PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Enhanced antioxidant formula based on a selenium-supplemented carotenoid-producing yeast biomass

Carotenoid-producing yeast species such as Rhodotorula glutinis and Sporobolomyces roseus efficiently accumulated selenium from the growth medium. It was observed that incorporation of selenium into yeast cells during the growth inhibited production of beta-carotenoid and other carotenoid precursors (torularhodin and torulene). The yeasts with high content of the carotenoid pigments and selenium may be used for the preparation of a new type of antioxidant formula that could be directly applied for various human and animal diets. We have demonstrated that such a formula can only be produced by separate processes of the cultivation of red yeasts and a subsequent sorption of selenium into the cells.     

The role of hereditary and environmental factors in the variability of the components comprising the mass of the human upper extremity at various stages of postnatal ontogeny

The investigation has been performed in mono- and dizygotic twins of the Ukrainian ethnic and territorial groups. As a whole 196 pairs of twins, among them 80 pairs of mono- and 116 pairs of dizygotes of both sex at the age of 12-60 years have been undergone the examination. The twins selected are divided into two age groups--period of sex maturation (12-17 years of age) and that of maturity (30-60 years of age). Quantitative parameters on composition of the components of the upper extremity mass (fatty, muscular and osseous) are studied in all the twins. Coefficients of intrapaired correlation and indices of Holzinger's heritability are calculated according to an original program, using the microcomputer "Electronika B3-34". The degree of hereditary and environmental influences for development and formation of the main components of the upper extremity mass at certain stages of individual development are characterized with ontogenic changeability. With age the degree of hereditary dependence of the development and formation of the upper extremity mass components decreases, and sex dimorphism according to the degree of the hereditary dependence of the components studied is displayed as a greater hereditary determination in male persons.     

Structural studies of mannans from the cell walls of the pathogenic yeasts Candida albicans serotypes A and B and Candida parapsilosis

A comparative study of three cell-wall mannans, of Candida albicans serotypes A and B and Candida parapsilosis, by means of methylation analysis supports a model of yeast mannans as having an alpha-(1----6)-linked backbone with some units (depending on the origin of the mannan) being substituted at O-2 with oligosaccharides joined by alpha-(1---2) and, to a lesser extent, by alpha-(1----3) glycosidic bonds. Branching points in the side chains of Candida albicans mannans were found in substantial proportions for the first time, and the corresponding branched hexasaccharides were isolated by means of acetolysis and subsequent gel filtration. 13C-N.m.r. spectroscopy of the mannans, as well as a 1H-n.m.r. spectroscopic study of the oligosaccharides obtained on acetolysis of the mannans, led to results that agreed with those of methylation analysis.     

Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent.

Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, can be used to direct selective killing of human immunodeficiency virus (HIV)-infected cells. This approach takes advantage of the ability of sCD4 to bind with high affinity to gp120, the envelope protein of HIV-1, which is expressed on actively infected cells. However, conjugation of sCD4 via reagents that target amino groups may reduce its affinity for gp120, since at least one such group is important for gp120 binding. Here, we describe a novel cross-linking reagent which enables the conjugation of sCD4 via its carbohydrate moieties rather than its free amino groups. This heterobifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), combines a nucleophilic hydrazide with an electrophilic maleimide, thereby allowing coupling of carbohydrate-derived aldehydes to free thiols. We describe conditions by which MPBH is coupled selectively to the sialic acid residues of sCD4, and exemplify the use of MPBH by conjugating sCD4 to hemoglobin and to beta-galactosidase. We show that, whereas conjugation of sCD4 via amino groups markedly reduces its gp120 binding affinity, conjugation via the carbohydrate chains using MPBH does not affect binding. Moreover, we demonstrate the ability of a sCD4-MPBH-fluorescein conjugate to label HIV-infected human CEM cells selectively. These results indicate that, by targeting its carbohydrate moieties, sCD4 can be cross-linked to other molecules without compromising its function. The approach described here can be useful for glycoproteins in which amino groups, but not carbohydrates, are important for function. More generally, this approach can be considered for use in cross-linking glycoconjugates to compounds which either contain thiols, or to which thiols can be added.     

Do consensus conferences influence their participants?

OBJECTIVE: To determine whether participation in a consensus conference on the assessment of dementia would influence conference participants with respect to their recommendations to primary care physicians for the assessment of dementia. DESIGN: Questionnaire before and after the conference. SETTING: Canadian Consensus Conference on the Assessment of Dementia, held in Montreal, Oct. 5 and 6, 1989. PARTICIPANTS: All 38 experts representing relevant health disciplines who participated in the consensus conference; 36 completed both questionnaires. OUTCOME MEASURES: Participants'' opinion before and after the conference as to how frequently each of 28 manoeuvres (12 blood tests, 4 neurologic imaging procedures, 4 types of consultation and 8 "other" tests) should be ordered by primary care physicians as part of an assessment of a patient with dementia suspected in clinical grounds. RESULTS: For 18 (64%) of the 28 manoeuvres (10 of the 12 blood tests, 3 of the 4 neurologic imaging procedures and 5 of the 8 "other" tests), there was a shift in opinion after the conference toward recommending that primary care physicians order them less often; for 10 of these 18 (5 blood tests and 5 "other" tests) the shift was statistically significant. For the remaining 10 manoeuvres (36%) the shift in opinion was toward a recommendation that primary care physicians order them more often; the shift was not statistically significant for any of these 10 manoeuvres. CONCLUSION: Expert members of a consensus conference are influenced by the process of having participated in such a conference and are capable and willing to chance their initial recommendations when confronted with relevant data.     

Preparation of isolated cells by alkaline dissociation of formalin fixed tissue]

A procedure is described for preparation of isolated cells by treating formaldehyde fixed tissues with a 50%-solution of KOH. This results in complete yield of cells from a variety of organs (liver, kidney, heart, spleen, etc.). The alkali-treated cells entirely retain their morphological and tinctorial peculiarities. It was shown that preparations derived from alkali-treated tissues were useful for a series of quantitative cytological and cytochemical techniques: cytofluorometric estimation of nuclear DNA; interferometric determination of dry cell mass; autoradiographic studies of nuclear DNA synthesis; cell number counts; evaluation of cell distribution according to the number of their nuclei; estimation of the mitotic index.