In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.     

Developmental Expression of Neural Cell Adhesion Molecules of Oligodendrocytes In Vivo and in Culture

Abstract: Previously, we have shown that oligodendrocyte adhesion molecules are related to the 120,000–M_r neural cell adhesion molecule (NCAM-120). In this report, we present further evidence that the oligodendrocyte adhesion molecule is NCAM-120. Studies on the expression of NCAM-120 and other molecular forms of NCAM in vivo in rat brain, in vitro in primary mixed cultures, and in cultures enriched for oligodendrocytes are described. Western blot analysis of rat brain using anti-NCAM showed that NCAM-120 first appears at postnatal day 7 and increases in quantity thereafter, coincident with the development of oligodendrocytes in vivo and comparable to the expression of myelin basic protein. Purified oligodendrocytes from 4-week-old rat brains expressed only NCAM-120. Quantitation of various forms of NCAMs in rat brain showed marked age-related differences in the expression of three molecular forms of NCAM. Immunofluorescence analysis showed that oligodendrocytes, at all ages tested, expressed NCAM, but in older oligodendrocytes, the intensity of staining was less. Western blot analysis of oligodendrocyte-enriched cultures showed that from day 1 after isolation (12 days of age) through day 7 after isolation (18 days of age) only NCAM-120 is seen. A possible role for NCAM in myelination and remyelination is discussed.     

Homogenization of geographical variants at the nontranscribed spacer of rDNA in Drosophila mercatorum

rDNA nontranscribed spacer (NTS) lengths of Drosophila mercatorum have been measured in individuals from several geographic regions. Individuals from the different geographic subpopulations share some length fragments but are in general distinct. The length differences, both within and between individuals, arise from different copy numbers of a 250-bp repeating unit that is localized to one part of the NTS. In addition to the length differences caused by the 250-bp repeat, there is a Y chromosome (male)-specific length variant elsewhere in the NTS that is approximately 70 bp shorter than the NTS fragment from the X chromosome. Sexual dimorphism seems to be present in all Drosophila. Also, D. mercatorum has fewer NTS length variants per individual than does D. melanogaster while possessing comparable levels of restriction- site polymorphism. The mechanisms that may cause this pattern of variation are selection, gene conversion, and unequal recombination.      

Exploring the Structural Mechanism of Covalently Bound E3 Ubiquitin Ligase: Catalytic or Allosteric Inhibition?

Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The Nedd4-1, an E3 ubiquitin ligase, is characterized by two covalent binding sites, of which catalytic Cys^cat and allosteric Cys^allo are enclosed. This enzyme has demonstrated inhibition at both the above-mentioned binding sites; however, a detailed molecular understanding of the structural mechanism of inhibition upon Cys^cat and Cys^allo binding remains vague. This prompted us to provide the first account of investigating the preferential covalent binding mode and the underlying structural and molecular dynamic implications. Based on the molecular dynamic analyses, it was evident that although both catalytic and allosteric covalent binding led to greater stability of the enzyme, a preferential covalent mechanism of inhibition was seen in the allosteric-targeted system. This was supported by a more favorable binding energy in the allosteric site compared to the catalytic site, in addition to the larger number of residue interactions and stabilizing hydrogen bonds occurring in the allosteric covalent bound complex. The fundamental dynamic analysis presented in this report compliments, as well as adds to previous experimental findings, thus leading to a crucial understanding of the structural mechanism by which Nedd4-1 is inhibited. The findings from this study may assist in the design of more target-specific Nedd4-1 covalent inhibitors exploring the surface-exposed cysteine residues.     

Novel nicotinamide analog as inhibitor of nicotinamide N-methyltransferase

Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by ～80% at 2?h when dosed in mice orally at 50?mg/kg.     

The influence of optimization target selection on the structure of arterial tree models generated by constrained constructive optimization

The computational method of constrained constructive optimization was used to generate complex arterial model trees by optimization with respect to a target function. Changing the target function also changes the tree structure obtained. For a parameterized family of target functions a series of trees was created, showing visually striking differences in structure that can also be quantified by appropriately chosen numerical indexes. Blood transport path length, pressure profile, and an index for relative segment orientation show clear dependencies on the optimization target, and the nature of changes can be explained on theoretical grounds. The main goal was to display, quantify, and explain the structural changes induced by different optimization target functions.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.