Immunoproteomic analyses of outer membrane antigens of Actinobacillus pleuropneumoniae grown under iron-restricted conditions.

Actinobacillus pleuropneumoniae, a bacterial pathogen of swine and agent of porcine pneumonia, causes a highly infectious disease of economic importance in the pig industry. Commercial vaccines for A. pleuropneumoniae include whole-cell bacterins and second generation subunit vaccines but they only confer partial protective immunity.

Role of Aspergillus niger acrA in arsenic resistance and its use as the basis for an arsenic biosensor.

Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS).

"Combi-targeting" mitozolomide: conferring novel signaling inhibitory properties to an abandoned DNA alkylating agent in the treatment of advanced prostate cancer.

Purpose: At the preclinical stage, mitozolomide (MTZ) showed exciting preclinical activity but failed later in clinical trial due to toxic side effects. We surmised that by targeting MTZ to epidermal growth factor receptor (EGFR), we may not only alter its toxicity profile, but also enhance its potency in EGFR-overexpressing tumors. To test this hypothesis, we designed JDF12, studied its mechanism of action in human prostate cancer (PCa) cells and determined its potency in vivo.

OmpT outer membrane proteases of enterohemorrhagic and enteropathogenic Escherichia coli contribute differently to the degradation of human LL-37.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are food-borne pathogens that cause serious diarrheal diseases. To colonize the human intestine, these pathogens must overcome innate immune defenses such as antimicrobial peptides (AMPs).

Interaction between MMACHC and MMADHC, two human proteins participating in intracellular vitamin B₁₂ metabolism.

The identification of eight genes involved in inherited cobalamin (Cbl) disorders has provided insight into the complexity of the vitamin B₁₂ trafficking pathway. Detailed knowledge about the structure, interaction, and physiological functions for many of the gene products, including the MMACHC and MMADHC proteins, is lacking. Having cloned, expressed, and purified MMACHC in Escherichia coli, we demonstrated its monodispersity by dynamic light scattering and measured its hydrodynamic radius, either alone or in complex with each of four vitamin B₁₂ derivatives.

Control of cell survival and proliferation by mammalian eukaryotic initiation factor 4B.

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5' untranslated region (5'UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi).

Transcobalamin in cultured fibroblasts from patients with inborn errors of vitamin B12 metabolism.

Derivatives of vitamin B(12) (cobalamin, Cbl) are required for activity of the mitochondrial enzyme L-methylmalonyl-CoA mutase and the cytoplasmic enzyme methionine synthase in human cells. We recently described a putative novel Cbl-binding protein in crude mitochondrial fractions isolated from cultured fibroblasts. The amount of Cbl bound to this protein varied in fibroblasts from patients with different genetic defects affecting cobalamin metabolism.

Outer membrane proteome of Actinobacillus pleuropneumoniae: LC-MS/MS analyses validate in silico predictions.

The Gram-negative bacterial pathogen Actinobacillus pleuropneumoniae causes porcine pneumonia, a highly infectious respiratory disease that contributes to major economic losses in the swine industry. Outer membrane (OM) proteins play key roles in infection and may be targets for drug and vaccine research. Exploiting the genome sequence of A.

Tryptophan 32 potentiates aggregation and cytotoxicity of a copper/zinc superoxide dismutase mutant associated with familial amyotrophic lateral sclerosis.

One familial form of the neurodegenerative disease, amyotrophic lateral sclerosis, is caused by gain-of-function mutations in the gene encoding copper/zinc superoxide dismutase (SOD-1). This study provides in vivo evidence that normally occurring oxidative modification to SOD-1 promotes aggregation and toxicity of mutant proteins. The oxidation of Trp-32 was identified as a normal modification being present in both wild-type enzyme and SOD-1 with the disease-causing mutation, G93A, isolated from erythrocytes.

The combi-targeting concept: in vitro and in vivo fragmentation of a stable combi-nitrosourea engineered to interact with the epidermal growth factor receptor while remaining DNA reactive.

Purpose: JDA58 (NSC 741282), a "combi-molecule" optimized in the context of the "combi-targeting concept," is a nitrosourea moiety tethered to an anilinoquinazoline. Here, we sought to show its binary epidermal growth factor receptor (EGFR)/DNA targeting property and to study its fragmentation in vitro and in vivo.

Experimental Design: The fragmentation of JDA58 was detected in cells in vitro and in vivo by fluorescence microscopy and tandem mass spectrometry.

