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RNA accessible sites are the regions in an RNA molecule that are available for hybridization with cDNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of ~75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array composed of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner. 10.1021/ac9015962

African sleeping sickness is a fatal disease that is caused by the protozoan parasite Trypanosoma brucei. Polyamine biosynthesis is an essential pathway in the parasite and is a validated drug target for treatment of the disease. S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in polyamine biosynthesis. Here, we show that trypanosomatids uniquely contain both a functional AdoMetDC and a paralog designated prozyme that has lost catalytic activity. The T. brucei prozyme forms a high-affinity heterodimer with AdoMetDC that stimulates its activity by 1,200-fold. Both genes are expressed in T. brucei, and analysis of AdoMetDC activity in T. brucei extracts supports the finding that the heterodimer is the functional enzyme in vivo. Thus, prozyme has evolved to be a catalytically dead but allosterically active subunit of AdoMetDC, providing an example of how regulators of multimeric enzymes can evolve through gene duplication and mutational drift. These data identify a distinct mechanism for regulating AdoMetDC in the parasite that suggests new strategies for the development of parasite-specific inhibitors of the polyamine biosynthetic pathway. gene duplication | S-adenyosylmethionine decarboxylase | prozyme | Trypanosoma brucei

A powerful approach to relative quantification by mass spectrometry is to employ labeling reagents that target specific functional groups in molecules of interest. A quantitative comparison of two or more samples may be readily accomplished by using a chemically identical but isotopically distinct labeling reagent for each sample. The samples may then be combined, subjected to purification steps, and mass analyzed. Comparison of the signal intensities obtained from the isotopically labeled variants of the target analyte(s) provides quantitative information on their relative concentrations in the sample. In this report, we describe the synthesis and use of heavy and light isotopic forms of methyl acetimidate for the relative quantification of amine-containing species. The principal advantages of methyl acetimidate as a labeling reagent are that the reaction product is positively charged and hydrophobicity is increased, both of which enhance electrospray ionization efficiency and increase detection sensitivity. The quantitative nature of the analysis was demonstrated in model metabolomics experiments in which heavy and light labeled Arabidopsis extracts were combined in different ratios. Finally, the labeling strategy was employed to determine differences in the amounts of amine-containing metabolites for Arabidopsis seeds germinated under two different conditions.

Mutations in both the putrescine-binding site and the distant enzyme active site are generated to probe the structural basis by which putrescine stimulates Trypanosoma cruzi S-adenosylmethionine decarboxylase (AdoMetDC). Mutations of residues in the putrescine-binding site that results in reduced and enhanced catalytic activity are identified, providing evidence for coupling between residues in the putrescine-binding site and the active site, indicating allosteric regulation.

Records of restaurant inspections by public health departments provide sequential "snapshots" of conditions in retail food service establishments that can be used to identify risk factors and evaluate the effectiveness of interventions. Data from a random 10 percent sample of restaurant inspection files from 31 counties in Oklahoma, including 4,044 inspections conducted during 1996-2000 in "medium-risk" and "high-risk" establishments, were analyzed to determine rates of critical violations and recurrent violations for different categories of establishments. Repeat violations accounted for about half of all violations. Establishments subjectively designated as high risk by health department personnel were in fact found to have higher violation rates than those described as medium-risk establishments. Outside Oklahoma County, regional chain restaurants were significantly more likely than other restaurants to have recurrent violations of critical items related to food-holding temperature, hygiene practices, sanitization, and hygiene facilities. Differences observed in violation rates among individual establishments were not primarily attributable to inconsistent enforcement by individual inspectors; rather, they appeared to be indicative of real differences in hygienic conditions and practices., Introduction Restaurant inspection is a public health activity intended to prevent foodborne illness. In addition to its primary function as a public health intervention, the inspection process generates a written [...]

Ornithine decarboxylase (ODC) is an obligate homodimer that catalyzes the pyridoxal 5'-phosphate-dependent decarboxylation of L-ornithine to putrescine in polyamine biosynthesis. It is reported that ODC is investigated as a therapeutic target for a number of proliferative diseases and has proved to be an effective target in the treatment of African sleeping sickness, a fatal tropical disease.

Research has been conducted on S-adenosylmethionine decarboxylase from Trypanosoma cruzi. The role of putrescine in polypeptide processing reaction activation and enzyme activity in S-adenosylmethionine decarboxylase has been investigated and the details are presented.

Research has been conducted on the gamma-glutamylcysteine synthetase which catalyzes ATP-dependent L-Glu and L-Cys ligation. The role of the amino acid residues in the kinetic properties of the substrates in the reaction catalyzed by Trypanosoma brucei gamma-glutamylcysteine synthetase has been examined and discussed.

Results support that long-range energetic coupling of interface residues to the active site is necessary for ornithine decarboxylase function. Further, the catalytic function of the enzyme is governed by the enzyme's dimer interface, enabling access by small molecules to the enzyme interface without energy barriers.

