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.     

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.     

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.     

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.     

Fine needle aspiration cytology of spermatocytic seminoma. Report of a case

The clinical, radiologic, cytopathologic and histologic features of a case of spermatocytic seminoma in a 56-year-old man are reported and discussed. The cytologic findings in fine needle aspiration (FNA) smears suggested a diagnosis of spermatocytic seminoma, which was confirmed by subsequent histopathologic studies. The case suggests that spermatocytic seminoma can be suspected and distinguished from other types of seminoma and germ cell neoplasms by FNA cytology. The following criteria appear to be the most useful in making the cytodiagnosis: the presence of three different types of cells (large, medium and small), a clean background, an absence of a lymphoid component and "tigroid" substances and the relative paucity and clumping tendency of the neoplastic cells in the smears.     

Interaction of retinol and retinoic acid with phospholipid membranes. A differential scanning calorimetry study.

The influence of retinol and retinoic acid, two retinoids of major interest, on the main gel to liquid-crystalline phase transition of different phospholipid membranes has been studied by means of differential scanning calorimetry. Both compounds exerted perturbing effects on the phase transition of membranes composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine. At concentrations up to 42.5 mol% of retinoid in the membrane, the delta H was not much affected with respect to the pure phospholipid, indicating a rather slight interaction. As the concentration of retinol was increased the Tc transition temperature decreased. A fluid-phase immiscibility was observed for the system DPPC/retinol at concentrations between 0 and 33 mol%. Almost ideal phase diagrams were obtained for the mixture DPPE/retinol. At concentrations of 33 mol% and higher retinol was able to induce phase separations in DPPC membranes, but not in DPPE. The effect of retinoic acid was much weaker, the Tc and delta H remaining almost unaltered and equal to that of the pure phospholipid up to concentrations of 30 mol%, at neutral pH. Retinoic acid exerted a pH-dependent effect. As the pH decreased, and therefore increased the extent of protonation of retinoic acid, the pertubation of the membrane induced by this compound was less. A strong effect, both on Tc and delta H, was observed at pH 10, where the retinoic acid moiety will be mainly unprotonated and the negative charge will generate repulsive forces thus destabilizing the membrane. The mixture DPPC/retinoic acid presents a region of fluid-phase immiscibility. At low pH, when the retinoic acid moiety was fully protonated, this fluid-immiscibility region extended from 0 to 36 mol% of retinoic acid, but its size decreased with increasing pH, and at pH 10 it was only found from 0 to 3 mol%. These results are discussed in terms of the possible retinoid/phospholipid interactions and the disposition of the retinoid moiety in the bilayer.     

Effect of beta-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk.

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.     

Substance P affects the GABAergic system in the hypothalamo-pituitary axis

In the present work we examined the effect of the neutralization of endogenous substance P by the administration of an anti-substance P serum (ASPS) on GABA concentration in the anterior pituitary in hyperprolactinemic conditions induced by 5-hydroxytryptophan or by grafting anterior pituitaries. ASPS reduced the increase in the anterior pituitary GABA concentration induced by hyperprolactinemia. In vitro experiments showed that substance P inhibited K⁺-evoked GABA efflux from hypothalamic fragments and decreased GABA concentration in the anterior pituitary but ASPS increased it. Our results demonstrate that substance P modifies hypothalamic GABA release and anterior pituitary GABA concentration and suggest that an interaction exists between substance P and GABA.