Papers by James J. Valdes
Psychology, Health & Medicine, 2015
Frontiers in microbiology, 2016
Protein phosphatase 1 (PP1c) is one of the main phosphatases whose function is shaped by many reg... more Protein phosphatase 1 (PP1c) is one of the main phosphatases whose function is shaped by many regulators to confer a specific location and a selective function for this enzyme. Here, we report that eukaryotic initiation factor 2β of Plasmodium falciparum (PfeIF2β) is an interactor of PfPP1c. Sequence analysis of PfeIF2β revealed a deletion of 111 amino acids when compared to its human counterpart and the presence of two potential binding motifs to PfPP1 ((29)FGEKKK(34), (103)KVAW(106)). As expected, we showed that PfeIF2β binds PfeIF2γ and PfeIF5, confirming its canonical interaction with partners of the translation complex. Studies of the PfeIF2β-PfPP1 interaction using wild-type, single and double mutated versions of PfeIF2β revealed that both binding motifs are critical. We next showed that PfeIF2β is able to induce Germinal Vesicle Break Down (GVBD) when expressed in Xenopus oocytes, an indicator of its capacity to regulate PP1. Only combined mutations of both binding motifs abo...
Analytical Letters, Aug 18, 2006
Small molecules, peptides, and proteins can be imprinted using mixtures of organic silanes. Molec... more Small molecules, peptides, and proteins can be imprinted using mixtures of organic silanes. Molecular imprints may serve as artificial receptors, i.e., biosensor sensing elements for detection of chemical and biological toxins, drugs, and environmental hazards. One method for detection of imprint-bound molecules is fluorescence. Molecular imprints to N-acetyltryptophanamide (NATA) and fluorescein were prepared, and their respective binding constants determined using
Toxicol Lett, 1998
Non-cholinesterase effects of organophosphate anticholinesterases were investigated using a Cytos... more Non-cholinesterase effects of organophosphate anticholinesterases were investigated using a Cytosensor microphysiometer, which continuously monitors potential cytotoxic effects of compounds on cell metabolic activity. Human neuroblastoma and liver cells exposed to ...
Analytical Biochemistry, Nov 1, 1989
An acetylcholine receptor-based optical biosensor was developed, which uses the evanescent wave t... more An acetylcholine receptor-based optical biosensor was developed, which uses the evanescent wave to excite fluorescein isothiocyanate-labeled alpha-bungarotoxin (FITC-alpha-BGT), that is bound to a receptor protein immobilized on the surface of a quartz fiber. After excitation of the fluorophore just outside the waveguide boundary, the resultant fluorescence was trapped by and propagated back up the fiber. Pure nicotinic acetylcholine receptor (nAChR) protein, isolated from Torpedo electric organ, was immobilized noncovalently on quartz optic fibers and its density was quantitated by 125I-BGT binding. In the absence of nAChR, FITC-alpha-BGT alone bound to the quartz fiber. This nonspecific (i.e., nonreceptor) binding of FITC-alpha-BGT was totally eliminated by the addition of 0.1 mg/ml bovine serum albumin to the phosphate-buffered saline medium. This solution did not interfere with FITC-alpha-BGT binding to the nAChR that was immobilized on the fiber. Thus, only specific binding was observed under these conditions, resulting in a very high signal-to-noise ratio of greater than 99. Specific FITC-alpha-BGT binding to the nAChR protein on the optic fibers was inhibited by agonists (e.g., acetylcholine, nicotine, and carbamylcholine) and antagonists (e.g., pancuronium and d-tubocurarine) of the nAChR and was insensitive to high salt concentrations (e.g., 154 mM NaCl). The binding was most sensitive to the highly nAChR-specific alpha-BGT and alpha-cobratoxin. The optic-fiber sensor in its present form does not distinguish between receptor agonists or antagonist and cannot be regenerated for repeated use.(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular and Cellular Probes, Apr 1, 2003
The bioterrorism threat is perceived to be a real challenge to our nation's security. This threat... more The bioterrorism threat is perceived to be a real challenge to our nation's security. This threat has necessitated the design of better and faster assays for the detection of biothreat agents including staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. This study describes a simple, fast and highly sensitive fluorescence-based immunoassay, in which the antibody is fluorescently-labeled for use in this assay. Use of labeled antibodies resulted in very low level of detection of SEB, 100 pg/well. This method is four times faster than classical and conventional enzyme-linked immunosorbent assay (ELISA). q
Molecular Biotechnology, Apr 17, 2007
Infection of insect cells with baculovirus expression constructs is commonly used to produce reco... more Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress TM ) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (preoccluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress TM system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.
