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At least 541 records · Page 30Linked to original sources
PubMed2006

Cluster of DnaA boxes involved in regulation of Streptomyces chromosome replication: from in silico to in vivo studies.

In Streptomyces coelicolor, replication is initiated by the DnaA protein in the centrally located oriC region and proceeds bidirectionally until the replication forks reach the ends of the linear chromosome. We identified three clusters of DnaA boxes (H69, H24, and D78) which are in a relatively short segment of the chromosome centered on the oriC region. Of the clusters analyzed, D78 exhibited the highest affinity for the DnaA protein; the affinity of DnaA for the D78 cluster was about eightfold higher than the affinity for oriC. The high-affinity DnaA boxes appear to be involved in the control of chromosome replication. Deletion of D78 resulted in more frequent chromosome replication (an elevated ratio of origins to chromosome ends was observed) and activated aerial mycelium formation, leading to earlier colony maturation. In contrast, extra copies of D78 (delivered on a plasmid) caused slow colony growth, presumably because of a reduction in the frequency of initiation of chromosome replication. This suggests that the number of high-affinity DnaA boxes is relatively constant in hyphal compartments and that deletion of D78 therefore permits an increased copy number of either the chromosomal origin region or a plasmid harboring the D78 cluster. This system conceivably influences the timing of decisions to initiate aerial mycelial formation and sporulation.

Bacterial Proteins↗
PubMed2006

Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation.

The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant (DeltaUL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with DeltaUL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented DeltaUL32-BAC replication but murine CMV M32 failed to complement, consistent with evolutionary divergence of rodent and primate cytomegaloviruses. Infection with DeltaUL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.

Animals↗
PubMed2006

Complete replication cycle and acquisition of tegument in nucleus of human herpesvirus 6A in astrocytes and in T-cells.

The ultrastructural replication cycle of human herpesvirus 6A and 6B, both T-lymphotropic viruses, with tropism for the central nervous system, was compared by electron microscopy in the same cells, that is, in the T-lymphoblastoid cell line SupT-1 and in human astrocytes. Both HHV-6A and HHV-6B replicated efficiently in SupT-1 and formed viral particles. The tegument is the least characterized structure of the herpesviral particle and both variants were able to form intranuclear membrane compartments called tegusomes in SupT-1 where tegumentation occurred. Also, tegumentation occurred in HHV-6A infected cells in the nucleoplasm without the presence of a tegusome. This suggests that there is more than one possible route of tegumentation. Differences in the replication cycles between HHV-6A and HHV-6B were also observed in the cytoplasm. One such difference was that prominent annulate lamellae were only found in the cytoplasm of HHV-6A infected cells. In astrocytes a successful formation of viral particles was only seen with the HHV-6A variant. The HHV-6A virus life cycle in astrocytes resembled the life cycle in the T-cell line SupT-1, except that no annulate lamellae were found. Complete viral particles were found extracellularly around the astrocytes and the supernatant of infected astrocytes were able to re-infect SupT-1 cells. This suggests that HHV-6A infection in astrocytes can generate complete, viable, and infectious viral particles. The HHV-6 variants behave differently in the same type of cells and have different tropisms for astrocytes, supporting the notion that the variants might induce different diseases.

Astrocytes↗
PubMed2000

Rapid clearance of human immunodeficiency virus type 1 from ventricular cerebrospinal fluid during antiretroviral treatment.

To understand the pathogenesis of human immunodeficiency virus-induced neuropathology, it is critical to know the dynamics of viral replication in the central nervous system. Viral decay kinetics were mathematically analyzed from multiple serial specimens of ventricular cerebrospinal fluid and plasma during antiretroviral therapy in a patient with asymptomatic human immunodeficiency virus infection and an external ventricular catheter for hydrocephalus. A rapid exponential decay of virus with an elimination half-life of 4.2 days in ventricular cerebrospinal fluid and 2.3 days in plasma was found. Sequencing the V3 loop-encoding envelope gene of virus in both compartments revealed high sequence homology. The combined data suggest that virus in ventricular cerebrospinal fluid is at least partly contributed by rapidly replicating virus-producing cells recruited from the circulation.