Mass spectrometric quantitation of C-reactive protein using labeled tryptic peptides.

Given the extensive efforts applied toward proteomics and research in biomarkers, methods for the simultaneous measurement of proteins, peptides, metabolic intermediates, hormones, etc. in a complex sample may be required in the foreseeable future. Assays based on mass spectrometric detection may be suitable for meeting the demands of such complex samples with sensitivity and specificity.

Regulation of cytochrome P450 by posttranslational modification.

Cytochrome P450s are a family of enzymes represented in all kingdoms with expression in many species. Over 3,000 enzymes have been identified in nature. Humans express 57 putatively functional enzymes with a variety of critical physiological roles.

Thrombospondin 1, produced by endothelial cells under the action of erythropoietin, stimulates thymidine incorporation into erythroid cells and counteracts the inhibitory action of insulin-like growth factor binding protein 3.

The nature of erythropoietin (EPO)-dependent, erythroid cell regulatory factors secreted by endothelial cells is largely unknown. The production of thrombospondin 1 (TSP-1) and insulin-like growth factor binding protein 3 (IGFBP-3) is increased in cultures of human umbilical vein endothelial cells (HUVEC) incubated with erythropoietin (EPO). Simultaneous incubation of HUVEC with EPO and interleukin 3 (IL-3) resulted in a decreased production, suggesting that both TSP-1 and IGFBP-3 belong to the EPO- and IL-3-dependent erythroid regulatory factors previously described in cultures of bone marrow endothelial cells.

Complete chemical and enzymatic treatment of phosphorylated and glycosylated proteins on ProteinChip arrays.

A simple and quick protocol for chemical treatment, enzymatic digestion, and subsequent identification of proteins on ProteinChip arrays is presented. Chicken ovalbumin, bovine fetuin and a heavily posttranslationally modified protein, human epidermal growth factor receptor extracellular domain, were employed as model proteins to evaluate the novel protocol. The chemical treatment includes denaturation, reduction, and alkylation, while enzymatic digestion encompasses deglycosylation, dephosphorylation, and digestion by various proteases.

Sensitive capillary chromatography mass spectrometric methods for the determination of salcatonin in human biological matrices.

New methods employing capillary liquid chromatography in combination with time-of-flight mass spectrometry (microLC-TOF/MS) were developed for the rapid determination of salcatonin in human urine and plasma. The present approaches utilize (13)C(6)-leucine (19)-labeled salcatonin as internal standard, small matrix volumes and simple sample preparation procedures. They allow TOF/MS to be used as a highly selective detector for providing accurate quantitation of salcatonin.

Utilization of a new biotinylation reagent in the development of a nondiscriminatory investigative approach for the study of cell surface proteins.

In order to circumvent the various problems encountered during the study of membrane-bound proteins, we designed and synthesized a novel membrane-impermeable biotinylation reagent incorporating chemical properties compatible with this goal. We then developed a nondiscriminatory analytical procedure for such studies which overcomes possible selectivity, contamination and solubility problems. The necessary steps (labeling, limited in situ proteolysis, affinity purification) are all conducted in mild or near native conditions.

The C-terminal peptide of thrombospondin-4 stimulates erythroid cell proliferation.

Erythropoietin (EPO) stimulates the production of small erythroid cell stimulating factors (molecular weight <5 kDa) in cultures of bone marrow endothelial cells. We identified a fragment of thrombospondin-4 (TSP-4) as an EPO-stimulated protein in endothelial cell lysates. Pre-incubation of the low molecular weight fractions from supernatants of EPO-treated umbilical cord endothelial cells (HUVEC) with antibodies against the C-terminal residues of TSP-1,2 and TSP-4 decreased the erythroid cell stimulating activity.

Substance P and neurotensin are up-regulated in the lumbar spinal cord of animals with neuropathic pain.

Many neuropeptides have been shown to be up-regulated in response to pain. The purpose of this study was to identify pain-related peptides in a rat model of neuropathic pain induced by sciatic nerve cuff implantation. Rats were tested for touch sensitivity prior to and 7 days following cuff implantation or sham surgeries.

Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae.

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of approximately 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A.

Bioleaching of heavy metals from a low-grade mining ore using Aspergillus niger.

The main concern of this study is to develop a feasible and economical technique to microbially recover metals from oxide low-grade ores. Owing to the significant quantities of metals that are embodied in low-grade ores and mining residues, these are potential viable sources of metals. In addition, they potentially endanger the environment, as the metals they contain may be released to the environment in hazardous form.