Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents

Drug resistance is a major obstacle in the successful treatment of cancer. Thus, elucidation of the mechanisms responsible is a critical first step in trying to prevent or delay such manifestations of resistance. In this regard, three-dimensional multicellular tumor cell spheroids are intrinsically more resistant to virtually all anticancer cytotoxic drugs than conventional monolayer cultures. We have employed the EMT-6 subline PC5T, which forms highly compact spheroids, and differential display to identify candidate genes whose expression differs between monolayer and spheroids. Approximately 5,000 bands were analyzed, revealing 26 to be differentially expressed. Analysis of EMT-6 tumor variants selected in vivo for acquired resistance to alkylating agents identified eight genes whose expression correlated with drug resistance in tumor spheroids. Four genes (encoding Nop56, the NADH SDAP subunit, and two novel sequences) were found to be down-regulated in EMT-6 spheroids and four (encoding 2-oxoglutarate carrier protein, JTV-1, and two novel sequences) were up-regulated. Analysis of the DNA mismatch repair-associated PMS2 gene, which overlaps at the genomic level with the JTV-1 gene, revealed PMS2 mRNA to be down-regulated in tumor spheroids, which was confirmed at the protein level. Analysis of PMS2(-/-) mouse embryo fibroblasts confirmed a role for PMS2 in sensitivity to cisplatin, and DNA mismatch repair activity was found to be reduced in EMT-6 spheroids compared to monolayers. Dominant negative PMS2 transfection caused increased resistance to cisplatin in EMT-6 and CHO cells. Our results implicate reduced DNA mismatch repair as a determinant factor of reversible multicellular resistance of tumor cells to alkylating agents.     

Tuber borchii fruit body: 2-dimensional profile and protein identification

The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified.     

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.     

Role of PGE2 on gallbladder muscle cytoprotection of guinea pigs

H2O2 and taurochenodeoxycholic acid (TCDC) impair the contraction induced by CCK-8, ACh, and KCl without affecting the actions of PGE2 and damage functions of membrane proteins except for PGE2 receptors. The aim of this study was to examine whether the preserved PGE2 actions contribute to cytoprotective mechanisms against reactive oxygen species. Muscle cells from guinea pig gallbladder were obtained by enzymatic digestion. Levels of lipid peroxidation and activities of SOD and catalase were determined by spectrophotometry. Pretreatment with PGE2 prevented the inhibition of H2O2 or TCDC on agonist (CCK-8, ACh, and KCl)-induced contraction and reduced the expected increase in lipid peroxidation and activities of catalase and SOD caused by H2O2 and TCDC. Incubation with CCK-8 for 60 min desensitized CCK-1 receptors up to 30 min, whereas no receptor desensitization was observed after PGE2 pretreatment. Cholesterol-rich liposome treatment enhanced the inhibition of H2O2 and TCDC on agonists-induced contraction, including that of PGE2. Pretreatment with PGE2 before H2O2 and TCDC did not completely block their inhibition on agonist-induced contraction. Cholesterol-rich liposome treatment impaired the expected increase in catalase activities in response to PGE2. We conclude that pretreatment with PGE2 prevents the muscle cell damage caused by H2O2 and TCDC due to the resistance of PGE2 receptors to agonist-induced desensitization. The preservation of PGE2 receptors may be designed to conserve these cytoprotective functions that are, however, impaired by the presence of excess cholesterol in the plasma membrane.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.     

Physiological responses to fitness activities: a comparison between land-based and water aerobics exercise

This study compared the heart rate (HR) and blood lactate (BL) responses in young healthy women performing the same routine of aerobics exercise in 3 different conditions: on land, in shallow water (0.8 m), and in deep water (1.4 m). The average age and body mass index (BMI) of the group were 27.4 years and 22.6 kg.m(-2), respectively. The highest HR and BL values were reached during land aerobics (median HR values were 138.0 and 161.5 b.min(-1), and lactate values were 3.10 and 5.65 mmol.L(-1) at slow and at faster pace, respectively). These parameters were progressively reduced going from shallow water (121.5 and 154.0 b.min(-1), 1.75 and 3.15 mmol.L(-1)) to deep water (97.5 and 113.5 b.min(-1), 1.70 and 1.75 mmol.L(-1)). The HR measured as percentage of maximum HR varied from 48.43% to 77.53% depending on the water depth and the pace. These data indicate that exercise in water significantly reduces HR and BL production compared with the same exercise performed on land.     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

ortho-Halogen naphthaleins as specific inhibitors of Lactobacillus casei thymidylate synthase. Conformational properties and biological activity

Thymidylate synthase (TS) (EC 2.1.1.45), an enzyme involved in the DNA synthesis of both prokaryotic and eukaryotic cells, is a potential target for the development of anticancer and antinfective agents. Recently, we described a series of phthalein and naphthalein derivatives as TS inhibitors. These compounds have structures unrelated to the folate (Non-Analogue Antifolate Inhibitors, NAAIs) and were selective for the bacterial versus the human TS (hTS). In particular, halogen-substituted molecules were the most interesting. In the present paper the halogen derivatives of variously substituted 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[2,3-c]furan-1-one (1-5) and 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-c,d]pyran-1-one (6-14) were synthesized to investigate the biological effect of halogen substitution on the inhibition and selectivity for the TS enzymes. Conformational properties of the naphthalein series were explored in order to highlight possible differences between molecules that show species-specific biological profile with respect to non species-specific ones. With this aim, the conformational properties of the synthesized compounds were investigated by NMR, in various solvents and at different temperatures, and by computational analysis. The apparent inhibition constants (K(i)) for Lactobacillus casei TS (LcTS) were found to range from 0.7 to 7.0 microM, with the exception of the weakly active iodo-derivatives (4, 10, 13); all] the compounds were poorly active against hTS. The di-halogenated compounds 7, 8, 14 showed the highest specificity towards LcTS, their specificity index (SI) ranging between 40 and >558. The di-halogenated 1,8-naphthalein derivatives (7-10) exhibited different conformational properties with respect to the tetra-haloderivatives. Though a clear explanation for the observed specificity by means of conformational analysis is difficult to find, some interesting conformational effects are discussed in the context of selective recognition of the compounds investigated by the LcTS enzyme.