Adult↗
PubMed2003

Mucosal priming of simian immunodeficiency virus-specific cytotoxic T-lymphocyte responses in rhesus macaques by the Salmonella type III secretion antigen delivery system.

Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. The Salmonella type III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore tested DeltaphoP-phoQ attenuated strains of Salmonella enterica serovar Typhimurium and S. enterica serovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques. Mamu-A*01(+) macaques were inoculated with three oral doses of recombinant Salmonella, followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag(181-189) epitope were detected following each dose of Salmonella. After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag(181-189)-specific CD8(+) T-cell responses in peripheral blood. A significant percentage of the Gag(181-189)-specific T-cell population in each animal also expressed the intestinal homing receptor alpha4beta7. Additionally, Gag(181-189)-specific CD8(+) T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses, Salmonella-primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by the Salmonella type III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.

AIDS Vaccines↗
PubMedDate not supplied

A new mechanism for the neutralization of enveloped viruses by antiviral antibody.

Despite the considerable research that has been carried out into viral neutralization by antiviral antibody, its mechanisms remain poorly understood. Cases have been reported in which antiviral antibody can inhibit viral replication without inhibiting the binding and uptake of virus by susceptible cells. It has been shown that many enveloped viruses enter their target cells by endocytosis and are subsequently located in cellular compartments of increasing acidity. With several enveloped viruses this acidic pH can catalyse a fusion reaction between the membrane of the virus particle and that of a prelysosomal endosome, thus enabling the viral core to enter the cytosol and replication to commence. We have recently demonstrated that such an endosomal fusion event at mild acidic pH is involved in the entry pathway of the enveloped flavivirus, West Nile virus (WNV), into macrophages. We now show that antiviral antibody can neutralize WNV by inhibiting this intraendosomal acid-catalysed fusion step and we speculate on possible implications for the future design of antiviral vaccines.

Animals↗
PubMedDate not supplied

Differences between semen and plasma of nucleoside reverse transcriptase resistance mutations in HIV-infected patients, using a rapid assay.

The emergence of HIV-resistant strains generally occurs during treatment failure. A high rate of viral replication, low patient adherence to therapy, poor drug absorption or increased drug metabolism may be related to treatment failure and favour the onset of resistance. Recently, despite the absence of detectable HIV viral load in plasma, the importance of the persistence of HIV replication in other reservoirs has been underlined. Paired semen and plasma specimens stored from HIV male patients were tested using the LiPA assay, detecting genotype mutations related to Nucleoside Reverse Transciptase Inhibitors (NRTI) resistance. Overall, 32 paired semen and plasma samples were analyzed. The presence of HIVsequences was determined in 13 paired specimens (40.6%). Sixteen semen and 10 plasma specimens failed to amplify. At least one mutation was identified in the plasma of 77.8% of treated and 50.0% of naive patients. Similarly, mutations were identified in the semen of 66.7% of treated and 50.0% of untreated patients. Different mutation patterns between the two compartments were found in 46.2% of patients. The use of rapid assessment of HIV resistance in plasma and semen may be useful in some situations, such as HIV infection transmitted by sexual contact. Our data reinforce the evidence that, regarding HIV replication, the male genital tract must be considered as a separate compartment from the plasma.

Anti-HIV Agents↗
PubMed2006

Cryoelectron microscopy structures of rotavirus NSP2-NSP5 and NSP2-RNA complexes: implications for genome replication.

