Hypoxanthine: Guanine phosphoribosyltransferase mutants in Saccharomyces cerevisiae

Summary Yeast mutants lacking activity of the enzyme hypoxanthine: guanine phosphoribosyltransferase (H:GPRT) have been isolated by selecting for resistance to 8-azaguanine in a strain carrying the wild type allele, ade4 ⁺ of the gene coding for amidophosphoribosyltransferase (PRPPAT), the first enzyme of de novo purine synthesis. The mutants excrete purines and are cross-resistant to 8-azaadenine. They are recessive and represent a single complementation group, designated hpt1. Ade4-su, a prototrophic allele of ade4 with reduced activity of PRPPAT, is epistatic to hpt1, suppressing purine excretion and resistance to azaadenine but not resistance to azaguanine. The genotype ade2 hpt1 does not respond to hypoxanthine. Hpt1 complements and is not closely linked to the purine excreting mutants pur1 to pur5. Hpt1 and pur6, a regulatory mutant of PRPPAT, are also unlinked but do not complement, suggesting a protein-protein interaction between H:G-PRT and PRPPAT. Mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis, inhibits the growth of hpt1 and hpt1 ⁺. Xanthine allows both genotypes to grow in the presence of MPA whereas guanine only allows growth of hpt1 ⁺. Activity of A-PRT, X-PRT and H:G-PRT is present in hpt ⁺. Hpt1 lacks activity of H:G-PRT but has normal A-PRT and X-PRT.     

The learning skills of primates: The rhesus macaque in comparative perspective

Twenty-month-old rhesus monkeys were tested in a modified discrimination-reversal paradigm, which was designed to distinguish abstract learning from stimulus-response associational learning. Previous studies indicate that talapoin monkeys learn associationally and great apes via forming abstract concepts. Adult rhesus monkeys are apparently capable of forming simple abstractions, but learn primarily through associational process. The results of this study show the adolescent rhesus monkeys to be associational learners, with their response patterns indicating more complexity than the talapoins but less than the adult rhesus monkeys. The data suggest that rhesus monkeys develop their low-level capacity of abstract learning with maturation.     

Plasmodium berghei: Osmotic fragility of malaria parasites and mouse host erythrocytes

The osmotic properties of intraerythrocytic and ultrasonically liberated malaria parasites (Plasmodium berghei) were analyzed and compared with those of mouse host erythrocytes utilizing a multiple tube fragility test. Cells were incubated in phosphate buffered saline solutions of varying osmolalities ranging from 20–4000 mOsm. Changes in cell ultrastructure and parasite infectivity were used as indicators of osmotic damage. Intraerythrocytic and host cell-free plasmodia showed similar patterns of cell alteration and changes in infectivity following osmotic stress. The various developmental forms within each of the preparations responded somewhat differently to hypo-osmotic stress, however. The majority of merozoites seemed to be more sensitive than many trophozoites, schizonts, and segmenters. Small trophozoites were, on the average, more resistant than other developmental forms. Incubation of parasite populations in hypotonic salt solutions with osmolalities slightly greater than the infectivity threshold of 100 mOsm lysed the majority of the merozoites, whereas many small trophozoites were still intact. While normal erythrocytes were more resistant to hypo-osmotic stress than were either intracellular or free parasites, the majority of parasitized erythrocytes was less resistant than normal erythrocytes. The predominant alteration induced by hyperosmotic stress appears in the parasite's nuclear region with myelination of the nuclear membranes and chromatin clumping. The infectivity threshold in the hypertonic range was found to be approximately 2500 mOsm. Results indicate that these obligate intracellular parasites have a wide range of osmotic sensitivities and that they are capable of existing for short periods in various osmotic environments ranging from 100–2500 mOsm without complete loss of infectivity. This suggests that these parasites have osmotic regulatory capabilities at least comparable to those of host cells.     

Severe Painful Vaso-Occlusive Crises and Mortality in a Contemporary Adult Sickle Cell Anemia Cohort Study

Background

Frequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort.

Methods

Two hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation.

Results

Higher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p < 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p < 0.0001) were also independent risk factors for mortality.

Conclusions

Severe painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies.

Trial Registration

ClinicalTrials.gov NCT00011648 http://clinicaltrials.gov/     

Pharmacological inhibition of the MEK5/ERK5 and PI3K/Akt signaling pathways synergistically reduces viability in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.     

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.     

QTL mapping and confirmation for tolerance of anaerobic conditions during germination derived from the rice landrace Ma-Zhan Red

Wide adoption of direct-seeded rice practices has been hindered by poorly leveled fields, heavy rainfall and poor drainage, which cause accumulation of water in the fields shortly after sowing, leading to poor crop establishment. This is due to the inability of most rice varieties to germinate and reach the water surface under complete submergence. Hence, tolerance of anaerobic conditions during germination is an essential trait for direct-seeded rice cultivation in both rainfed and irrigated ecosystems. A QTL study was conducted to unravel the genetic basis of tolerance of anaerobic conditions during germination using a population derived from a cross between IR42, a susceptible variety, and Ma-Zhan Red, a tolerant landrace from China. Phenotypic data was collected based on the survival rates of the seedlings at 21 days after sowing of dry seeds under 10 cm of water. QTL analysis of the mapping population consisting of 175 F_2:3 families genotyped with 118 SSR markers identified six significant QTLs on chromosomes 2, 5, 6, and 7, and in all cases the tolerant alleles were contributed by Ma-Zhan Red. The largest QTL on chromosome 7, having a LOD score of 14.5 and an R ² of 31.7 %, was confirmed using a BC₂F₃ population. The QTLs detected in this study provide promising targets for further genetic characterization and for use in marker-assisted selection to rapidly develop varieties with improved tolerance to anaerobic condition during germination. Ultimately, this trait can be combined with other abiotic stress tolerance QTLs to provide resilient varieties for direct-seeded systems.