HLA-restricted lysis of herpes simplex virus-infected monocytes and macrophages mediated by CD4+ and CD8+ T lymphocytes

Freshly isolated human peripheral blood monocytes and in vitro monocyte-derived macrophages were infected with HSV type 1 and used as target cells in a cell-mediated cytotoxicity assay. PBMC from both HSV-immune and non-immune donors were stimulated in vitro for 5 days with UV-inactivated HSV Ag and used as effector cells. Effectors from HSV-immune donors mediated virus-specific lysis of both monocyte and macrophage targets, whereas effectors from non-immune donors failed to mediate target cell lysis. Mean virus-specific lysis of autologous monocytes was (8.5 +/- (+/- 2.0)%) compared to a threefold greater virus-specific lysis of autologous macrophages (24.7 (+/- 4.3)%). More than 70% of this lysis was mediated by CD16- T lymphocytes. Further analysis demonstrated that the majority of the lysis against autologous and allogeneic targets was HLA-DR-restricted and mediated by CD4+ CTL. However, CD8+ CTL also contributed to the lysis of autologous targets as well as allogeneic targets having a common HLA-A and/or -B determinant. The HLA-restricted cytotoxicity was virus-specific as HSV-infected, but not CMV-infected, cells were lysed. CTL-mediated lysis of HSV-infected monocytes and macrophages may be of significance in the anti-viral and immunoregulatory host response.     

A comparative study of FMS tool allocation and part type selection approaches for a varying part type mix

Hankins and Rovito (1984) examined the impact of different tool policies on cutting tool inventory levels and spindle utilization for a flexible manufacturing system (FMS). This study provides a broader perspective of the impact of tool allocation approaches on flow times, tardiness, percent of orders tardy, machine utilization, and robot utilization. Part type selection procedures have been suggested for the FMS prerelease planning problem. However, very little research has specifically evaluated the part type selection procedures across different tool allocation approaches. Also, with the exception of Stecke and Kim (1988, 1991) no other known study has provided any insights on what tool allocation approaches are appropriate when processing different mixes of part types. This research is devoted to addressing those issues. Three tool allocation approaches, three production scheduling rules, and three levels of part mix are evaluated in this study through a similation model of a flexible manufacturing system. The specific impacts of the tool approaches, their interaction effects with the part type selection rules, and their effectiveness at different part type mix levels are provided through the use of a regression metamodel.     

Reasons for Missing Antiretroviral Therapy: Results from a Multi-Country Study in Tanzania,Uganda, and Zambia

Objectives

To identify the reasons patients miss taking their antiretroviral therapy (ART) and the proportion who miss their ART because of symptoms; and to explore the association between symptoms and incomplete adherence.

Methods

Secondary analysis of data collected during a cross-sectional study that examined ART adherence among adults from 18 purposefully selected sites in Tanzania, Uganda, and Zambia. We interviewed 250 systematically selected patients per facility (≥18 years) on reasons for missing ART and symptoms they had experienced (using the HIV Symptom Index). We abstracted clinical data from the patients’ medical, pharmacy, and laboratory records. Incomplete adherence was defined as having missed ART for at least 48 consecutive hours during the past 3 months.

Results

Twenty-nine percent of participants reported at least one reason for having ever missed ART (1278/4425). The most frequent reason was simply forgetting (681/1278 or 53%), followed by ART-related hunger or not having enough food (30%), and symptoms (12%). The median number of symptoms reported by participants was 4 (IQR: 2–7). Every additional symptom increased the odds of incomplete adherence by 12% (OR: 1.1, 95% CI: 1.1–1.2). Female participants and participants initiated on a regimen containing stavudine were more likely to report greater numbers of symptoms.

Conclusions

Symptoms were a common reason for missing ART, together with simply forgetting and food insecurity. A combination of ART regimens with fewer side effects, use of mobile phone text message reminders, and integration of food supplementation and livelihood programmes into HIV programmes, have the potential to decrease missed ART and hence to improve adherence and the outcomes of ART programmes.     

Caenorhabditis elegans,a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown.Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C. elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans.This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.     

