Expression of Betapapillomavirus Oncogenes Increases the Number of Keratinocytes with Stem Cell-Like Properties

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a “stem cell-like” state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44^high EpCAM^high cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.     

OXIDIZED LIPIDS DID NOT REDUCE LIFESPAN IN THE FRUIT FLY,Drosophila melanogaster

Aging is often associated with accumulation of oxidative damage in proteins and lipids. However, some studies do not support this view, raising the question of whether high levels of oxidative damage are associated with lifespan. In the current investigation, Drosophila melanogaster flies were kept on diets with 2 or 10% of either glucose or fructose. The lifespan, fecundity, and feeding as well as amounts of protein carbonyls (PC) and lipid peroxides (LOOH), activities of superoxide dismutase (SOD), catalase, glutathione‐S‐transferase (GST), and glutathione reductase activity of thioredoxin reductase (TrxR) were measured in “young” (10‐day old) and “aged” (50‐day old) flies. Flies maintained on diets with 10% carbohydrate lived longer than those on the 2% diets. However, neither lifespan nor fecundity was affected by the type of carbohydrate. The amount of PC was unaffected by diet and age, whereas flies fed on diets with 10% carbohydrate had about fivefold higher amounts of LOOH compared to flies maintained on the 2% carbohydrate diets. Catalase activity was significantly lower in flies fed on diets with 10% carbohydrates compared to flies on 2% carbohydrate diets. The activities of SOD, GST, and TrxR were not affected by the diet or age of the flies. The higher levels of LOOH in flies maintained on 10% carbohydrate did not reduce their lifespan, from which we infer that oxidative damage to only one class of biomolecules, particularly lipids, is not sufficient to influence lifespan.     

Nest-mate recognition template of guard honeybees (Apis mellifera) is modified by wax comb transfer

In recognition, discriminators use sensory information to make decisions. For example, honeybee (Apis mellifera) entrance guards discriminate between nest-mates and intruders by comparing their odours with a template of the colony odour. Comb wax plays a major role in honeybee recognition. We measured the rejection rates of nest-mate and non-nest-mate worker bees by entrance guards before and after a unidirectional transfer of wax comb from a 'comb donor' hive to a 'comb receiver' hive. Our results showed a significant effect that occurred in one direction. Guards in the comb receiver hive became more accepting of non-nest-mates from the comb donor hive (rejection decreased from 70 to 47%); however, guards in the comb donor hive did not become more accepting of bees from the comb receiver hive. These data strongly support the hypothesis that the transfer of wax comb increases the acceptance of non-nest-mates not by changing the odour of the bees, but by changing the template used by guards.     

Capturing heterogeneity in gene expression studies by surrogate variable analysis

It has unambiguously been shown that genetic, environmental, demographic, and technical factors may have substantial effects on gene expression levels. In addition to the measured variable(s) of interest, there will tend to be sources of signal due to factors that are unknown, unmeasured, or too complicated to capture through simple models. We show that failing to incorporate these sources of heterogeneity into an analysis can have widespread and detrimental effects on the study. Not only can this reduce power or induce unwanted dependence across genes, but it can also introduce sources of spurious signal to many genes. This phenomenon is true even for well-designed, randomized studies. We introduce "surrogate variable analysis" (SVA) to overcome the problems caused by heterogeneity in expression studies. SVA can be applied in conjunction with standard analysis techniques to accurately capture the relationship between expression and any modeled variables of interest. We apply SVA to disease class, time course, and genetics of gene expression studies. We show that SVA increases the biological accuracy and reproducibility of analyses in genome-wide expression studies.     

Regulation of Apoptosis and Autophagy During Anoxia in the Freshwater Crayfish,Faxonius virilis

Marine Biotechnology - The ability of an animal to survive prolonged periods of oxygen deprivation is a critical area of study, both in terms of its importance to better understanding the...     

Dissociation-association of lactate dehydrogenase isozymes: influences on the formation of tetramers versus dimers of M4-LDH and H4-LDH

1. A molecular sieve membrane was used to separate active dimer vs active tetramer fractions of M4-LDH and H4-LDH. 2. Dissociation of both enzymes was influenced by enzyme protein concentration and by the concentration of added substrates, pyruvate or lactate. 3. Increasing lactate concentrations increased the fraction of tetrameric enzyme whereas increasing pyruvate (up to saturating levels) had the opposite effect, raising the content of dimer fraction. 4. For H4-LDH, levels of pyruvate that caused substrate inhibition reversed the effect of lower concentrations of pyruvate and reduced the dimer content. 5. The data suggest that dissociation-association of LDH may have functional importance, the dimer having a preferential role in pyruvate reduction and the tetramer a preferred function in lactate oxidation.     

A genetic screen for ethanolamine auxotrophs in Saccharomyces cerevisiae identifies a novel mutation in Mcd4p, a protein implicated in glycosylphosphatidylinositol anchor synthesis

A genetic screen for ethanolamine auxotrophs has identified a novel mutant allele of the morphogenesis checkpoint dependent (MCD)-4 gene, designated mcd4-P301L. In the presence of a null allele for the phosphatidylserine (PtdSer) decarboxylase 1 gene (psd1 Delta), the mcd4-P301L mutation confers temperature sensitivity for growth on minimal medium. This growth defect is reversed by either ethanolamine or choline supplementation. Incubation of mutant cells with [(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrated a 60% decrease in phosphatidylethanolamine (PtdEtn) formation compared to parental cells. Chemical analysis of phospholipid content after culture under non-permissive conditions also demonstrated a 60% decrease in the PtdEtn pool compared to the parental strain. Although the morphogenesis checkpoint dependent (MCD)-4 gene and its homologues have been shown to play a role in glycosylphosphatidylinositol (GPI) anchor synthesis, the mcd4-P301L strain displayed normal incorporation of [(3)H]inositol into both proteins and lipids. Thus, a defect in GPI anchor synthesis does not explain either the ethanolamine auxotrophy or biochemical phenotype of this mutant. We also examined the growth characteristics and PtdSer metabolism of a previously described mcd4-174 mutant strain, with defects in GPI anchor synthesis, protein modification and cell wall maintenance. The mcd4-174, psd1 Delta strain is a temperature sensitive ethanolamine auxotroph that requires osmotic support for growth, and displays normal PtdEtn formation compared to parental cells. These results reveal important genetic interactions between PSD1 and MCD4 genes, and provide evidence that Mcd4p can modulate aminoglycerophospholipid metabolism, in a way independent of its role in GPI anchor synthesis.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (11-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol P_i/mol of -subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P_i into the -subunit and the loss of activity of the Na,K-ATPase.