In Vitro Cellular Developmental Biology Animal, Nov 1, 1999
Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neu... more Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatoeytes, which was detectable by the Cytosensor ® microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 [aM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose-and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 [aM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma ceils. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LCs0 of 65, 775, 640, 340, or 672 [aM, respectively, whereas 24 h gave an LCs0 of 0.7, 3.7, 2.5, 29, and 31 [aM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxieity. There was no correlation between OP in vivo neurotoxieity and in vitro eytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.
Polymerase chain reaction (PCR) is an exquisitely sensitive method for the amplification and dete... more Polymerase chain reaction (PCR) is an exquisitely sensitive method for the amplification and detection of genetic material (DNA and RNA sequences). We have developed several assays using a variation of PCR that proceeds very quickly and allows the monitoring of the progress of the reaction in real time. We describe here assays for the detection of the gene encoding staphylococcal enterotoxin A (SEA), a toxin produced by the bacterium Staphylococcus aureus and an agent responsible for a significant fraction of food poisoning incidents worldwide. We also describe several assays for the detection of a simulant of viral pathogens, the bacteriophage MS2.
Expert Review of Vaccines, 2016
Journal of Bacteriology, Aug 15, 2007
The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a ... more The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Because LsrR and LsrK directly regulate AI-2 uptake, we hypothesized that they might play a wider role in regulating other QS-related cellular functions. In this study, we characterized physiological changes due to the genomic deletion of lsrR and lsrK. We discovered that many genes were coregulated by lsrK and lsrR but in a distinctly different manner than that for the lsr operon (where LsrR serves as a repressor that is derepressed by the binding of phospho-AI-2 to the LsrR protein). An extended model for AI-2 signaling that is consistent with all current data on AI-2, LuxS, and the LuxS regulon is proposed. Additionally, we found that both the quantity and architecture of biofilms were regulated by this distinct mechanism, as lsrK and lsrR knockouts behaved identically. Similar biofilm architectures probably resulted from the concerted response of a set of genes including flu and wza, the expression of which is influenced by lsrRK. We also found for the first time that the generation of several small RNAs (including DsrA, which was previously linked to QS systems in Vibrio harveyi) was affected by LsrR. Our results suggest that AI-2 is indeed a QS signal in E. coli, especially when it acts through the transcriptional regulator LsrR.
Toxicon, Mar 1, 1998
... Directorate, US Army, Edgewood Research Development and Engineering Center, Aberdeen Proving ... more ... Directorate, US Army, Edgewood Research Development and Engineering Center, Aberdeen Proving Ground, Aberdeen, MD 21010, USA. Received 26 June 1997; accepted 5 September 1997. Available online 06 September 2005. Abstract. The sea nettle jellyfish toxin (SNTX ...
Epigenetics, Jan 28, 2016
Epigenetic mechanisms have not been characterized in ticks despite their importance as vectors of... more Epigenetic mechanisms have not been characterized in ticks despite their importance as vectors of human and animal diseases worldwide. The objective of this study was to characterize the histones and histone modifying enzymes (HMEs) of the tick vector Ixodes scapularis and their role during Anaplasma phagocytophilum infection. We first identified 5 histones and 34 HMEs in I. scapularis in comparison with similar proteins in model organisms. Then, we used transcriptomic and proteomic data to analyze the mRNA and protein levels of I. scapularis histones and HMEs in response to A. phagocytophilum infection of tick tissues and cultured cells. Finally, selected HMEs were functionally characterized by pharmacological studies in cultured tick cells. The results suggest that A. phagocytophilum manipulates tick cell epigenetics to increase I. scapularis p300/CBP, histone deacetylase, and Sirtuin levels, resulting in an inhibition of cell apoptosis that in turn facilitates pathogen infection ...
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Papers by James J. Valdes