The chromolipoids: Dependance of histochemical characteristics on the degree of polymerization and their differentiation from DNA

Summary Chromolipoid pigments prepared in vitro were found to be a mixture of compounds of different degrees of polymerization and different histochemical characteristics. Some fractions give positive Feulgen nucleal reaction, stain with methyl-green and absorb ultraviolet light, thus closely mimicking DNA. Ways of differentiating such chromolipoids from DNA are pointed out.Similar chromolipoids have been produced in rats by injecting subcutaneously cod liver oil, or by chronic poisoning by lead.With 4 Figures in the Text, of which 2 in ColourThis study is part of a thesis submitted by Mr.S. Shoshan to the Hebrew University Hadassah Medical School in partial fulfilment of the requirements for the title of Master of Medical Sciences.     

Two distinct steps of Bak regulation during apoptotic stress signaling: different roles of MEKK1 and JNK1

Stress-activated protein (SAP) kinases and the mitochondrial pro-apoptotic Bcl-2 protein Bak are important regulators of apoptosis. Reduced expression of Bak increases cellular resistance to the anticancer agent cisplatin, and we report here that mouse embryo fibroblasts deficient in the SAP kinase jnk1 are highly resistant to apoptosis induced by cisplatin. When human melanoma cells were treated with cisplatin, Bak function was found to be regulated in two distinct steps by two SAP kinases, MEKK1 and JNK1. The first of these steps involves MEKK1-controlled conformational activation of Bak. The second step leads to formation of 80-170 kDa Bak complexes correlating with apoptosis, and is controlled by JNK1. Inhibition of MEKK1 blocked the initial Bak conformational activation but did not block JNK1 activation, and deficiency in, or inhibition of, JNK1 did not prevent conformational activation of Bak. Furthermore, inducible expression of a constitutively active form of MEKK1 led to Bak conformational activation, but not to 80-170 kDa complexes. Consequently, apoptosis was delayed unless JNK was exogenously stimulated, indicating that Bak conformational activation is not necessarily an apoptotic marker. The two-step regulation of Bak revealed here may be important for tight control of mitochondrial factor release and apoptosis.     

Expression of Mitochondrial Regulators PGC1α and TFAM as Putative Markers of Subtype and Chemoresistance in Epithelial Ovarian Carcinoma

Epithelial ovarian carcinoma (EOC), the major cause of gynaecological cancer death, is a heterogeneous disease classified into five subtypes. Each subtype has distinct clinical characteristics and is associated with different genetic risk factors and molecular events, but all are treated with surgery and platinum/taxane regimes. Tumour progression and chemoresistance is generally associated with major metabolic alterations, notably altered mitochondrial function(s). Here, we report for the first time that the expression of the mitochondrial regulators PGC1α and TFAM varies between EOC subtypes; furthermore, we have identified a profile in clear-cell carcinoma consisting of undetectability of PGC1α/TFAM, and low ERα/Ki-67. By contrast, high-grade serous carcinomas were characterised by a converse state of PGC1α/TFAM, ERα positivity and a high Ki-67 index. Interestingly, loss of PGC1α/TFAM and ERα was found also in a non-clear cell EOC cell line made highly resistant to platinum in vitro. Similar to clear-cell carcinomas, these resistant cells also showed accumulation of glycogen. Altogether, our data provide mechanistic insights into the chemoresistant nature of ovarian clear-cell carcinomas. Furthermore, these findings corroborate the need to take into account the diversity of EOC and to develop subtype specific treatment strategies.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins

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