E; Stewart, Kevin M; Nelson, Lauren M; Chen, Albert C; Masuda, Koichi; Hazelwood, Scott J; Klisch, Stephen M; Sah, Robert L

2010-09-17

Mechanisms of articular cartilage growth and maturation have been elucidated by studying composition-function dynamics during in vivo development and in vitro culture with stimuli such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta 1 (TGF-beta1). This study tested the hypothesis that IGF-1 and TGF-beta1 regulate immature cartilage compressive moduli and Poisson's ratios in a manner consistent with known effects on tensile properties. Bovine calf articular cartilage from superficial-articular (S) and middle-growth (M) regions were analyzed fresh or following culture in medium with IGF-1 or TGF-beta1. Mechanical properties in confined (CC) and unconfined (UCC) compression, cartilage matrix composition, and explant size were assessed. Culture with IGF-1 resulted in softening in CC and UCC, increased Poisson's ratios, substantially increased tissue volume, and accumulation of glycosaminoglycan (GAG) and collagen (COL). Culture with TGF-beta1 promoted maturational changes in the S layer, including stiffening in CC and UCC and increased concentrations of GAG, COL, and pyridinoline crosslinks (PYR), but little growth. Culture of M layer explants with TGF-beta1 was nearly homeostatic. Across treatment groups, compressive moduli in CC and UCC were positively related to GAG, COL, and PYR concentrations, while Poisson's ratios were negatively related to concentrations of these matrix components. Thus, IGF-1 and TGF-beta1 differentially regulate the compressive mechanical properties and size of immature articular cartilage in vitro. Prescribing tissue growth, maturation, or homeostasis by controlling the in vitro biochemical environment with such growth factors may have applications in cartilage repair and tissue engineering.

Matrix with Prescribed Eigenvectors

ERIC Educational Resources Information Center

Ahmad, Faiz

2011-01-01

It is a routine matter for undergraduates to find eigenvalues and eigenvectors of a given matrix. But the converse problem of finding a matrix with prescribed eigenvalues and eigenvectors is rarely discussed in elementary texts on linear algebra. This problem is related to the "spectral" decomposition of a matrix and has important technical…

Grassmann matrix quantum mechanics

DOE PAGES

Anninos, Dionysios; Denef, Frederik; Monten, Ruben

2016-04-21

We explore quantum mechanical theories whose fundamental degrees of freedom are rectangular matrices with Grassmann valued matrix elements. We study particular models where the low energy sector can be described in terms of a bosonic Hermitian matrix quantum mechanics. We describe the classical curved phase space that emerges in the low energy sector. The phase space lives on a compact Kähler manifold parameterized by a complex matrix, of the type discovered some time ago by Berezin. The emergence of a semiclassical bosonic matrix quantum mechanics at low energies requires that the original Grassmann matrices be in the long rectangular limit.more » In conclusion, we discuss possible holographic interpretations of such matrix models which, by construction, are endowed with a finite dimensional Hilbert space.« less

Fragility Fracture Incidence in Chronic Obstructive Pulmonary Disease (COPD) Patients Associates With Nanoporosity, Mineral/Matrix Ratio, and Pyridinoline Content at Actively Bone-Forming Trabecular Surfaces.

PubMed

Paschalis, Eleftherios P; Gamsjaeger, Sonja; Dempster, David; Jorgetti, Vanda; Borba, Victoria; Boguszewski, Cesar L; Klaushofer, Klaus; Moreira, Carolina A

