Characterization of serum biomarkers for detection of early stage ovarian cancer

We have previously reported the identification of three ovarian cancer biomarker panels comprised of SELDI-TOF-MS peaks representing 14 differentially expressed serum proteins for the diagnosis of ovarian cancer. Using micro-LC-MS/MS, we identified five m/z peaks as transthyretin (TTR 13.9 kDa, TTR fragment 12.9 kDa), beta-hemoglobin (Hb, 15.9 kDa), apolipoprotein AI (ApoAI, 28 kDa) and transferrin (TF, 79 kDa). Western and/or ELISA methods confirmed the differential expression of TTR, Hb, and TF, and multivariate analyses resulted in improving the detection of early stage ovarian tumors (low malignant potential and malignant; receiver operating characteristic, ROC 0.933) as compared to cancer antigen CA125 alone (ROC 0.833). Interestingly, when CA125 was included with our markers in the multivariate analysis, the ROC increased to 0.959. Furthermore, multivariate analysis with only the mucinous subtype of early stage ovarian tumors, showed our markers to greatly improve the detection of disease (ROC 0.959) as compared to CA125 alone (ROC 0.613). Interestingly, the combination of CA125 with our markers did not seem to further improve the detection of mucinous tumors (ROC 0.955). We conclude that TTR, Hb, ApoAI and TF, when combined with CA125 should significantly improve the detection of early stage ovarian cancer.     

Differential bile-pancreatic secretory effects of CCK-58 and CCK-8

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCK(A) and CCK(B) receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCK(A) and CCK(B) receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity.     

Synthesis and evaluation of 2-amino-8-alkoxy quinolines as MCHr1 antagonists. Part 2

The continued SAR investigation of 2-amino-8-alkoxy quinolines as melanin concentrating hormone receptor-1 (MCHr1) antagonists is reported. Prior hit-to-lead efforts resulted in the identification of 1 as a robust MCHr1 antagonist. Further delineation of the structural parameters essential for MCHr1-binding affinity of this class of nontraditional GPCR ligands resulted in the identification of compounds such as 33, 34 and 37, which demonstrate single digit nanomolar antagonism of MCHr1-mediated Ca(2+) release. The synthesis and biological evaluation of these compounds are reported.     

Aminopiperidine indazoles as orally efficacious melanin concentrating hormone receptor-1 antagonists

The synthesis and biological evaluation of novel 3-amino indazole melanin concentrating hormone receptor-1 antagonists are reported, several of which demonstrated functional activity of less than 100nM. Compounds 19 and 28, two of the more potent compounds identified in this study, were characterized by high exposure in the brain and demonstrated robust efficacy when dosed in diet-induced obese mice.     

Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt mice

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt^+/− mice was quantified to be 406.06 ± 23.63 μg/g testis and 0.13 ± 0.02 μg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt^−/− males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt^+/− males sire offspring. The higher than 50% expression level of SGG in Cgt^+/− animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt^+/− mice were sufficient for normal spermatogenesis.     

Spatially Extensive Standardized Surveys Reveal Widespread,Multi-Decadal Increase in East Antarctic Adélie Penguin Populations

Seabirds are considered to be useful and practical indicators of the state of marine ecosystems because they integrate across changes in the lower trophic levels and the physical environment. Signals from this key group of species can indicate broad scale impacts or response to environmental change. Recent studies of penguin populations, the most commonly abundant Antarctic seabirds in the west Antarctic Peninsula and western Ross Sea, have demonstrated that physical changes in Antarctic marine environments have profound effects on biota at high trophic levels. Large populations of the circumpolar-breeding Adélie penguin occur in East Antarctica, but direct, standardized population data across much of this vast coastline have been more limited than in other Antarctic regions. We combine extensive new population survey data, new population estimation methods, and re-interpreted historical survey data to assess decadal-scale change in East Antarctic Adélie penguin breeding populations. We show that, in contrast to the west Antarctic Peninsula and western Ross Sea where breeding populations have decreased or shown variable trends over the last 30 years, East Antarctic regional populations have almost doubled in abundance since the 1980’s and have been increasing since the earliest counts in the 1960’s. The population changes are associated with five-year lagged changes in the physical environment, suggesting that the changing environment impacts primarily on the pre-breeding age classes. East Antarctic marine ecosystems have been subject to a number of changes over the last 50 years which may have influenced Adélie penguin population growth, including decadal-scale climate variation, an inferred mid-20th century sea-ice contraction, and early-to-mid 20^th century exploitation of fish and whale populations.     

Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export,PfEMP1 Display,and Survival of Malaria Parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.     

Complete genome sequence of Treponema paraluiscuniculi, strain Cuniculi A: the loss of infectivity to humans is associated with genome decay

Treponema paraluiscuniculi is the causative agent of rabbit venereal spirochetosis. It is not infectious to humans, although its genome structure is very closely related to other pathogenic Treponema species including Treponema pallidum subspecies pallidum, the etiological agent of syphilis. In this study, the genome sequence of Treponema paraluiscuniculi, strain Cuniculi A, was determined by a combination of several high-throughput sequencing strategies. Whereas the overall size (1,133,390 bp), arrangement, and gene content of the Cuniculi A genome closely resembled those of the T. pallidum genome, the T. paraluiscuniculi genome contained a markedly higher number of pseudogenes and gene fragments (51). In addition to pseudogenes, 33 divergent genes were also found in the T. paraluiscuniculi genome. A set of 32 (out of 84) affected genes encoded proteins of known or predicted function in the Nichols genome. These proteins included virulence factors, gene regulators and components of DNA repair and recombination. The majority (52 or 61.9%) of the Cuniculi A pseudogenes and divergent genes were of unknown function. Our results indicate that T. paraluiscuniculi has evolved from a T. pallidum-like ancestor and adapted to a specialized host-associated niche (rabbits) during loss of infectivity to humans. The genes that are inactivated or altered in T. paraluiscuniculi are candidates for virulence factors important in the infectivity and pathogenesis of T. pallidum subspecies.