The replication and packaging of the rotavirus genome, comprising 11 segments of double-stranded RNA, take place in specialized compartments called viroplasms, which are formed during infection and involve a coordinated interplay of multiple components. Two rotavirus nonstructural proteins, NSP2 (with nucleoside triphosphatase, single-stranded RNA [ssRNA] binding and helix-destabilizing activities) and NSP5, are essential in these events. Previous structural analysis of NSP2 showed that it is an octamer in crystals, obeying 4-2-2 crystal symmetry, with a large 35-A central hole along the fourfold axis and deep grooves at one of the twofold axes. To ascertain that the solution structure of NSP2 is the same as that in the crystals and investigate how NSP2 interacts with NSP5 and RNA, we carried out single-particle cryoelectron microscopy (cryo-EM) analysis of NSP2 alone and in complexes with NSP5 and ssRNA at subnanometer resolution. Because full-length NSP5 caused severe aggregation upon mixing with NSP2, the deletion construct NSP566-188 was used in these studies. Our studies show that the solution structure of NSP2 is same as the crystallographic octamer and that both NSP566-188 and ssRNA bind to the grooves in the octamer, which are lined by positively charged residues. The fitting of the NSP2 crystal structure to cryo-EM reconstructions of the complexes indicates that, in contrast to the binding of NSP566-188, the binding of RNA induces noticeable conformational changes in the NSP2 octamer. Consistent with the observation that both NSP5 and RNA share the same binding site on the NSP2 octamer, filter binding assays showed that NSP5 competes with ssRNA binding, indicating that one of the functions of NSP5 is to regulate NSP2-RNA interactions during genome replication.

Binding Sites↗
PubMed1992

Successive negative contrast in one-way avoidance learning in rats.

In three experiments, successive negative contrast was examined in one-way avoidance learning. Reward magnitude in first (pre-shift) and second (post-shift) phases was manipulated by time spent in the safe compartment. Experiment 1 demonstrated that when time in the danger compartment was held constant, a group shifted from a large reward--30 sec spent in the safe compartment--to a small reward--1 sec--showed poor performance and longer response latency than a group conditioned with the small reward in both phases. Experiment 2 replicated this effect with a less intense shock and also demonstrated that a group shifted from large to small reward performed more poorly than a group exposed to large reward--30 sec--in both phases. Finally, Experiment 3 showed that changes in intertrial interval, defined as total time spent in the safe compartment and the danger compartment before the onset of the warning signal, were not responsible for this contrast effect. These results suggest that time spent in a safe place can act as appetitive incentive during one-way avoidance learning.

Animals↗
PubMed2004

Steroid hormones, polypeptide growth factors, hormone refractory prostate cancer, and the neuroendocrine phenotype.

The growth, development, and differentiation of the prostate gland is largely dependent on the action of androgens and peptide growth factors that act differentially at the level of the mesenchymal and epithelial compartments. It is our premise that to understand the emergence of metastatic and hormone refractory prostate cancer we need to investigate: (1) how androgen action at the level of the mesenchyme induces the production of peptide growth factors that in turn can facilitate the growth and development of the epithelial compartment; (2) how androgen action at the level of the epithelium induces and maintains cellular differentiation, function, and replicative senescence; and (3) how transformation of the prostate gland can corrupt androgen and growth factor signaling homeostasis. To this end, we focus our discussion on how deregulation of the growth factor signaling axis can cooperate with deregulation of the androgen signaling axis to facilitate transformation, metastasis, and the emergence of the hormone refractory and neuroendocrine phenotypes associated with progressive androgen-independent prostate cancer. Finally, we suggest a working hypothesis to explain why hormone ablation therapy works to control early disease but fails to control, and may even facilitate, advanced prostate cancer.

Animals↗
PubMed2002

Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons.

For many years our knowledge on hepatitis C virus (HCV) replication has been based on in vitro experiments or transfection studies. Recently, the first reliable system for studying viral replication in tissue culture cells was developed. Taking advantage of this system, we examined in detail the localization of viral nonstructural (NS) proteins in cells containing functional replication complexes. By fractionation experiments and immunomicroscopy, we observed that all NS proteins were associated with the endoplasmic reticulum (ER) membranes, confirming the hypothesis that the ER is the site of membrane-associated HCV RNA replication. Interestingly, NS3 and NS4A were preferentially localized in endoplasmic reticulum cisternae surrounding mitochondria, suggesting additional subcellular compartment-related functions for these viral proteins. Furthermore, the immunoelectron microscopy revealed the loss of the organization and other morphological alterations of the ER (convoluted cisternae and paracrystalline structures), resembling alterations observed in liver biopsies of HCV-infected individuals and in flavivirus-infected cells.