2017-01-01

Chronic obstructive pulmonary disease (COPD) is associated with low areal bone mineral density (aBMD) by dual-energy X-ray absorptiometry (DXA) and altered microstructure by bone histomorphometry and micro-computed tomography. Nevertheless, not all COPD patients sustain fragility fractures. In the present study, we used Raman microspectroscopic analysis to determine bone compositional properties at actively forming trabecular surfaces (based on double fluorescent labels) in iliac crest biopsies from 19 postmenopausal COPD patients (aged 62.1 ± 7.3 years). Additionally, we analyzed trabecular geometrical centers, representing tissue much older than the forming surfaces. Eight of the patients had sustained fragility fractures, and 13 had received treatment with inhaled glucocorticoids. None of the patients had taken oral glucocorticoids. The monitored parameters were mineral/matrix ratio (MM), nanoporosity, and relative glycosaminoglycan (GAG), lipid, and pyridinoline contents (PYD). There were no significant differences between the glucocorticoid-treated patients and those who did not receive any. On the other hand, COPD patients sustaining fragility fractures had significantly lower nanoporosity and higher MM and PYD values compared with COPD patients without fragility fractures. To the best of our knowledge, this is the first study to discriminate between fracture and non-fracture COPD patients based on differences in the material properties of bone matrix. Given that these bone material compositional differences are evident close to the cement line (a major bone interface), they may contribute to the inferior bone toughness and coupled with the lower lumbar spine bone mineral density values result in the fragility fractures prevalent in these patients. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

A new fracture mechanics model for multiple matrix cracks of SiC fiber reinforced brittle-matrix composites

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

Okabe, T.; Takeda, N.; Komotori, J.

1999-11-26

A new model is proposed for multiple matrix cracking in order to take into account the role of matrix-rich regions in the cross section in initiating crack growth. The model is used to predict the matrix cracking stress and the total number of matrix cracks. The model converts the matrix-rich regions into equivalent penny shape crack sizes and predicts the matrix cracking stress with a fracture mechanics crack-bridging model. The estimated distribution of matrix cracking stresses is used as statistical input to predict the number of matrix cracks. The results show good agreement with the experimental results by replica observations.more » Therefore, it is found that the matrix cracking behavior mainly depends on the distribution of matrix-rich regions in the composite.« less

Effect of Fiber Poisson Contraction on Matrix Multicracking Evolution of Fiber-Reinforced Ceramic-Matrix Composites

NASA Astrophysics Data System (ADS)

Longbiao, Li

2015-12-01

An analytical methodology has been developed to investigate the effect of fiber Poisson contraction on matrix multicracking evolution of fiber-reinforced ceramic-matrix composites (CMCs). The modified shear-lag model incorporated with the Coulomb friction law is adopted to solve the stress distribution in the interface slip region and intact region of the damaged composite. The critical matrix strain energy criterion which presupposes the existence of an ultimate or critical strain energy limit beyond which the matrix fails has been adopted to describe matrix multicracking of CMCs. As more energy is placed into the composite, matrix fractures and the interface debonding occurs to dissipate the extra energy. The interface debonded length under the process of matrix multicracking is obtained by treating the interface debonding as a particular crack propagation problem along the fiber/matrix interface. The effects of the interfacial frictional coefficient, fiber Poisson ratio, fiber volume fraction, interface debonded energy and cycle number on the interface debonding and matrix multicracking evolution have been analyzed. The theoretical results are compared with experimental data of unidirectional SiC/CAS, SiC/CAS-II and SiC/Borosilicate composites.

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation

NASA Astrophysics Data System (ADS)

Zhong, Zhensheng; Yang, Lixia; Zhang, Haiping; Shi, Jiahao; Vandana, J. Jeya; Lam, Do Thuy Uyen Ha; Olsthoorn, René C. L.; Lu, Lanyuan; Chen, Gang

2016-12-01

Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The -1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between -1 frameshifting efficiency and unfolding rate at forces of 15-35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and -1 frameshifting efficiency.

Hybrid matrix amplifier

DOEpatents

Martens, J.S.; Hietala, V.M.; Plut, T.A.

1995-01-03

The present invention comprises a novel matrix amplifier. The matrix amplifier includes an active superconducting power divider (ASPD) having N output ports; N distributed amplifiers each operatively connected to one of the N output ports of the ASPD; and a power combiner having N input ports each operatively connected to one of the N distributed amplifiers. The distributed amplifier can included M stages of amplification by cascading superconducting active devices. The power combiner can include N active elements. The resulting (N[times]M) matrix amplifier can produce signals of high output power, large bandwidth, and low noise. 6 figures.

Hybrid matrix amplifier