Kinetics study of the biotransformation of an oligonucleotide prodrug in cells extract by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. The oligonucleotide prodrug was fully metabolized to yield the T12 phosphorothioate likely through a carboxyesterase mediated mechanism.     

Synthesis of carbocyclic phosphononucleosides

Synthesis of carbocyclic analogs of phosphononucleosides are described by two different methods (introduction of the heterocycle under Mitsunobu conditions or build-up of the base around a cyclopentylamine moiety).     

Chemical and biochemical issues related to X-ray crystallography of the ligand-binding domain of estrogen receptor alpha

Careful attention to technical issues preceded successful crystallography of the ligand-binding domain of estrogen receptor alpha (ERalpha) complexed with CP-336156, a nonsteroidal estrogen agonist/antagonist. An affinity column based on immobilized estradiol was prepared according to the scheme of Greene et al. (Greene, G. L., Nolan, C., Engler, J. P., and Jensen, E. V. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5115-5119). It was shown by X-ray crystallography that the major and less polar isomer of the affinity column precursor was 17alpha-((S)-2',3'-epoxyprop-1'-yl)estra-1,3,5(10)-triene-3,17beta-diol. This diastereomer was coupled to Thiopropyl Sepharose, with coupling monitored by observing loss of the phenolic absorption band of estradiol from the reaction supernatant, and gave an affinity matrix containing about 9 micromol of estradiol per milliliter of wet gel. Recombinant ERalpha ligand binding domain was selectively removed from E. coli cell lysate by binding to the column and was partly S-carboxymethylated by treatment with iodoacetic acid while bound to the column as described by previous workers. After being eluted from the column as a complex with drug, the receptor fragment was shown by mass spectrometry to be a mixture of differently modified forms. It was further S-carboxymethylated in solution, after which anion-exchange chromatography was used to isolate protein in which two of the four cysteine residues were S-carboxymethylated. This material, which afforded diffraction-quality crystals, was subjected to digestion with trypsin and peptide mapping analysis by HPLC coupled with mass spectrometry. For this experiment, the two previously unmodified cysteines were alkylated with 4-vinylpyridine to allow definitive identification. It was shown that Cys-417 and Cys-530 were S-carboxymethylated in the crystallized protein, while Cys-381 and Cys-447 remained unmodified. Close attention to such technical issues may be important in structural studies of other nuclear receptors, a very important class of potential drug targets.     

Phylogeny and evolution of the neotropical rodent genus Calomys: inferences from mitochondrial DNA sequence data

Field mice of the genus Calomys are small, mostly granivorous rodents common to several habitats in South America. To date, phylogenies for the genus have been proposed on the basis of morphological, chromosomal, and biochemical data, often with contradictory results due to incomplete species sampling or methodological shortcomings. In this paper, we propose relationships among 10 species of Calomys based on the complete cytochrome b gene sequence. Our analyses show that Calomys is constituted by two major clades, one mostly associated with mountain habitats with subsequent invasions to lowland habitats and another with species restricted to lowland habitats both north and south of the Amazon basin. The evolution of the genus was likely accompanied by a reduction of chromosome diploid numbers that occurred independently in each of the two evolutionary lineages. A "clock" calibrated on the split between Auliscomys and Loxodontomys suggests that the almost nonexistent fossil record for the genus greatly underestimates divergence times among its species.     

Hemodynamics for medical students

The flow of blood through the cardiovascular system depends on basic principles of liquid flow in tubes elucidated by Bernoulli and Poiseuille. The elementary equations are described involving pressures related to velocity, acceleration/deceleration, gravity, and viscous resistance to flow (Bernoulli-Poiseuille equation). The roles of vascular diameter and number of branches are emphasized. In the closed vascular system, the importance of gravity is deemphasized, and the occurrence of turbulence in large vessels is pointed out.     

Effects of structure of beta-cyclodextrin-containing polymers on gene delivery

Linear cationic beta-cyclodextrin-based polymers (betaCDPs) are capable of forming polyplexes with nucleic acids and transfecting cultured cells. The betaCDPs are synthesized by the condensation of a diamino-cyclodextrin monomer A with a diimidate comonomer B. In this paper, the effects of polymer structure on polyplex formation, in vitro transfection efficiency and toxicity are elucidated. By comparison of the betaCDPs to polyamidines lacking cyclodextrins, the inclusion of a cyclodextrin moiety in the comonomer A units reduces the IC50s of the polymer by up to 3 orders of magnitude. The spacing between the cationic amidine groups is also important. Different polymers with 4, 5, 6, 7, 8, and 10 methylene units (betaCDP4, 5, 6, 7, 8, and 10) in the comonomer B molecule are synthesized. Transfection efficiency is dependent on comonomer B length with up to 20-fold difference between polymers. Optimum transfection is achieved with the betaCDP6 polymer. In vitro toxicity varied by 1 order of magnitude and the lowest toxicity is observed with betaCDP8. The LD40 of the betaCDP6 to mice is 200 mg/kg, making this polymer a promising agent for in vivo gene delivery applications.     

Estimating the Productivity of the Black Sea Bivalve Chamelea gallinafrom the Size and Biomass of Its Populations

Characteristics of annual production based on various mass data (whole animal mass; shell mass; and wet, dry, and ash-free dry soft tissue mass), the P/B coefficient, and specific production were estimated for the bivalve mollusk Chamelea gallinasampled from 21 localities in the northwestern and eastern Black Sea. The annual production correlated with population biomass (r= 0.994) and mean individual mass (r= –0.737), while the P/Bcoefficient depended mainly on mean individual mass (r= –0.719). Estimates of the P/Bcoefficient in Ch. gallinafrom the Black Sea were smaller than those of other marine invertebrates found worldwide with the same energy equivalent. Multiple linear regression equations were calculated for empirical relationships of various characteristics of annual production to the population biomass and mean individual biomass of Ch. gallina.     

Dynamics of the Biological Activity of Litter as It Is Transformed by Earthworms

The dynamics of the biological activity of litter during its transformation were studied under laboratory conditions. The model group of soil saprophages was represented by earthworms (Eisenia genus). The system litter–droppings—litter + droppings–soil was investigated. The feeding activity of earthworms intensified biological processes in the decomposing plant residues. A fraction of readily hydrolyzable nitrogen was accumulated in the worm droppings. Dressing the litter with droppings shifted transformation of the plant materials towards mineralization.     

Putting a face on poverty--a tale of two counties

Disparities in social and economic status create a wall between the well and well-off and those living on the other side of town: the poor, the chronically ill, and the dying. In some cases, the needy to whom we turn a blind eye, those who need healthcare the most, live next door to us or across the street, only a zip code away. The case studies presented in this article draw attention to these members of our community and the imperfections in our present system.     

Dystrophin in limb skeletal muscle fibers and creatinkinase leakage after acute +3 Gz acceleration in rhesus monkeys

Intensive muscle tension induces significant blood accumulation of enzymes and structural proteins of the muscle origin. Altered macromolecular permeability of the sarcolemma is attributed to integrity of sarcolemmal cytoskeleton, mainly to dystrophin-sarcoglycan (DSG) complex. It is known that intensive tension of the antigravity extensor muscles is observed under conditions of gravitational overloading. We assumed that acute exposure to hypergravity would lead to serum accumulation of creatine phosphokinase (CK) associated with considerably altered integrity of the dystrophin layer in fibers of extensor muscles.