Animals↗
PubMed2026

A reproducible computational transcriptomic framework for cell-type-resolved fibroinflammatory-AKT remodeling in human heart failure.

BACKGROUND: Human heart failure involves multicellular transcriptional remodeling, but public transcriptomic studies often remain disconnected from cell-type localization and perturbational interpretation. METHODS: We developed a reproducible computational workflow integrating human left-ventricular bulk transcriptomes, donor-level cell-type pseudobulk results from a human heart-failure single-cell/single-nucleus atlas, external snRNA-seq support, curated module scoring, focused ligand-receptor prioritization and LINCS/L1000 perturbational matching. RESULTS: Cross-cohort analysis identified 14,358 same-direction HF-associated genes, including 1633 replicated HF-up and 785 replicated HF-down genes. Donor-level pseudobulk analysis localized disease remodeling to cardiomyocyte, fibroblast and myeloid compartments. Activated fibroblast and inflammatory myeloid programs defined a fibroinflammatory remodeling axis connected to context-dependent AKT-associated transcriptional shifts. External snRNA-seq support was strongest for fibroblast activation and AKT-associated remodeling, with etiology-dependent heterogeneity across validation resources. L1000FWD screening prioritized safety-aware perturbational hypotheses, including glimepiride and simvastatin as interpretable candidates requiring experimental validation. CONCLUSIONS: This study provides a computational transcriptomic framework linking reproducible human HF signatures, cell-type-resolved fibroinflammatory remodeling and perturbational genomic prioritization without claiming drug efficacy or AKT causality.

Humans↗
PubMedDate not supplied

Total hip arthroplasty as an overnight-stay procedure using an ambulatory continuous psoas compartment nerve block: a prospective feasibility study.

OBJECTIVE: Total hip arthroplasty (THA) results in severe postoperative pain requiring hospitalization to provide potent analgesia. Consequently, the average duration of hospitalization after THA in the United States is 4 to 5 days. This prospective study investigated the feasibility of converting THA into an overnight-stay procedure using a continuous psoas compartment nerve block provided at home with a portable infusion pump. CASE REPORT: Preoperatively, patients undergoing THA had a psoas compartment perineural catheter placed. Postoperatively, perineural ropivacaine 0.2% was delivered through postoperative day (POD) 4. Patients were discharged home when they met specific, prospectively defined criteria, as early as POD 3 for the first phase and POD 1 for the second phase. Of the patients in the first phase (n = 7) who remained hospitalized for at least 3 postoperative nights, 5 met discharge criteria on POD 1 and the remainder on POD 2. Of the patients in phase 2 (n = 5), all but 1 met discharge criteria on POD 1 and 3 were discharged directly home on POD 1. Postoperative pain was well controlled, opioid requirements and sleep disturbances were minimal, and patient satisfaction high. CONCLUSIONS: These results suggest that for a subset of patients without major comorbidities, it is feasible to convert THA into an overnight-stay procedure using an ambulatory continuous psoas compartment nerve block as part of a multimodal analgesic regimen provided at home. Additional research is required to replicate these results in a controlled trial, define the appropriate subset of patients, and assess the incidence of complications associated with this practice before its mainstream use.

Aged↗
PubMed2000

Acceleration of lymphomagenesis in mismatch-repair deficient mice by exposure to genotoxic agents.

Hemizygosity for genes that are essential for DNA mismatch repair (MMR) was found to underlie cancer predisposition in hereditary nonpolypsis colorectal cancer (HNPCC). Loss of the wild-type allele generates a MMR-deficient cell compartment with a high propensity to oncogenic transformation. MMR deficiency not only accelerates spontaneous mutagenesis resulting from DNA replication errors, but also affects the cellular response to genotoxic agents. To study the consequences of MMR deficiency in vitro and to provide experimental access to HNPCC we have generated MMR-deficient cell lines and mice. The combination of MMR deficiency and exposure to genotoxic agents strongly accelerated lymphomagenesis.

Animals↗
PubMed2026

SARS-CoV-2 Orf3a protein interaction mapping using unnatural amino acid incorporation.

