Capillary electrophoresis of methotrexate polyglutamates and its application in evaluation of γ-glutamyl hydrolase activity

A rapid and sensitive procedure for the separation of methotrexate (MTX) polyglutamates² using capillary electrophoresis (CE) is described as it applies to the in vitro assay of the enzyme γ-glutamyl hydrolase (GGH, EC 3.4.22.12). Distinct separation of MTX and polyglutamylated forms (up to glu₄) is achieved within 10 min using a 75 μm I.D. capillary (50 cm, +25 kV), and enables quantitation of both reactant and enzyme products. As activity can be reliably determined using less than 5×10⁵ eukaryotic cells, this new technique can be used to measure GGH in patient tumor samples and investigate the relationship between GGH levels and clinical MTX resistance.     

Effects of α,β-methylene-adenosine-5′-diphosphate on blood platelet aggregation

The effects on platelet aggregation of α,β-methylene-adenosine-5′-diphosphate (Ado-PCP) have been investigated. Using human citrated platelet-rich plasma it has been shown that: (i) at concentrations of 10^?3 M or higher Ado-PCP is able to induce platelet aggregation; (ii) the rate of Ado-PCP-induced aggregation increases on raising the pH of platelet-rich plasma above the pK_a for the secondary phosphonyl dissociation of Ado-PCP; (iii) at concentrations from 1 · 10^?4 to 5 · 10^?4 M Ado-PCP does not cause platelet aggregation itself, but it inhibits ADP-induced aggregation. This inhibition is also observed in washed platelet suspensions. The data suggest that Ado-PCP acts at the same site on the platelet membrane as does ADP and that ADP to AMP transformation is not a prerequisite for the process of aggregation. The observed effect of pH on the rate of Ado-PCP induced aggregation suggests that the ionization state of a nucleotide terminal acid group is important in the process of aggregation.     

Free and esterified necines in Heliotropium species from mexico and texas

Fourteen species of Heliotropium from Mexico and Texas contained 3-7 necines per species. In most cases there were no qualitative differences in th     

Behavioral changes during weaning in captive chimpanzees

Behavioral changes during weaning of chimpanzee infants in captive social groups were compared with those of infants in the natural habitat. Results of the weaning process were the same for the mother-infant pairs in captivity and the natural habitat, i.e., an infant independent of suckling, regular maternal transport and sleeping with the mother. The captive infants, however, did not respond to weaning with depression or regression to infantile behaviors as did infants in the natural habitat. Quite probably the social and physical environmental differences between the two habitats faciliated a less stressful weaning period for the captive mother-infant pairs.     

Estimating intrinsic growth rates of arthropods from partial life tables using predatory mites as examples

The intrinsic rate of natural increase of a population (r_m) has been in focus as a key parameter in entomology and acarology. It is considered especially important in studies of predators that are potential biological control agents of fast-growing pests such as mites, whiteflies and thrips. Life-table experiments under controlled laboratory conditions are standard procedures to estimate r_m. However, such experiments are often time consuming and may critically depend on the precise assessment of the developmental time and the fecundity rate early in the reproductive phase. Using selected studies of predatory mites with suitable life-table data, we investigated whether and how measurements of growth rates can be simplified. We propose a new method for estimating r_m from partial life tables, in which the researcher can choose a level of precision based on a stand-in measure of relative error. Based on this choice, the procedure helps the researcher to decide when a life-table experiment can be terminated. Depending on the chosen precision, significant amounts of experimental time can be saved without seriously compromising the reliability of the estimated growth parameter.

     

Luminescence properties of neodymium,samarium, and europium niobate and tantalate thin films

Thin films of lanthanide orthoniobate LnNbO₄ (LnNO) and orthotantalate LnTaO₄ (LnTO), (Ln = Nd, Sm, Eu) were fabricated using the sol–gel method with subsequent spin-coating on the PbZrO₃/Al₂O₃ substrate and annealing at 1000°C. X-ray diffraction patterns showed monoclinic M-LnNbO₄ or M´-LnTaO₄, which coexists with the orthorhombic or tetragonal phase. X-ray photoelectron spectroscopy demonstrated the presence of Nd³⁺, Sm³⁺/Sm²⁺ and Eu³⁺/Eu²⁺ ions. The luminescence properties of polymorphic films were investigated. Excitation spectra of PbZrO₃ interlayer represented broad bands at 410 and 550 nm that were assigned to charge transfer bands (CTB). In all films, the CTB broad band at ~275 nm related to charge transfer transition of Ln³⁺→O²⁻ and NbO₄³⁻ or TaO₄³⁻ groups. In excitation spectra, ⁴I_9/2→⁴G_5/2 (Nd³⁺), ⁶H_5/2→⁶P_3/2 (Sm³⁺) and ⁷F₀→⁵L₆ (Eu³⁺) transitions (at 585, 402 and 395 nm), respectively were found to be more intense than any other Ln³⁺ transition. The emission spectra showed narrow and intense bands at 1065, 600, and 614 nm that were ascribed to Nd³⁺, Sm³⁺, and Eu³⁺ 4f–f intraconfigurational transitions ⁴F_3/2→⁴I_11/2, ⁴G_5/2→⁶H_7/2, and ⁵D₀→⁷F₂, respectively. The excellent luminescence properties of films make them new potential groups for visible and/or near-infrared applications such as sensors and imaging equipment.     

Applying water scarcity footprint methodologies to milk production in Finland

The International Journal of Life Cycle Assessment - Food production without consuming scarce local freshwater resources in an unsustainable way needs to be ensured. A robust method to assess water...     

The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.     

Chlorpromazine and apigenin reduce adenovirus replication and decrease replication associated toxicity

BACKGROUND: Adenoviruses can cause severe toxicity in immunocompromised individuals. Although clinical trials have confirmed the potency and safety of selectively oncolytic adenoviruses for treatment of advanced cancers, increasingly effective agents could result in more toxicity and therefore it would be useful if replication could be abrogated if necessary. METHODS: We analyzed the effect of chlorpromazine, an inhibitor of clathrin-dependent endocytosis and apigenin, a cell cycle regulator, on adenovirus replication and toxicity. First, we evaluated the in vitro replication of a tumor targeted Rb-p16 pathway selective oncolytic adenovirus (Ad5/3-Delta24) and a wild-type adenovirus in normal cells, fresh liver samples and in ovarian cancer cell lines. Further, we analyzed the in vitro cell killing efficacy of adenoviruses in the presence and absence of the substances. Moreover, the effect on in vivo efficacy, replication and liver toxicity of the adenoviruses was evaluated. RESULTS: We demonstrate in vitro and in vivo reduction of adenovirus replication and associated toxicity with chlorpromazine and apigenin. Effective doses were well within what would be predicted safe in humans. CONCLUSIONS: Chlorpromazine and apigenin might reduce the replication of adenovirus, which could provide a safety switch in case replication-associated side effects are encountered in patients. In addition, these substances could be useful for the treatment of systemic adenoviral infections in immunosuppressed patients.     

Mutation in MRPS34 Compromises Protein Synthesis and Causes Mitochondrial Dysfunction

The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The Mrps34 mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The Mrps34 mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age.