Gene expression patterns in ovarian carcinomas

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.     

Multimodal assessment of pain in the esophagus: a new experimental model

A new multimodal pain assessment model was developed integrating electrical, mechanical, cold, and warmth stimuli into the same device. The device, with a bag and electrodes for electrical stimulation, was positioned in the lower part of the esophagus in 11 healthy subjects. Mechanical stimuli were delivered with an impedance planimetric system. Thermal stimuli were performed by circulating water of different temperatures (5-50 degrees C) inside the bag. All subjects reported both nonpainful and painful local and referred sensations to all stimuli. Temporal summation to repeated electrical stimuli could be studied. For all stimuli, there was a relationship between stimulus intensity and pain intensity. The referred pain area increased with increasing intensity of the electrical and mechanical stimuli. There were several differences between the sensations evoked by the four stimulus modalities, indicating activation of different visceral nerve pathways. This model offers the possibility for controlled multimodal stimuli activating the superficial and deeper layers of the human gut and should be used in basic, clinical, and pharmacological pain studies.     

No evidence of inhibition of horizontal gene transfer by CRISPR–Cas on evolutionary timescales

The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)–Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR–Cas maintenance and one of the causes of the patchy distribution of CRISPR–Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR–Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR–Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.     

The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers

Transformation and cancer growth are regulated by the coordinate actions of oncogenes and tumor suppressors. Here, we show that the novel E3 ubiquitin ligase HACE1 is frequently downregulated in human tumors and maps to a region of chromosome 6q21 implicated in multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late-onset cancer. A second hit from either environmental triggers or genetic heterozygosity of another tumor suppressor, p53, markedly increased tumor incidence in a Hace1-deficient background. Re-expression of HACE1 in human tumor cells directly abrogates in vitro and in vivo tumor growth, whereas downregulation of HACE1 via siRNA allows non-tumorigenic human cells to form tumors in vivo. Mechanistically, the tumor-suppressor function of HACE1 is dependent on its E3 ligase activity and HACE1 controls adhesion-dependent growth and cell cycle progression during cell stress through degradation of cyclin D1. Thus, HACE1 is a candidate chromosome 6q21 tumor-suppressor gene involved in multiple cancers.     

FAS-Dependent Cell Death in α-Synuclein Transgenic Oligodendrocyte Models of Multiple System Atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.     

Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.     

CO2- and anaerobiosis-induced changes in physiology and gene expression of different Listeria monocytogenes strains

Although carbon dioxide (CO(2)) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO(2) concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N(2), and 100% CO(2). The CO(2)-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO(2) as to other anaerobic, slightly acidic environments (100% N(2), pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO(2) and N(2) at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO(2)-containing atmospheres. Together, our findings demonstrate that the CO(2)-response is a partly strain-dependent complex mechanism. The possible links between the CO(2)-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.     

Phase I and II metabolism and carbohydrate metabolism in cultured cryopreserved porcine hepatocytes

Primary porcine hepatocytes were cryopreserved using freezing boxes or a programmable freezer (PF). Upon thawing and culturing in 12-well plates cryopreserved hepatocytes were compared with their fresh controls on days 1 and 2 after plating. Cryopreserved hepatocytes attached approximately as well as fresh hepatocytes and useful cultures were obtained. In cryopreserved hepatocytes, coumarin 7-hydroxylation, 6beta-testosterone hydroxylation and p-nitrophenol glucuronidation were reduced to about 10-40, 35 and 40%, respectively, compared to their fresh counterparts. Glycogen synthesis in cryopreserved hepatocytes was reduced to about 30% on day 1 of culture and about 47% on day 2 of culture compared to the synthesis in fresh hepatocytes. Both fresh and cryopreserved hepatocytes increased the synthesis by twofold in response to stimulation with insulin. Reduced basal levels of glycogen and of glycogen synthesis could be explained by an increased energy demand in cryopreserved hepatocytes needing to repair damages caused by cryopreservation. Glycogenolysis was reduced to about 50% in cryopreserved hepatocytes and gluconeogenesis to about 40% of the glucose production in fresh hepatocytes. In both fresh and cryopreserved hepatocytes the glucose production from glycogenolysis and gluconeogenesis, respectively, was increased fourfold in response to stimulation with glucagon. Overall, the hepatocytes cryopreserved in boxes had a tendency to perform better than hepatocytes cryopreserved in a programmable freezer. In conclusion, the cryopreserved hepatocytes were metabolic active; however, to a lower extent than the fresh hepatocytes, although, the cryopreserved hepatocytes responded as well as the fresh hepatocytes to insulin and glucagon.     

Effects of inbreeding and rate of inbreeding in Drosophila melanogaster- Hsp70 expression and fitness

Induction of heat shock proteins (Hsp) is a well-known mechanism through which cells cope with stressful conditions. Hsp are induced by a variety of extrinsic stressors. However, recently intrinsic stressors (aging and inbreeding) have been shown to affect expression of Hsp. Increased homozygosity due to inbreeding may disrupt cellular homeostasis by causing increased expression of recessive deleterious mutations and breakdown of epistatic interactions. We investigated the effect of inbreeding and the rate of inbreeding on the expression of Hsp70, larval heat resistance and fecundity. In Drosophila melanogaster we found that inbred lines (F approximately 0.67) had significantly up-regulated expression of Hsp70, and reduced heat resistance and fecundity as compared with outbred control lines. A significant negative correlation was observed between Hsp70 expression and resistance to an extreme heat stress in inbred lines. We interpreted this as an increased requirement for Hsp70 in the lines suffering most from inbreeding depression. Inbreeding depression for fecundity was reduced with a slower rate of inbreeding compared with a fast rate of inbreeding. Thus, the effectiveness of purging seems to be improved with a slower rate of inbreeding.     

The presence of intact mitochondrial DNA in HeLa cell nuclei.

Restriction analysis of DNA labelled with [32P]dCTP in an in vitro replication system with isolated nuclei from early S phase cells showed preferential labelling of restriction fragments derived from mitochondrial DNA (mtDNA) by a replication machinery distinct from that responsible for bulk nuclear DNA replication. Use of restriction nucleases with one recognition site in mtDNA gave rise to 16.5 kbp long fragments corresponding to full-length linearized mtDNA, indicating the presence of intact mtDNA in the isolated cell nuclei. Incorporation of dNTPs into mtDNA was not restricted to the S phase of the cell cycle. We were unable to increase the labelling of mtDNA by the addition of purified mitochondria or mtDNA to the nuclear replication system. These and other results presented is evidenced that the presence of mtDNA in the isolated nuclei was not due to uptake during preparation, thus indicating its presence in the cell nucleus in vivo.