Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.     

Chromosome evolution and phylogeny in Ronderosia (Orthoptera,Acrididae, Melanoplinae): clues of survivors to the challenge of sympatry?

In an attempt to unveil the origin of neo‐sex chromosomes in Ronderosia Cigliano grasshoppers, we performed a combined phylogenetic analysis based on morphological (external morphology and male genitalia) and molecular data (COI, COII, 16S and ITS2) to explore the chromosome evolution within the genus. We also analysed the distributional patterns of the various Ronderosia species and considered the possible role of chromosome rearrangements (CRs) in speciation processes within the genus in the light of ‘suppressed‐recombination’ models. We mapped the states of three chromosomal characters on the combined tree topology. The combined evidence supported Ronderosia as a monophyletic group. The cytogenetic analyses of the genus demonstrated the importance of rearranged karyotypes with single, complex and multiples neo‐sex chromosome determination systems in all species. The chromosome character optimisation suggests X‐autosome centric fusion as the mechanism responsible for neo‐sex chromosome formation in most Ronderosia species, except in R. dubia and R. bergii. Similar autosomes were involved in fusions with the ancestral X chromosome in Ronderosia, supporting previous hypotheses on the unique origin of X‐autosome fusion for the sex chromosome in the genus. As a source of chromosome variation, autosome‐autosome centric fusion played a secondary role in Ronderosia compared with other Dichroplini. Given the homogeneity in the morphological features, the sympatric distribution of closely related species and the intrinsic property of centric fusion as suppressors of the crossing over, we suggest that CRs may have played a key role during the speciation process within Ronderosia.     

A New Real-Time PCR for the Detection of Plasmodium ovale wallikeri

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite’s small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.     

Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (p_O2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14–16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14–16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both prophobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogen-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found to differ in several aspects.     

Transbilayer movement of bile acids in model membranes

The ability of bile acids to traverse membranes has important implications for their reabsorption from the gut, recirculation to and uptake into the liver, and resecretion into bile. The rate constant for transbilayer movement, or "flip-flop", of three common, unconjugated bile acids was determined by 13C nuclear magnetic resonance spectroscopy. At high pH, the sodium salts of the bile acids did not appreciably traverse the bilayer; however, upon protonation a rapid equilibration between the inner and outer monolayers occurred. The rate of flip-flop of each bile acid at 37 degrees C was found to be dependent on both number and location of hydroxyl groups but not on concentration in the bilayer over the range studied (2-4 wt%) nor on the presence of a different bile acid in the same bilayer.     

Production of ethanol by immobilized Saccharomyces bayanus in an extractive fermentation system

An extractive fermentation system using immobilized yeast cells was developed to study the ethanol production at high sugar concentrations. Organic acids were used as extracting solvents of ethanol and their toxicity was tested in free and k-carrageenan entrapped cell preparations. Immobilization seems to protect cells against solvent toxicity, when long-chain organic acids, e.g., oleic acid, were used, probably due to steric and diffusional limitations, the free cells not being viable at high oleic acid concentrations. The entrapped cells also present a higher metabolic activity than their free counterparts at high glucose concentrations. A solution of 300 g/L of glucose was totally fermented by the immobilized yeast cells, which when free cannot normally convert more than 200 g/L. In situ recovery of ethanol by oleic acid in a batch immobilized cell system led to higher ethanol productivities and to the fermentation of 400 g/L, when an oleic acid/medium ratio of 5 was used.     

Molecular characterization of β-thalassemia in Czechoslovakia

Summary We have identified different -thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific ³²P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The GA mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (GA), IVS-II-745 (CG), IVS-I-110 (GA), and codon 39 (CT); these were present in 9 additional families. The GT mutation at codon 121, known to cause Heinzbody -thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One -thalassemia allele was incompletely characterized. We observed in 2 families a TC mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism, -Thalassemia was rare; only one person carried the -^3.7 heterozygosity, and one other had a yet to be identified -thalassemia-1, while seven had the ^{anti 3.7} triplication.     

Steroid bioconversion in a novel aqueous two-phase system

A novel aqueous two-phase system, based on polyethyleneglycol (PEG) and monosodium glutamate, was tested for the 1-dehydrogenation of hydrocortisone-based substrates. This system led to higher substrate solubilities and biocatalyst/steroid separation levels when compared with alternative systems. The addition of short-chain monohydric alcohols resulted in higher solubilities and more favourable partition coefficients for the tested substrates. Bioconversion activities in PEG/glutamate systems with 2,5% (v/v) methanol were comparable to those measured in monophasic buffer-methanol medium.     

Rapid Activation of Tyrosine Hydroxylase in Response to Nerve Growth Factor

Abstract: Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC 12 pheochromocytoma cells. PC 12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 ± 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2–5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 n M ). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron-like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF-induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent K_m of the enzyme for tyrosine or for co-factor (6-methyltetrahydropteridine), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.