Mapping transient protein-protein interactions remain a major challenge in studying viral host-pathogen interfaces. While some virus-host interactions are stable and readily captured, the majority are highly dynamic, reflecting the need for viral proteins to engage distinct host factors at different stages of the life cycle. Here, we employ a protein engineering strategy based on the site-specific incorporation of the unnatural acid p-azido-L-phenylalanine (AzF) to enable photo-crosslinking proteomic analysis of the SARS-CoV-2 accessory protein Orf3a in live cells. Genetic installation of AzF at residue K198 of Orf3a permitted UV-induced covalent capture of proximal host interacting proteins, overcoming challenges associated with membrane localization and limited protein abundance. A total of 248 high-confidence Orf3a-interacting proteins were reproducibly identified and subjected to gene ontology analysis, revealing enrichment in innate immune signaling, antiviral defense, RNA processing, and viral replication-associated pathways. Orf3a is an accessory protein that functions as a viroporin and traffics across multiple cellular compartments, and was found to interact with host RNA helicases, RNA-binding proteins, immune regulators, and metabolic enzymes implicated in SARS-CoV-2 infection. Together, these results demonstrate that genetically encoded, site-specific photo-crosslinking enables selective capture of transient interactions that are often missed by nonspecific 254 nm UV crosslinking approaches and highlights Orf3a as a multifunctional protein that engages diverse host pathways. More broadly, this study establishes a generalizable framework for leveraging unnatural amino acid-based protein engineering approaches to interrogate dynamic host-pathogen interactions.

Humans↗
PubMed2001

Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients.

Posttransplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplantation recipients that is caused by iatrogenic suppression of T-cell function. The diagnostic value of weekly EBV DNA load monitoring was investigated in prospectively collected unfractionated whole blood and serum samples of lung transplantation (LTx) recipients with and without PTLD. In PTLD patients, 78% of tested whole blood samples were above the cut-off value of quantitative competitive polymerase chain reaction (Q-PCR) (greater than 2000 EBV DNA copies per mL blood), with the majority of patients having high viral loads before and at PTLD diagnosis. Especially in a primary EBV-infected patient and in patients with conversion of immunosuppressive treatment, rapid increases in peripheral blood EBV DNA load diagnosed and predicted PTLD. In non-PTLD transplantation recipients, only 3.4% of the whole blood samples was above the cut-off value (P <.0001) despite heavy immune suppression and cytomegalovirus (CMV)-related disease. These findings illustrate the clinical importance of frequent EBV DNA load monitoring in LTx recipients. The increased EBV DNA loads in PTLD patients were restricted to the cellular blood compartment, as parallel serum samples were all below cut-off value, which indicates absence of lytic viral replication. EBV(+) cells in PTLD patients have a very short doubling time, which can be as low as 56 hours, thereby creating the need for high screening frequency in high-risk patients. Furthermore, it is shown that EBV and CMV can reactivate independently in LTx recipients and that EBV DNA load monitoring may be useful in discriminating PTLD from rejection.

Adult↗
PubMed1998

At the onset of transformation polyomavirus middle-T recruits shc and src to a perinuclear compartment coincident with condensation of endosomes.

To examine the early cellular changes that accompany transformation, middle-T antigen (the transforming protein of polyomavirus), was expressed from an inducible promoter in Rat2 fibroblasts and in normal diploid human fibroblast (MRC5) cells using a replication defective adenovirus vector. The earliest detectable expression of middle-T antigen correlated closely with the initial changes in cellular morphology. At this time, expression of middle-T antigen resulted in the redistribution of shc and src to a brefeldin A resistant perinuclear compartment coincident with the formation of kinase active complexes containing middle-T antigen, shc and src. The first detectable changes directly related to altered morphology was reorganization of actin filaments, and the condensation of tubular endosomes in the perinuclear region of the cell. These results suggest a potential role for perinuclear localized mT molecules in some of the morphologic changes associated with the initial phase of cellular transformation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects.

Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4(+) and/or CD8(+) T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average approximately 10% of both the CD4(+) and CD8(+) memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8(+) T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

Adult↗