Results show that formation of putrescine by ornithine decarboxylase is dependent on the alpha-carbon protonation as indicated by the mutation of cysteine-360 residue of the enzyme to alanine or serine. The rate-limiting step in the enzyme steady-state reaction involves Schiff base hydrolysis.

Research has been conducted on the S-adenosylmethionine decarboxylase from Trypanosoma cruzi. The catalysis of the pyruvoyl-dependent S-adenosylmethionine decarboxylation has been carried out.

The structure of ornithine decarboxylase from Trypanosoma brucei is described, as well as the structure of its interaction with alpha-difluoromethylornithine (DFMO). DFMO blocks the activity of the enzyme, and is used to treat trypanosoma infections.

The effects of carbon-13 kinetic isotope on the wild-type Trypanosoma brucei ornithine decarboxylase (ODC) and the active site mutants of the enzyme were investigated. ODC is responsible for the first step in polyamine biosynthesis and carbon-13 isotope effects are utilized to examine the pathway mechanisms of several pyridoxal 5'-phosphate-dependent decarboxylases. It was revealed that the carbon isotope effect affected the irreversible decarboxylation step while the wild-type ODC with L-Lys as substrate indicated rate-limiting decarboxylation.

The characterization of the reaction mechanism for ornithine decarboxylase (ODC) was conducted based on multiwavelength stopped-flow analysis over the spectral range of 300-500 nm. Four intermediates including a species with quinoid-like characteristics were detected in the reaction of ODC with Orn. Results indicate that the formation of the quinoid species upon decarboxylation is not the rate-limiting step but product release is, at least, partially rate limiting.

The Arg-277 of Trypanosoma brucei ornithine decarboxylase (ODC) was mutated to Ala to determine the role of the amino acid residue in the binding of pyridoxal 5-phosphate (PLP). The mutant ODC or R277A ODC exhibited attenuated PLP binding capabilities and altered spectral properties at high hydrogen-ion concentrations compared to wild-type ODC. PLP was also bound to the active site through a network of interactions which maintained a strained conformation that was conducive to catalysis. Furthermore, loss of PLP binding in R277A ODC altered the conformation of the ODC-bound cofactor.

The precursor of rat pancreatic carboxypeptidase A1 (proCPA1) with intact N-terminal prodomain can renature after denaturation but the mature enzyme without prodomain region fails to do so. In yeast, proCPA1 is expressed and secreted while mature CPA1 is degraded rather than secreted. Thus, apart from maintaining CPA1 in an inactive form, the prodomain is crucial for its folding and secretion. Site-directed mutagenesis of residues involved in ionic interaction between the active site and the prodomain inhibits secretion, indicating that these interactions are needed for proper folding.

A chimeric enzyme consisting of T. brucei and mouse ornithine decarboxylase when kinetically analyzed revealed that the substrate carboxylate binding determinant is situated between residues 1 and 170. The N- and C- terminal peptides of this enzyme are firmly attached into a fully active tetramer having the same molecular weight as the native dimer. A surface loop at amino acids 160-170 is identified by proteolysis with trypsin or chymotrypsin. Kinetic constant measurement indicate that the enzyme has two structural domains with a domain boundary in the region of amino acid 305.

Analysis of the formation kinetics of ornithine decarboxylase (ODC) from mice and two parasitic protozoa, Leishmania donovani and Trypanosoma brucei, reveals the spontaneous formation of cross-species heterodimers in mice and T. brucei. The dimer interface of ODC has a highly conserved amino acid composition in T. brucei and mice and the substrate binding characteristics of the heterodimers resemble those of the wild-type enzyme. No cross-species heterodimers form between ODC of mouse or T. brucei and L. donovani and the ODC subunits in L. donovani do not exhibit rapid equilibrium as those of the other two species.

Cyclic peptides do not hydrolyze ester and amide bonds with chymotrypsin-like or trypsin-like specificity. The trypsin-mimic peptide that was produced does not exhibit any activity toward either ester or peptide substrates. These cyclic peptides lacked subsite interactions, a defined SI specificity pocket, an oxyanion hole and a rigid structure.

Adults, defined here as people between 15 and 59 years of age, in developing countries have a high risk of premature death and suffer from frequent morbidity and high rates of chronic impairment. Their ill health imposes a major burden on health services and large negative consequences on families, communities, and societies. This paper describes the level and impact of adult mortality and morbidity, and highlights some of its characteristics and causes, which in some cases contradict commonly held beliefs. It concludes that 'adult health' is a legitimate public health concern for developing countries that is not being addressed. An agenda for remedial research and action is proposed., Programs that address adult health problems in developing countries are scarce, because of an emphasis on children's health and safety, lack of knowledge about the causes of ill health in adults and lack of understanding of how timely prevention and treatment could cut healthcare costs. Research has focused on specific diseases rather than accurate surveys of all illness among adults. What is known is that poverty increases illness and mortality rates. However, this relationship does not hold absolutely. The US, for example, has a higher mortality rate than its per capita income would suggest, while Spain has a lower one. Except for tropical diseases, the leading causes of death for adults in developing countries are noncommunicable diseases. Campaigns that reduce smoking; promote traffic safety, safe sex, prenatal care, cancer screening, disease management and vaccinations against communicable diseases, and clean up pollution are designed to lower the incidence of adult morbidity and mortality rates.

