<p>The marine sponge <i>Geodia barretti</i> produces a range of secondary metabolites. Two of these compounds were isolated and elucidated guided by their ability to inhibit settlement of cypris larvae of the barnacle <i>Balanus improvisus</i>. The compounds barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]) as E/Z mixture and 8,9-dihydrobarettin (cyclo-[6-bromo-tryptophan)-arginine]) were determined by using mass spectrometry, nuclear magnetic resonance and quantitative amino acid analysis.The bioactivity of these brominated dipeptides is in the range of antifouling substances used today: EC₅₀ values of 0.9 µM (barettin) and 7.9 µM (8,9-dihydrobarettin). The compounds were successfully synthesised and then tested in a field experiment to evaluate their antifouling properties. The compounds were incorporated in four different commerical, non-toxic marine coatings. The concentrations of the compounds were 0.1 and 0.01% (w/w) and coated panels were exposed to field conditions for eight weeks. The experiment evaluated the effect of barettin and 8,9-dihydrobarettin on recruitment of the barnacle <i>B. improvisus</i> and the blue mussel <i>Mytilus edulis</i> (major Swedish foulers). The most efficient paint was a SPC polymer, for which the reduction of recruitment of <i>B. improvisus</i> was 89% with barettin (0.1%) and 61% with 8,9-dihydrobarettin (0.1%). For <i>M. edulis</i> the reduction of recruitment was 81% with barettin (0.1%) and 72% with 8,9-dihydrobarettin (0.1%) with the same SPC paint. Furthermore, 14 analogs of barettin and dipodazine were synthesised and tested for their ability to inhibit larval settlement. Two of the analogs have a barettin scaffold and twelve have a dipodazine scaffold. Six of the analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine (EC₅₀ value 0.034 µM). The effect of benzo[g]dipodazine was also shown to be reversible. Finally, an investigation of the mode of action was performed on 5-HT receptors. Barettin demonstrated a specific affinity to 5-HT_2A, 5-HT_2C and 5-HT₄, while 8,9-dihydrobarettin interacted only with 5-HT_2C of the receptor subtypes tested (5-HT₁-5-HT₇).</p>

The marine sponge Geodia barretti produces a range of secondary metabolites. Two of these compounds were isolated and elucidated guided by their ability to inhibit settlement of cypris larvae of the barnacle Balanus improvisus. The compounds barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]) as E/Z mixture and 8,9-dihydrobarettin (cyclo-[6-bromo-tryptophan)-arginine]) were determined by using mass spectrometry, nuclear magnetic resonance and quantitative amino acid analysis.The bioactivity of these brominated dipeptides is in the range of antifouling substances used today: EC50 values of 0.9 µM (barettin) and 7.9 µM (8,9-dihydrobarettin). The compounds were successfully synthesised and then tested in a field experiment to evaluate their antifouling properties. The compounds were incorporated in four different commerical, non-toxic marine coatings. The concentrations of the compounds were 0.1 and 0.01% (w/w) and coated panels were exposed to field conditions for eight weeks. The experiment evaluated the effect of barettin and 8,9-dihydrobarettin on recruitment of the barnacle B. improvisus and the blue mussel Mytilus edulis (major Swedish foulers). The most efficient paint was a SPC polymer, for which the reduction of recruitment of B. improvisus was 89% with barettin (0.1%) and 61% with 8,9-dihydrobarettin (0.1%). For M. edulis the reduction of recruitment was 81% with barettin (0.1%) and 72% with 8,9-dihydrobarettin (0.1%) with the same SPC paint. Furthermore, 14 analogs of barettin and dipodazine were synthesised and tested for their ability to inhibit larval settlement. Two of the analogs have a barettin scaffold and twelve have a dipodazine scaffold. Six of the analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine (EC50 value 0.034 µM). The effect of benzo[g]dipodazine was also shown to be reversible. Finally, an investigation of the mode of action was performed on 5-HT receptors. Barettin demonstrated a specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted only with 5-HT2C of the receptor subtypes tested (5-HT1-5-HT7).

