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.     

Type II collagen defects in the chondrodysplasias. I. Spondyloepiphyseal dysplasias.

The spondyloepiphyseal dysplasias (SEDs) and spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of skeletal dysplasias (dwarfing disorders) characterized by abnormal epiphyses, with and without varying degrees of metaphyseal irregularities, flattened vertebral bodies, and myopia. To better define the underlying cause of these disorders, we have analyzed the collagens from costal cartilage from several of these patients, using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) of intact chains and cyanogen bromide (CNBr) peptides and amino acid analysis. In almost all of the patients in this study group, the type II collagen exhibited a slower electrophoretic mobility when compared with that in normal controls. The mobility of many, but not all, of the CNBr peptides was also retarded. Peptides near the amino terminus were almost always altered, while the mobility of peptides close to the carboxyl terminus were normal in all but the severely affected cases. Analysis of the CNBr peptides on an HPLC sieving column confirmed that the electrophoretically abnormal peptides were of a higher molecular weight than were control peptides. Amino acid analysis indicated that the abnormal collagens have a higher ratio of hydroxylysine to lysine than does control collagen, suggesting that overmodification may be involved in the altered mobility. Our results are consistent with a defect in the collagen helix that results in overmodification of the molecule from that point toward the amino terminus. We propose that some forms of SED and SEMD are associated with abnormalities in type II collagen that results in delayed helix formation and consequent overmodification of the collagen. Cases of SED fit onto a continuous spectrum of clinical severity that correlates positively with both the extent of alteration and the proximity of the defect to the carboxyl terminus.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

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.     

Anti-adenovirus type 5 cytotoxic T lymphocytes: immunodominant epitopes are encoded by the E1A gene.

Virus specific, major histocompatibility complex-restricted, cytotoxic T lymphocytes (CTL) generated in Fischer strain rats infected with human adenovirus type 5 (Ad5) were found to recognize antigenic determinants encoded within the Ad5 early region 1A (E1A) gene. Preliminary mapping studies suggest that the E1A CTL epitopes are encoded within the regions between bp 625 to 810 and 916 to 974 in the first exon of this gene. These epitope-coding regions occur within subregions of E1A that are conserved functionally, and to some extent structurally (approximately 50% sequence homology), among adenoviruses of different groups. Nevertheless, Ad5-specific CTL lysed only targets infected with adenoviruses of the same group (group C; e.g., Ad2) and not targets infected with adenoviruses of different groups (groups A, B, and E). These results suggest that virus-specific CTL may limit adenoviral dissemination by destroying virus-infected cells at an early stage in the viral replicative cycle, during E1A gene expression. Expression of other adenovirus genes does not appear to be required to target infected cells for elimination by CTL.     

Benzoate modulates renal and extrarenal nitrogen flow: metabolic mechanisms.

The mechanism by which benzoate enhances total nitrogen excretion was investigated in-situ and in separated rat renal proximal tubules. Orally administered benzoate augmented NH4+, urea and hippurate excretion 2, 1.9 and 76 fold respectively, as compared to baseline for control. Hippurate had similar effects. Benzoate augmented renal blood flow, glutamine extraction and total NH4+ production. Arterio-venous concentration differences of glutamine, glutamate, and NH4+ across the kidney, liver and gut demonstrated an increase in glutamine uptake by the kidney despite reduced release and uptake by the liver and gut, respectively; glutamate release by the kidney and gut was increased; NH4+ handling was unchanged at these three organs. Studies in separated rat renal proximal tubules demonstrated that benzoate stimulated glutamine dependent ammonia-genesis by activation of gamma-glutamyltransferase, via the synthesis of hippurate. The results demonstrate that benzoate can modulate the interorgan partitioning of nitrogen metabolites across several organs, the net effect of which is physiologically expressed as enhanced NH4+ , urea and hippurate excretion.     

In situ diel variation in gut pigment contents of Ceriodaphnia sp. in stratification and destratification periods

The short-term, in situ diel grazing of Ceriodaphnia sp. duringperiods of stratification and mixing was investigated usingthe technique of fluorimetric analysis of the gut pigments.There were considerable seasonal differences in feeding behaviourIn mixing, when the concentration of chlorophyll a in the watercolumn was high and Ceriodaphnia abundance was low, gut pigmentcontents showed clear diel variation patterns, probably dueto diel variations of the high values of feeding activity observedin the 24 hour cycle The maximum values were found at dawn.On the other hand, no diel variations in gut pigment were observedduring periods of stratification and while the amounts of pigmentsin the water and in the gut were very low, species abundancewas high. Taking into account the ambient conditions, the authorsdiscuss the possibility that the change of feeding of the Ceriodaphniasp. observed when the environment changed from a mixing periodto one of stratification represents a change from herbivorousto detritivorous behavior.