The contribution of naturally occurring polymorphisms in altering the biochemical and structural characteristics of HIV-1 subtype C protease

Fourteen subtype B and C protease variants have been engineered in an effort to study whether the preexistent baseline polymorphisms, by themselves or in combination with drug resistance mutations, differentially alter the biochemical and structural features of the subtype C protease when compared with those of subtype B protease. The kinetic studies performed in this work showed that the preexistent polymorphisms in subtype C protease, by themselves, do not provide for a greater level of resistance. Inhibition analysis with eight clinically used protease inhibitors revealed that the natural polymorphisms found in subtype C protease, in combination with drug resistance mutations, can influence enzymatic catalytic efficiency and inhibitor resistance. Structural analyses of the subtype C protease bound to nelfinavir and indinavir showed that these inhibitors form similar interactions with the residues in the active site of subtype B and C proteases. It also revealed that the naturally occurring polymorphisms could alter the position of the outer loops of the subtype C protease, especially the 60's loop.     

Inhibition of carbonic anhydrase II by thioxolone: a mechanistic and structural study

This paper examines the functional mechanism of thioxolone, a compound recently identified as a weak inhibitor of human carbonic anhydrase II by Iyer et al. (2006) J. Biomol. Screening 11, 782-791 . Thioxolone lacks sulfonamide, sulfamate, or hydroxamate functional groups that are typically found in therapeutic carbonic anhydrase (CA) inhibitors, such as acetazolamide. Analytical chemistry and biochemical methods were used to investigate the fate of thioxolone upon binding to CA II, including Michaelis-Menten kinetics of 4-nitrophenyl acetate esterase cleavage, liquid chromatography-mass spectrometry (LC-MS), oxygen-18 isotope exchange studies, and X-ray crystallography. Thioxolone is proposed to be a prodrug inhibitor that is cleaved via a CA II zinc-hydroxide mechanism known to catalyze the hydrolysis of esters. When thioxolone binds in the active site of CA II, it is cleaved and forms 4-mercaptobenzene-1,3-diol via the intermediate S-(2,4-thiophenyl)hydrogen thiocarbonate. The esterase cleavage product binds to the zinc active site via the thiol group and is therefore the active CA inhibitor, while the intermediate is located at the rim of the active-site cavity. The time-dependence of this inhibition reaction was investigated in detail. Because this type of prodrug inhibitor mechanism depends on cleavage of ester bonds, this class of inhibitors may have advantages over sulfonamides in determining isozyme specificity. A preliminary structure-activity relationship study with a series of structural analogues of thioxolone yielded similar estimates of inhibition constants for most compounds, although two compounds with bromine groups at the C1 carbon of thioxolone were not inhibitory, suggesting a possible steric effect.     

Mixed lineage kinase activity of indolocarbazole analogues.

The MLK1-3 activity for a series of analogues of the indolocarbazole K-252a is reported. Addition of 3,9-bis-alkylthiomethyl groups to K-252a results in potent and selective MLK inhibitors. The in vitro and in vivo survival promoting activity of bis-isopropylthiomethyl-K-252a (16, CEP-11004/KT-8138) is reported.     

Mutation at the APRT locus in Friend erythroleukaemia cells 1. Mutation rates and properties of mutants

Spontaneous mutation at the adenine phosphoribosyl transferase (APRT) locus in clone 707 of the Friend cell line was examined. The frequency of cells resistant to 2,6-diaminopurine (DAP) was found to be 2.6 × 10⁻⁵ with a mutation rate of 1.81 × 10⁻⁶ cell⁻¹ generation⁻¹. APRT activities in 9 DAP-resistant clones were found to vary between 0 and 27% the level observed in wild-type cells. It is suggested that clone 707 cells are heterozygous or functionally hemizygous at the APRT locus.     

Fluid compartmentation in skeletal muscle and carcass of Mus musculus acclimated to water scarcity

1. Total water (TW), and extracellular water (ECW) (as sodium and chloride space) were determined in skeletal muscle and carcass of Mus musculus acclimated to long-term water shortage. 2. The presence of fat in control mice and those in early stages of acclimation resulted in an apparent increase in TW and ECW as acclimation proceeded. 3. In contrast, fluid volumes per fat-free weight were either unchanged from controls or reduced. 4. Sodium space exceeded chloride space. 5. Muscle and carcass had essentially the same pattern of fluid shifts. 6. We conclude that ECW maintenance is a preeminent component of the acclimation process in this species.     

Generating a T cell tumor-specific immune response in vivo: can flt3-ligand-generated dendritic cells tip the balance?

flt3 ligand (FL) is a growth factor that induces hematopoietic progenitor cell and dendritic cell (DC) expansion when administered to mice. Lymphoid-related (CD8α⁺) and myeloid-related (CD8α⁻) DC are transiently expanded in multiple tissues. Treatment of tumor-bearing mice with FL results in slower tumor growth and, in some cases, tumor rejection and the development of tumor-specific T cell immunity. The clinical use of DC as cellular vehicles for tumor antigen presentation to generate a tumor-specific T cell response is under investigation. DC are currently generated ex vivo, pulsed with antigen, and then infused into patients, and much effort is being directed toward optimizing each of these steps. Administration of FL to humans induces a profound increase in circulating DC. The availability of a large number of DC generated in vivo has important implications for tumor immunotherapy approaches. Received: 13 May 1999 / Accepted: 14 June 1999     

"To Be or Not to Be" Protonated: Atomic Details of Human Carbonic Anhydrase-Clinical Drug Complexes by Neutron Crystallography and Simulation

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Prediction of radiosensitivity in human bladder cell lines using nuclear chromatin phenotype.

BACKGROUND: Nuclear texture analysis measures phenotypic changes in chromatin distribution within a cell nucleus, while the alkaline Comet assay is a sensitive method for measuring the extent of DNA breakage in individual cells. The authors aim to use both methods to provide information about the sensitivity of cells to ionizing radiation. METHODS: The alkaline Comet assay was performed on six human bladder carcinoma cell lines and one human urothelial cell line exposed to gamma-radiation doses from 0 to 10 Gy. Nuclear chromatin texture analysis of 40 features was then performed in the same cell lines exposed to 0, 2, and 6 Gy to explore if nuclear phenotype was related to radiation sensitivity. RESULTS: Comet assay results demonstrated that the cell lines exhibited different levels of radiosensitivity and could be divided into a radiosensitive and a radioresistant group at >6 Gy. Using stepwise discriminant analysis, a subset of important nuclear texture features that best discriminated between sensitive and resistant cell lines were identified A classification function, defined using these features, correctly classified 81.75% of all cells into their radiosensitive or radioresistant groups based on their pretreatment chromatin phenotype. Posttreatment chromatin changes also varied between cell lines, with sensitive cell lines showing a relaxed chromatin conformation following radiation, whereas resistant cell lines exhibited chromatin condensation. CONCLUSIONS: The authors conclude that the alkaline Comet assay and nuclear texture methodologies may prove to be valuable aids in predicting the response of tumor cells to radiotherapy.