The objective of this study is to evaluate pseudo-substrates of pantothenate kinase (PanK) for the therapeutic treatment of multidrug resistant bacterial infections of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Pantothenate (Pan) analogs, including N- pentylpantothenamide (N5-Pan) and N-heptylpantothenamide (N7-Pan), hamper bacterial growth by utilizing the PanK enzymes, which normally catalyze the rate determining step of the Coenzyme A biosynthetic pathway. Here we report the structures of SaPanK, Human PanK3 and EcPanK complexed with N7-Pan or N5-Pan, all of which have provided the opportunity to investigate the structural differences of bacterial and human Pan binding sites. The MTT assay showed these analogs to exhibit no apparent cytotoxicity against Human A549 lung adenocarcinoma cells, human HepG2 hepatoma cells and human umbilical vein endothelial cells (HUVEC). The presented structural differences have the potential for aiding the development of species-specific antimicrobial compounds with minimal effects on human cells.

Histone acetyltransferases (HATs) are important enzymes in transcriptional control and potential targets for chemotherapeutic intervention in malignant diseases. Among different HAT members, the yeast Esa1 and human Tip60 (the HIV-1 Tat interactive protein, 60KDa) play multiple roles in normal cellular processes including transcription, cell cycle and checkpoint machinery, double strand DNA break repair, apoptosis, and cell cycle progression. Tip60 is also implicated in several human diseases such as prostate cancer, and gastric cancer. These studies suggest that Tip60 is a potential therapeutic target for new cancer treatment. So, we designed experimental work to synthesize and investigate organic inhibitors of Tip60 using different strategies, including substrate analogs, small molecule screening, and modification of the natural product anacardic acid. These studies provide important chemical agents for basic biology research of HAT function, and produce potential lead compounds for future pharmacologic intervention of HAT deregulation in cancer. Currently, of the methods used for the measurement of acetyltransferase activities, many comprise tedious separation procedures and involve enzyme-coupled steps or radioactive materials. These shortcomings have limited their applications in high-throughput screening (HTS) of HAT inhibitors. To circumvent these problems, a homogenous fluorescent HAT assay based on engineered H4 peptide was designed, synthesized, and evaluated. The data showed that these fluorescent reporters can be used to detect the acetyltransferase activities.

No description available.

In this study, it was aimed to identify chickpea (Cicer arietinum) genes or gene fragments expressed upon Ascochyta rabiei infection using a tolerant chickpea cultivar ILC195 and fungal isolates with varying level of pathogenicity. PCR amplification of resistance gene analogs (RGA) and disease related genes, and mRNA differential display reverse transcription (DDRT) were used to get these expressed gene fragments in chickpea. The constitutively or differentially expressed PCR product fragments were cloned and sequenced. Out of nearly 300 clones, 160 sequences (expressed sequence tags, ESTs) could be analyzed and these sequences were disclosed in this study. About 100 of these ESTs were classified according to predicted &ldquo / molecular function&rdquo / , &ldquo / biological process&rdquo / and &ldquo / cellular component&rdquo / . The most common ppredicted functions of the products coded by these ESTs were &ldquo / Protein Fate&rdquo / , &ldquo / Metabolism&rdquo / , &ldquo / Cell Rescue, Defense and Virulence&rdquo / , &ldquo / Transcription&rdquo / , &ldquo / Transport&rdquo / , &ldquo / Energy&rdquo / , and &ldquo / Cell Fate&rdquo / . Six ESTs were subjected to Real-Time quantitative RT-PCR analysis to compare the response of ILC195 infected by one A.rabiei isolate with another resistant chickpea genotype (FLIP84-92C)/A.rabiei pathotype system. Some of these genes were differentially expressed among different chickpea/A.rabiei isolate combinations. Highly upregulated ESTs in all these combinations were a formate dehydrogenase (metabolism and detoxification), a serine carboxypeptidase (protein fate and communication) and a hypothetical protein probably similar to acyl-CoA synthetases. A genetic mapping study was carried out with EST specific primers and two EST markers were assigned in the current chickpea genetic map. However, no genetic linkage of them was detected with known chickpea quantitative trait loci for A.rabiei resistance.

