New approaches for biomarker discovery: the search for liver fibrosis markers in hepatitis C patients

Despite many shortcomings, liver biopsy is regarded as the gold standard for assessing liver fibrosis. A less invasive and equally or more reliable approach would constitute a major advancement in the field. Proteomics can aid discovery of novel serological markers and these proteins can be measured in patient blood. A major challenge of discovering biomarkers in serum is the presence of highly abundant serum proteins, which restricts the levels of total protein loaded onto gels and limits the detection of low abundance features. To overcome this problem, we used two-dimensional gel electrophoresis (2-DE) over a narrow pH 3-5.6 range since this lies outside the range of highly abundant albumin, transferrin and immunoglobulins. In addition, we used in-solution isoelectric focusing followed by SDS-PAGE to find biomarkers in hepatitis C induced liver cirrhosis. Using the pH 3-5.6 range for 2-DE, we achieved improved representation of low abundance features and enhanced separation. We found in-solution isoelectric focusing to be beneficial for analyzing basic, high molecular weight proteins. Using this method, the beta chains of both complement C3 and C4 were found to decrease in serum from hepatitis C patients with cirrhosis, a change not observed previously by 2-DE. We present two proteomics approaches that can aid in the discovery of clinical biomarkers in various diseases and discuss how these approaches have helped to identify 23 novel biomarkers for hepatic fibrosis.     

Predicting the points of interaction of small molecules in the NF-κB pathway

Background  

The similarity property principle has been used extensively in drug discovery to identify small compounds that interact with specific drug targets. Here we show it can be applied to identify the interactions of small molecules within the NF-κB signalling pathway.     

The parapatric distribution and contact zone of two forms showing different male-to-male aggressiveness in a social spider mite, Stigmaeopsis miscanthi (Acari: Tetranychidae)

Two forms showing different male-to-male aggressiveness, different male morphologies and different diapause attributes are known in Stigmaeopsis miscanthi (Saito), a social spider mite infesting Chinese silver grass (Miscanthus sinensis Anderss). Reproductive isolation exists between the forms, although it is not always complete, and the details of their distributional patterns are unknown, but expected to be parapatric. We searched for the contact zone at Mt. Unzen (Nagasaki Pref., Japan) where both forms are known to occur at different altitudes. We found the two forms together in several M. sinensis stands, suggesting there is frequent contact between the forms in their boundary area. We discuss the mechanism(s) that maintain the parapatry related to their frequent contact and the pattern of reproductive isolation between them.     

Why are there feeding and nonfeeding larvae in phytoseiid mites (Acari, Phytoseiidae)?

The larval feeding traits and oviposition behavior of ten phytoseiid mite species were investigated in the presence and absence of prey. Results showed a correlation between prey preferences, the manner of oviposition, and larval feeding behavior. Species with larvae that must feed to develop preferred prey species that were distributed sparsely and laid their eggs in a scattered fashion. Species with larvae that do not need to feed to develop preferred prey species with a high aggregation and laid their eggs in a clumped fashion. The results suggest that nonfeeding larval behavior may be an adaptation to avoid sib-cannibalism, which occurs when eggs are oviposited closer together. Received: October 13, 2000 / Accepted: December 15, 2000     

Ambulatory migration in mites (Acari: Tetranychidae, Phytoseiidae) to new leaves of moso bamboo shoots

The migratory behaviour of two tetranychid pest species, Aponychus corpuzae and Schizotetranychus nanjingensis, and one phytoseiid, Typhlodromus bambusae, was studied in several monocultural bamboo forests in Fujian Province, China. The aim of the study was to assess how the ambulatory immigration of tetranychid and phytoseiid mites from the ground to new leaves is affected by a sticky barrier around the stem, by the age of bamboo shoots or by shoot density. The results show that while the sticky barrier is particularly effective at disrupting the ambulatory immigration from the ground to new leaves of S. nanjinigensis to 1-year-old shoots and of A. corpuzae to 3-year-old shoots, it has no significant effect on the immigration of the phytoseiid mite.     

Synthesis of 1-deoxy-L-gulonojirimycin (L-guloDNJ) and 1-deoxy-D-talonojirimycin (D-taloDNJ)

Carbohydrate based syntheses of azasugars with unusual configurations viz. 1,5-dideoxy-1,5-imino-L-gulitol (L-guloDNJ) and 1,5-dideoxy-1,5-imino-L-talitol (L-taloDNJ) are reported, from D-mannose and D-fructose, respectively. The key steps in both syntheses involved reductive aminative cyclizations. Thus, L-guloDNJ was obtained by reduction of 2,3;4,6-di-O-isopropylidene-5-O-p-toluenesulfonyl-D-mannononitrile with LiAlH(4) in DME to give the protected azasugar which upon hydrolysis with HCl afforded crystalline L-guloDNJ as the HCl salt in 29% overall yield. Reduction of 6-azido-1-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-beta-D-ribohexulofuranose obtained from D-fructose in six steps, followed by treatment with HCl, afforded L-taloDNJ as an HCl salt in approximately 10% overall yield.     

ei24, a p53 response gene involved in growth suppression and apoptosis

DNA damage and/or hyperproliferative signals activate the wild-type p53 tumor suppressor protein, which induces a G(1) cell cycle arrest or apoptosis. Although the mechanism of p53-mediated cell cycle arrest is fairly well defined, the p53-dependent pathway regulating apoptosis is poorly understood. Here we report the functional characterization of murine ei24 (also known as PIG8), a gene directly regulated by p53, whose overexpression negatively controls cell growth and induces apoptotic cell death. Ectopic ei24 expression markedly inhibits cell colony formation, induces the morphological features of apoptosis, and reduces the number of beta-galactosidase-marked cells, which is efficiently blocked by coexpression of Bcl-X(L). The ei24/PIG8 gene is localized on human chromosome 11q23, a region frequently altered in human cancers. These results suggest that ei24 may play an important role in negative cell growth control by functioning as an apoptotic effector of p53 tumor suppressor activities.     

Some aspects of the isopropylidenation of d-glucitol under neutral conditions

Isopropylidenation of d-glucitol (1) under neutral conditions, by treatment with 2,2-dimethoxypropane in 1,2-dimethoxyethane, has been studied. An improved procedure for the isolation of 1,2:5,6-di-O-isopropylidene-d-glucitol, the main equilibrium product, by direct crystallisation or via the 3,4-dibenzoate is described. Some aspects of the reaction are discussed and compared with results obtained previously from the isopropylidenation of 1 in the presence of zinc chloride.