A novel transition-state (TS) analogy probing strategy for enzymatic systems was developed. The study also studied the extent to which phosphonate inhibitors function as TS analogues. This was determined for the wild-type enzyme and the mutants R127K, R127M and R127A. Results revealed that the mutations' effect on the inverse second-order rate constants for substrate hydrolysis corresponds is reflected by their effect on the phosphonate inhibitors' inhibition constants, indicatingthat certain electronic and geometric TS qualities are imitated by the phosphonate moiety.

A novel carbon-on-metal thin film substrate is described, which has enabled surface plasmon resonance detection with photolithographically fabricated DNA arrays for the analysis of biomolecular interactions. The utility of the technology is shown in the analysis of specific DNA-DNA and DNA-protein binding interactions and this group has used new substrates to determine the secondary structure of RNA molecules.

The position of the ubiquinone (CoQ) binding site relative to inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) is studied by using the site-directed mutagenesis of the species-selective inhibitor site. The results have identified residues involved in inhibitor binding and electron transfer to CoQ and have also shown that inhibitors act either by blocking the electron path between flavin and CoQ.

The X-ray structures of the Paramecium bursaria Chlorella virus arginine decarboxylase (cvADC) in both free and product-bound states are determined at 1.95 and 1.8 Angstrom resolution, respectively. The 'K148-loop' is observed in a closed, substrate-bound conformation.

Results point out that Phe-397 promotes the decarboxylation step of the Trypanosoma brucei ornithine decarboxylase via the binding of the enzyme to the carboxyl leaving group of L-Orn on the re face of the pyridoxal-5'-phosphate cofactor. By virtue of the localization of the leaving group in the buried side of the cofactor, the enzyme dcarboxylates by destabilizing the substrate carboxyl group.

Abstract On May 3, 1999, 58 tornadoes were reported in Oklahoma in one of the largest outbreaks of severe weather in Oklahoma history. The tornadoes killed 44 people and destroyed [...]

Most health care workers will not be exposed to significant amounts of nitric oxide or nitrogen dioxide when they treat patients with these gases. Researchers gave 28 nurses and 18 respiratory therapists a dosimeter to wear. These devices measure the amount of substances in the air to estimate the person's exposure to the substance. Most exposures were brief, uncommon, and below the standards of the Occupational Safety and Health Administration., Objective. This study was an assessment of potential exposures of medical personnel to nitrogen oxides during simulated and actual inhaled nitric oxide treatment of newborn and pediatric patients. Design. Breathing zone exposures to nitric oxide (NO) and nitrogen dioxide ([NO.sub.2]) were monitored using data-logging personal dosimeters during simulated and actual administration of NO gas to patients in an intensive care setting. Sample. A total of 28 bedside nurses and 18 respiratory therapists were monitored during 6 different patient treatments. Analysis. The highest measured concentrations of NO and [NO.sub.2] in the personal breathing zones of the nurses and respiratory therapists were peak readings ([is less than] 1 minute in duration) of 6.7 parts per million (ppm) NO and 3.1 ppm [NO.sub.2]. Exposures averaged throughout 15 minutes and throughout the work shift were below the limit of detection (0.8-ppm NO and 0.5-ppm [NO.sub.2]). Conclusion. Detectable exposures to NO and [NO.sub.2] were brief, infrequent, and well below Occupational Safety and Health Administration permissible exposure limits or any other exposure guideline, eg, American Conference of Governmental Hygienists Threshold Limit Values. Pediatrics 1999;104:1095-1100; occupational exposure., ABBREVIATIONS. I-NO, inhaled nitric oxide; NO, nitric oxide; [NO.sub.2], nitrogen dioxide; OSHA, Occupational Safety and Health Administration; NICU, neonatal intensive care unit; PICU, pediatric intensive care unit; STEL. short-term exposure [...]

Introduction Historically, protection of the health of the public from environmental hazards has been the main impetus for establishing the field of public health.[1] Today environmental health services account for […]

The National Library has recently conducted a review of its collecting of Australian online publications. Hard though it is to believe, we have now been selecting online publications for archiving [...]

Research was conducted to examine the mechanistic role of the Lys-69 residue in catalysis by Trypanosoma brucei ornithine decarboxylase (ODC) via an analysis of the mutant enzymes. It was found that the enzyme copurified with amines hat were tightly bound to the active site through a Schiff base with pyridoxal-5'-phosphate-dependent. A kinetic analysis of the binding of a series of diamines and amino acids to Arg ODC indicates that noncovalent interactions in the active site of Arg ODC promote selective ligand biding during Schiff base formation.