Interferon alpha but not interleukin 12 activates STAT4 signaling in human vascular endothelial cells

STAT4 signaling, activated by either interleukin 12 (IL12) or interferon alpha (IFNalpha), promotes T(H)1 responses in CD4(+) T cells. Vascular endothelial cells (EC) may also become polarized in response to various cytokines, favoring recruitment and activation of T(H)1 or T(H)2 effector cells. Here we have investigated the role of the STAT4 pathway in EC. Cultured human umbilical vein EC (HUVEC) express low levels of STAT4, which may be tyrosine-phosphorylated by treatment with IFNalpha but not IL12. This is because HUVEC lack both subunits of the IL12 receptor (IL12Rbeta1 and IL12Rbeta2), even following treatment with various cytokines. IL12 phosphorylation of STAT4 can be observed in HUVEC that have been transduced to express the IL12R. To identify STAT4-induced genes we pursued three approaches: analysis by DNA microarray and quantitative RT-PCR (Q-PCR) of the IL12 responses in IL12R-transduced EC; analysis by Q-PCR of IFNalpha responses in STAT4-overexpressing EC; and analysis of IFNalpha responses in U3A neuroblastoma cell lines that express either STAT1 or STAT4, but not both. In all three instances we observe STAT4-mediated induction of the chemokine monocyte chemoattractant protein 1 (MCP1) and suppressor of cytokine signaling 3 (SOCS3) mRNA, and we confirm the production of each protein in both IL12R-transduced EC and STAT4-transduced U3A cells. These observations reveal that there is a STAT4 response of EC, activated by IFNalpha but not IL12, and that it may modulate the pro-inflammatory behavior of EC.     

Genomic insights into Mn(II) oxidation by the marine alphaproteobacterium Aurantimonas sp. strain SI85-9A1

Microbial Mn(II) oxidation has important biogeochemical consequences in marine, freshwater, and terrestrial environments, but many aspects of the physiology and biochemistry of this process remain obscure. Here, we report genomic insights into Mn(II) oxidation by the marine alphaproteobacterium Aurantimonas sp. strain SI85-9A1, isolated from the oxic/anoxic interface of a stratified fjord. The SI85-9A1 genome harbors the genetic potential for metabolic versatility, with genes for organoheterotrophy, methylotrophy, oxidation of sulfur and carbon monoxide, the ability to grow over a wide range of O(2) concentrations (including microaerobic conditions), and the complete Calvin cycle for carbon fixation. Although no growth could be detected under autotrophic conditions with Mn(II) as the sole electron donor, cultures of SI85-9A1 grown on glycerol are dramatically stimulated by addition of Mn(II), suggesting an energetic benefit from Mn(II) oxidation. A putative Mn(II) oxidase is encoded by duplicated multicopper oxidase genes that have a complex evolutionary history including multiple gene duplication, loss, and ancient horizontal transfer events. The Mn(II) oxidase was most abundant in the extracellular fraction, where it cooccurs with a putative hemolysin-type Ca(2+)-binding peroxidase. Regulatory elements governing the cellular response to Fe and Mn concentration were identified, and 39 targets of these regulators were detected. The putative Mn(II) oxidase genes were not among the predicted targets, indicating that regulation of Mn(II) oxidation is controlled by other factors yet to be identified. Overall, our results provide novel insights into the physiology and biochemistry of Mn(II) oxidation and reveal a genome specialized for life at the oxic/anoxic interface.     

Tau Kinetics in Neurons and the Human Central Nervous System

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Cu(II) organizes beta-2-microglobulin oligomers but is released upon amyloid formation

beta-2-Microglobulin (beta2m) is deposited as amyloid fibrils in the bones and joints of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that biologically relevant amounts of Cu(II) can cause beta2m to be converted to amyloid fibrils under physiological conditions in vitro. In this work, dynamic light scattering, mass spectrometry, and size-exclusion chromatography are used to characterize the role that Cu plays in the formation of oligomeric intermediates that precede fibril formation. Cu(II) is found to be necessary for the stability of the dimer and an initial form of the tetramer. The initially formed tetramer then undergoes a structural change to a state that no longer binds Cu(II) before progressing to a hexameric state. Based on these results, we propose that the lag phase associated with beta2m fibril formation is partially accounted for by the structural transition of the tetramer that results in Cu(II) loss. Consistent with this observation is the determination that the mature beta2m amyloid fibrils do not contain Cu. Thus, Cu(II) appears to play a catalytic role by enabling the organization of the necessary oligomeric intermediates that precede beta2m amyloid formation.     

Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples.     

Mn(II) Oxidation Is Catalyzed by Heme Peroxidases in “Aurantimonas manganoxydans” Strain SI85-9A1 and Erythrobacter sp. Strain SD-21

A new type of manganese-oxidizing enzyme has been identified in two alphaproteobacteria, “Aurantimonas manganoxydans” strain SI85-9A1 and Erythrobacter sp. strain SD-21. These proteins were identified by tandem mass spectrometry of manganese-oxidizing bands visualized by native polyacrylamide gel electrophoresis in-gel activity assays and fast protein liquid chromatography-purified proteins. Proteins of both alphaproteobacteria contain animal heme peroxidase and hemolysin-type calcium binding domains, with the 350-kDa active Mn-oxidizing protein of A. manganoxydans containing stainable heme. The addition of both Ca²⁺ ions and H₂O₂ to the enriched protein from Aurantimonas increased manganese oxidation activity 5.9-fold, and the highest activity recorded was 700 μM min⁻¹ mg⁻¹. Mn(II) is oxidized to Mn(IV) via an Mn(III) intermediate, which is consistent with known manganese peroxidase activity in fungi. The Mn-oxidizing protein in Erythrobacter sp. strain SD-21 is 225 kDa and contains only one peroxidase domain with strong homology to the first 2,000 amino acids of the peroxidase protein from A. manganoxydans. The heme peroxidase has tentatively been named MopA (manganese-oxidizing peroxidase) and sheds new light on the molecular mechanism of Mn oxidation in prokaryotes.Mn(III,IV) oxides (Mn oxides) and soluble Mn(III) complexes are the strongest oxidizing agents in the environment after oxygen and play an important role in many biogeochemical cycles (25). At pH 7, they can oxidize metals, catalyze the formation of humic substances and organic nitrogen complexes, and oxidatively degrade humic and fulvic acids to bioavailable low-molecular-weight organic compounds (6, 38, 40). Geochemical cycling of Mn oxides can also control the distribution of many trace elements, as Mn minerals are highly charged and can adsorb and concentrate metals (22). In the pH range of aerobic natural waters (pH 6 to 8), chemical oxidation of Mn(II) is slow, but in the presence of Mn(II)-oxidizing microorganisms, the rate can be 4 to 5 orders of magnitude higher (30, 39, 48).Multicopper oxidases (MCOs) are the only identified proteins from bacteria capable of manganese oxidation. These enzymes are a class of proteins that utilize copper as a cofactor to perform four one-electron substrate oxidations, thereby reducing molecular oxygen to H₂O (37). Generally, MCOs oxidize organic compounds such as phenolics, but some fungal MCOs (laccases) that can oxidize Mn(II) to Mn(III) and Mn(IV) have been described previously (21, 36, 28). Bacterial MCOs involved with Mn oxidation have been genetically identified in Pseudomonas putida strains MnB1 and GB1 (cumA), Leptothrix discophora SS-1 (mofA), Bacillus sp. strain SG-1 (mnxG), and the alphaproteobacterium Pedomicrobium sp. strain ACM 3067 (moxA) (35). None of these MCOs share significant homology except for their copper binding motifs (35), and only in Bacillus sp. (11) have MCOs been directly linked to Mn oxidation.Another class of proteins known in eukaryotes to oxidize manganese, but not commonly identified to be involved in bacterial Mn oxidation, are heme-containing manganese peroxidases (MnPs) (5, 33). These enzymes are extremely important for the degradation activities of lignin-degrading fungi. The MnP from the basidiomycete Phanerochaete chrysosporium has a single Mn(II) binding site near the heme and produces two Mn(III) equivalents at the expense of one H₂O₂ equivalent (18, 34, 45). MnPs and MCOs are able to work in concert, with the MnP utilizing H₂O₂ produced by the MCO-catalyzed Mn(II) oxidation (36). Both types of protein produce Mn(III). While MnPs are best known to occur in fungi, a similar mechanism has been reported for a catalase/peroxidase from Mycobacterium (27), and a catalase-peroxide mechanism was suggested to be involved in Fe and Mn