Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G(1) phase. Aberrant expression of CDK4 and CDK6 is a hallmark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM (the calcein acetoxymethyl-ester) is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G(1) phase. The metabolic effects of calcein AM on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phosphate pathway was significantly altered. To elucidate whether these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.     

The late Neogene rodent succession of the Guadix–Baza Basin (south‐eastern Spain) and its magnetostratigraphic correlation

In this paper a magnetostratigraphically calibrated biozonation of the Miocene–Pliocene continental record of the Guadix–Baza Basin (south‐eastern Spain) is presented. This biozonation is based on a rodent succession which ranges from the latest Miocene (c. 6 Ma) to the latest Pliocene (c. 2.6 Ma). A total of nine biozones have been defined for the late Miocene and Pliocene, all of them based on the range or concurrent‐range of rodent species: Apodemus gudrunae – Apocricetus alberti Zone, Apocricetus barrierei Zone, Paraethomys aff. abaigari Zone, Trilophomys Zone, Mimomys davakosi Zone, Dolomys adroveri Zone, Mimomys hassiacus Zone, Mimomys polonicus Zone and Kislangia ischus Zone. A magnetobiostratigraphical correlation has been established between these biozones and the standard ATNTS scale, on the basis of the palaeomagnetic analysis carried out on the sections of Negratín, Botardo‐1 and Gorafe. The correlation has been completed with previous palaeomagnetic analysis in the sections of Galera and Zújar. The magnetobiostratigraphical correlation here established indicates a late Messinian age for the Apodemus gudrunae – Apocricetus alberti Zone, a Zanclean age for the Apocricetus barrierei, Paraethomys aff. abaigari, Trilophomys and Mimomys davakosi zones and a Piazencian age for the Mimomys hassiacus, Mimomys polonicus and Kislangia ischus zones. The Dolomys adroveri Zone is mostly Zanclean in age, but its uppermost part belongs to the Piazencian. Therefore, unit MN13 is correlated with the late Messinian, MN14 is correlated with the early Zanclean, most of MN15 is correlated with the late Zanclean, while the uppermost part of MN15 and MN16 are correlated with the Piazencian.     

Association between EBRT dose volume histograms and quality of life in prostate cancer patients

Aim

To evaluate the association between dose–volume histogram (DVH) values in organs at risk (OAR) and patient-reported HRQoL outcomes.

The specificity of alcohol dehydrogenase with cis-retinoids. Activity with 11-cis-retinol and localization in retina.

Studies in knockout mice support the involvement of alcohol dehydrogenases ADH1 and ADH4 in retinoid metabolism, although kinetics with retinoids are not known for the mouse enzymes. Moreover, a role of alcohol dehydrogenase (ADH) in the eye retinoid interconversions cannot be ascertained due to the lack of information on the kinetics with 11-cis-retinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with all-trans-, 7-cis-, 9-cis-, 11-cis- and 13-cis-isomers of retinol and retinal. These retinoids are substrates for all enzymes tested, except the 13-cis isomers which are not used by ADH1. In general, human and mouse ADH4 exhibit similar activity, higher than that of ADH1, while mouse ADH1 is more efficient than the homologous human enzymes. All tested ADHs use 11-cis-retinoids efficiently. ADH4 shows much higher k(cat)/K(m) values for 11-cis-retinol oxidation than for 11-cis-retinal reduction, a unique property among mammalian ADHs for any alcohol/aldehyde substrate pair. Docking simulations and the kinetic properties of the human ADH4 M141L mutant demonstrated that residue 141, in the middle region of the active site, is essential for such ADH4 specificity. The distinct kinetics of ADH4 with 11-cis-retinol, its wide specificity with retinol isomers and its immunolocalization in several retinal cell layers, including pigment epithelium, support a role of this enzyme in the various retinol oxidations that occur in the retina. Cytosolic ADH4 activity may complement the isomer-specific microsomal enzymes involved in photopigment regeneration and retinoic acid synthesis.     

Temporal genetic dynamics among mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) populations in invaded watersheds

The temporal components of genetic diversity and geographical structure of invasive mosquitofish populations are poorly known. Through the genetic monitoring of four consecutive cohorts of Gambusia holbrooki from three different river basins we aimed to determine temporal patterns of regional genetic variation and dispersal rates within invasive populations. Despite showing evidence of strong population size fluctuations, genetic diversity levels were maintained among local cohorts. We only detected temporal allele frequency changes associated with seasonal flooding that did not modify major trends on population structure among cohorts. Downstream gene flow coupled with increased connectivity at lowland locations to increase genetic diversity levels in these areas. A large proportion of local fish (up to 50 %) were dispersers, often originated from locations within the same river basin. High dispersal capability, ecological tolerance, and reproductive traits likely promote river colonization. Finally, our results also confirmed that human-assisted translocations promote within and among basin gene flow and maintained levels of genetic diversity, particularly in upstream locations.     

Biochemical changes in the equine capsule following prostaglandin-induced pregnancy failure

The equine embryonic capsule, an acellular covering that envelops the conceptus during the second and third weeks of pregnancy, is composed of mucin-like glycoproteins. Its structure is consistent with a dual role during early pregnancy: protection of the conceptus, and communication between the embryo and the mother. Loss of sialic acid from the capsular glycoproteins at day 16 correlates with the time of “fixation,” or loss of conceptus mobility throughout the uterine horns. This study investigated how the structure of the capsule is linked to the maintenance of pregnancy. Six pregnancies, confirmed by ultrasound, were terminated by prostaglandin injection on day 14, prior to the time of embryo fixation. These “defective” conceptuses were collected at day 17, and the structure and molecular properties of their capsules were compared to those of day 17 conceptuses collected from 5 normal pregnancies. Defective capsules were not significantly different from normal capsules in terms of dry weight, amino acid composition, and content of neutral and amino sugars. However, defective capsules failed to show the loss of sialic acid normally occurring around the time of embryo fixation. Analysis of the capsular mucins following trypsin digestion was carried out by radioactive labeling with ³H on sialyl-oligosaccharides and ¹²⁵I on tyrosine residues, followed by fast protein liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Differences in the trypsin fragmentation patterns indicated increased susceptibility of the defective capsules to proteolysis. We conclude that there is a temporal association between desialylation of the equine capsule and embryonic survival, and that failure to desialylate alters the properties of the capsule. Mol. Reprod. Dev. 46:286–295, 1997. © 1997 Wiley-Liss, Inc.     

Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low‐density lipoprotein receptor‐related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet‐free plasma by enzyme‐linked immunosorbent assay. Plasma‐derived EVs were extracted by size‐exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo‐transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin‐3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9‐ and CD81‐positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin‐3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.