Identification of differential expressions of proteins in proteomic profiles of biological samples shows great potential as a valuable technique for the early diagnosis of various diseases. An important challenge in modern protein profiling approaches is to reduce the complexity of the samples by limiting the number of proteins that need to be evaluated for distinction in the expression between normal and deceased cells. In this research, an affinity based approach for the enrichment of nucleotide and nucleoside binding proteins from a complex cell proteome has been developed. To achieve this goal, new N6-biotinylated-8-azido-adenosine probes (AdoRs) have been designed and synthesized to photolabel the nucleotide and nucleoside binding proteins. These probes contain a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Further, a mass spectrometric protein profiling approach is employed to quantitatively identify small variations in expression of nucleoside and nucleotide binding proteins in samples of interest. Mouse neuroblastoma N18TG2 cell proteome has been used as a model system for the development of the LC-MS/MS based proteomic analysis of these affinity enriched protein fractions. Upon enrichment, the photolabeled proteome exhibited an approximately four-fold abundance of nucleoside and nucleotide binding proteins over nonlabeled proteome. The approach was extended to compare the proteomic profiles of nucleotide and nucleoside binding proteins in cancerous (Hey) and non-cancerous (T-80) human ovarian cell proteome. Certain proteins that were not detected in cell lysate were also identified in labeled proteome, thereby demonstrating the strength of our approach in enriching low abundant proteins. To substantiate the qualitative analysis, we have employed the Stable Isotope Labeling in Amino Acid Cell Culture (SILAC) for the quantitative study of the protein expression in cancerous and non-cancerous human ovarian cells. A modest panel of proteins with differential expressions in these cell lines was identified, a few of which have been correlated to various forms of cancer. Vimentin, stress induced phosphoprotein-1, and heat shock protein 90 that were identified to have altered expressions in these cell lines are among some of the proteins associated with ovarian cancer.

The immune-reactive sperm are kept separate from the body by epithelial barriers such as the blood-testis barrier (BTB). While these barriers are beneficial for the protection of sperm from toxicants, they can make treating these areas difficult due to preventing the entry of pharmacological agents. This is especially an issue in the treatment of HIV and Ebola infection based on the ample evidence that these viruses are able to survive and spread from within the male genital tract (MGT), but only a few antiviral drugs are known to access the MGT. Transporters that line the epithelial barriers of the MGT, especially the BTB, are important for determining whether or not a drug is able to penetrate into the MGT through transepithelial transport. Several nucleoside analogs (NSA), which are used to treat HIV infection and leukemias, are known to be able to accumulate in seminal plasma, which makes them a useful tool for understanding transepithelial transport for the BTB. The purpose of these studies is to characterize the transport profile for the MGT, in particular the BTB, to gain a better understanding of how xenobiotics, especially ones based on nucleosides, can access the MGT. The chief finding of this work is the discovery of a transepithelial transport pathway expressed by Sertoli cells that allows for the entry of nucleosides (necessary for germ cell development) and NSA into the MGT. This pathway depends on equilibrative nucleoside transporter (ENT) 1 uptake and ENT2 efflux and occurs in both rats and humans. These studies provide the foundation for being able to predict the penetration of novel drugs into the MGT.

Cyclic guanosine 3', 5'-monophosphate (cGMP) has recently emerged as an endogenous regulator for controlling or reversing cardiac hypertrophy. Increased protein accumulation is a key feature of cardiac hypertrophy; thus, our study investigates the effects of a cGMP analog on protein accumulation in primary culture of adult rat cardiomyocytes and dissects out the mechanisms involved. We confirmed that a cGMP analog, 8-bromo-cGMP, inhibits phenylephrine (PE)-increased accumulation of newly synthesized proteins in cultured adult rat ventricular cardiomyocytes. Firstly, we have obtained data showing that 8-bromo-cGMP does not inhibit phosphorylation of S6K1 by PE during short time treatment (10 min to 2 h), but blocks phosphorylation of S6K1 by PE at 6 h; moreover this blocking effect is completely abolished by phosphatase inhibitor Tautomycin. Then, we have demonstrated that PE and cGMP induce sustained and transient increased phosphorylation of ERK, respectively. Moreover, cGMP inhibits PE-induced phosphorylation of ERK during long term treatment (3 and 6h). We have also shown that 8-bromo-cGMP inhibits ROS generation induced by PE. Other effects of PE that could be related to hypertrophy (i.e. increased concentration of upstream binding factor mRNA and decreased concentration of the mRNAs of Atrogin and muscle specific RING finger) were not abolished by 8-bromo-cGMP. We conclude that cGMP analog blocks protein accumulation by inhibiting the sustained phosphorylation of S6K1 via the activation of phosphatases.

Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets. To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15. To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells. In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.