oxidation in Arthrobacter (13).A search for MCO-like genes in the draft genome sequence of the Mn(II)-oxidizing marine alphaproteobacterium “Aurantimonas manganoxydans” strain SI85-9A1 revealed duplicate copies of moxA-like genes (12). Initial attempts to isolate the Mn oxidase enzyme focused on the secreted proteins. Two regions were identified by a native polyacrylamide gel electrophoresis (PAGE) in-gel activity assay to have active Mn(II) oxidation, one at >250 kDa and the other at approximately 50 kDa. The active areas were excised from the gel, digested with trypsin, and analyzed with tandem mass spectrometry (MS/MS) but no peptides could be identified (12). In the same study, five bands from a corresponding Coomassie-stained sodium dodecyl sulfate (SDS)-PAGE gel returned identifications of peptides from a putative Ca²⁺ binding heme peroxidase, but no attempt was made to connect this protein to the active bands from the in-gel activity assay because they would have migrated differently (Coomassie bands were denatured protein, the in-gel activity assay native proteins).Based on the genome data, the expected size of the MoxA-like proteins was approximately 50 kDa, similar to the size of MoxA from Pedomicrobium sp. strain ACM 3067 and similar in size to the most active region from the in-gel activity assay. It was then inferred that the Mn oxidase from A. manganoxydans strain SI85-9A1 could be a Mox ortholog with an estimated size of 50 kDa that may be part of a larger >250-kDa holoenzyme (12). The experimentally identified Ca²⁺ binding heme peroxidase was suggested to be involved with the biodegradation of complex organics utilizing H₂O₂ abiotically generated by the Mn(III) produced by the Mn(II)-oxidizing MoxA-like protein (after the mechanism described by Schlosser and Höfer [36]) (12).In Erythrobacter sp. strain SD-21, the Mn oxidase enzyme was found in both the soluble and excreted fractions, suggesting that the activity may be loosely associated with the cell surface (23). Protein chromatography was employed to identify the Mn oxidase in this organism but did not conclusively implicate an MCO. The enzyme was partially purified and was found to be associated with a quinone cofactor, PQQ, that stimulated manganese oxidation in partially pure protein extracts and rescued the manganese oxidation activity in a mutant strain of Pseudomonas putida MnB1. Mn oxidation was not stimulated in vitro when copper was added, and activity was vastly decreased in the presence of MCO and quinone inhibitors. The addition of o-phenanthroline, a copper chelator and potent inhibitor of Mn oxidation in P. putida GB-1 (32), inhibited Mn oxidation only partially at concentrations far in excess of those required for GB-1. The absorbance spectrum of the partially purified protein extract did not show characteristics of an MCO, and the activity of the cell extract was between 7- and 30-fold higher than the activity measured for Mn-oxidizing organisms containing MCOs. Although the evidence pointed away from MCO involvement, H₂O₂ did not stimulate activity as expected if the enzyme was an MnP (23).A. manganoxydans strain SI85-9A1 is not easily amenable to genetic techniques, and thus, isolation of the manganese oxidase was performed through protein chromatography techniques. Since early studies failed to conclusively identify the Mn oxidase, it was decided to fractionate the proteins in the organism to localize the activity and purify the protein from the active fraction. In this work, we report the significant purification of the Mn-oxidizing protein leading to its identification as an animal heme peroxidase with multiple calcium binding motifs, and localization of the protein to the outer membrane as a peripheral membrane protein. We revisit the Mn oxidase from Erythrobacter sp. strain SD-21 (also genetically recalcitrant) and identify the protein from an active Mn-oxidizing band with a native PAGE in-gel activity assay. This protein is also an animal heme peroxidase with calcium binding motifs.