Multiplex transcriptional analysis of paraffin-embedded liver needle biopsy from patients with liver fibrosis.

BACKGROUND: The possibility of extracting RNA and measuring RNA expression from paraffin sections can allow extensive investigations on stored paraffin samples obtained from diseased livers and could help with studies of the natural history of liver fibrosis and inflammation, and in particular, correlate basic mechanisms to clinical outcomes. RESULTS: To address this issue, a pilot study of multiplex gene expression using branched-chain DNA technology was conducted to directly measure mRNA expression in formalin-fixed paraffin-embedded needle biopsy samples of human liver. Twenty-five genes were selected for evaluation based on evidence obtained from human fibrotic liver, a rat BDL model and in vitro cultures of immortalized human hepatic stellate cells. The expression levels of these 25 genes were then correlated with liver fibrosis and inflammation activity scores. Statistical analysis revealed that three genes (COL3A1, KRT18, and TUBB) could separate fibrotic from non-fibrotic samples and that the expression of ten genes (ANXA2, TIMP1, CTGF, COL4A1, KRT18, COL1A1, COL3A1, ACTA2, TGFB1, LOXL2) were positively correlated with the level of liver inflammation activity. CONCLUSION: This is the first report describing this multiplex technique for liver fibrosis and has provided the proof of concept of the suitability of RNA extracted from paraffin sections for investigating the modulation of a panel of proinflammatory and profibrogenic genes. This pilot study suggests that this technique will allow extensive investigations on paraffin samples from diseased livers and possibly from any other tissue. Using identical or other genes, this multiplex expression technique could be applied to samples obtained from extensive patient cohorts with stored paraffin samples in order to correlate gene expression with valuable clinically relevant information. This method could be used to provide a better understanding of the mechanisms of liver fibrosis and inflammation, its progression, and help development of new therapeutic approaches for this indication.

Online immunoaffinity LC/MS/MS. A general method to increase sensitivity and specificity: How do you do it and what do you need?

There is an increased emphasis on hyphenated techniques such as immunoaffinity LC/MS/MS (IA-LC/MS/MS) or IA-LC/MRM. These techniques offer competitive advantages with respect to sensitivity and selectivity over traditional LC/MS and are complementary to ligand binding assays (LBA) or ELISA's. However, these techniques are not entirely straightforward and there are several tips and tricks to routine sample analysis. We describe here our methods and procedures for how to perform online IA-LC/MS/MS including a detailed protocol for the preparation of antibody (Ab) enrichment columns. We have included sample trapping and Ab methods. Furthermore, we highlight tips, tricks, minimal and optimal approaches. This technology has been shown to be viable for several applications, species and fluids from small molecules to proteins and biomarkers to PK assays.

Geldanamycin-induced PCNA degradation in isolated Hsp90 complex from cancer cells.

Hsp90 is a molecular chaperone involved in the folding and proteolytic turnover of many regulatory proteins associated with it. Some of the Hsp90 client proteins are known to be involved in tumorigenesis. An Hsp90-specific inhibitor, geldanamycin, is shown to bind to the ATP binding site of the chaperone to induce degradation of many client proteins, and results in antitumor activities. However, the mechanism of geldanamycin-induced client protein degradation is not fully understood. A large-scale immunoaffinity purification with anti-Hsp90 antibodies identified many Hsp90 client proteins from colon cancer cell line, HCT-116. One of the identified proteins, PCNA, was confirmed to be associated with Hsp90 in two additional cancer cell lines. After geldanamycin treatment, both PCNA and Hsp90 were shown to be degraded. More interestingly, this study demonstrated that in two different cancer cell lines, the degradation occurred in the isolated Hsp90 complex in vitro. This result indicated that the components responsible for the PCNA degradation are also associated with Hsp90. This finding provided a new mechanism for the Hsp90-mediated protein degradation induced by Hsp90-specific inhibitors.

A rat air pouch model for evaluating the efficacy and selectivity of 5-lipoxygenase inhibitors.

The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors. Inflammation was produced by adding carrageenan into a newly formed air pouch and prostaglandins produced. While macrophages and neutrophils are present in the inflamed pouch, little 5-LOX products are formed. Cellular 5-LOX activation was obtained by adding calcium ionophore (A23187) into the pouch thus providing a novel model to evaluate the efficacy and selectivity of 5-LOX inhibitors. Also, we described modifications to the in vitro 5-LOX enzyme and cell assays. These assays included a newly developed fluorescence-based enzyme assay, a 5-LOX redox assay, an ex vivo human whole blood assay and an IgE-stimulated rat mast cell assay, all designed for maximal production of leukotrienes. Zileuton and CJ-13,610, a competitive, non-redox inhibitor of 5-LOX, were evaluated for their pharmacological properties using these assays. Although both compounds achieved dose-dependent inhibition of 5-LOX enzyme activity, CJ-13,610 was 3-4 fold more potent than zileuton in all-assays. Evaluation of 5-LOX metabolites-by LC/MS/MS and ELISA confirmed that both compounds selectively inhibited all products downstream of 5-hydroperoxy eicosatetraenoic acid (5-HPETE), including 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE), without inhibition of 12-lipoxygenase (12-LOX), 15-lipoxygenase (15-LOX), or cyclooxygenase (COX) products. In the rat air pouch model, oral dosing of CJ-13,610 and zileuton resulted in selective inhibition 5-LOX activity from pouch exudate and ex vivo rat whole blood with similar potency to in vitro assay. These data show that the rat air pouch model is a reliable and useful tool for evaluating in vivo efficacy of 5-LOX inhibitors and may aid in the development of the next generation of 5-LOX inhibitors, such as the non-redox inhibitors similar to CJ-13,610.

Host limits to accurate human growth hormone production in multiple plant systems.

Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.

Selective site-specific fenton oxidation of methionine in model peptides: evidence for a metal-bound oxidant.

The metal-catalyzed oxidation (MCO) of proteins represents an important pathway for protein degradation. Although many mechanistic details of MCO are currently unknown, such mechanistic information would greatly benefit formulation scientists in the rational design and analysis of protein formulations. Here, we describe the Fenton oxidation (by Fe(2+)/H(2)O(2)) of several Met-, Tyr-, and His containing model peptides, including one derivative containing a conformationally restricted norbornyl Met analogue (Nor), Nor-Gly-His-Met-NH(2). Our results will provide evidence for a metal-bound reactive oxygen species selectively oxidizing Met to Met sulfoxide, indicating a Met-specific oxidant and arguing against the involvement of freely diffusible hydroxyl radicals. The Fenton oxidation of Nor-Gly-His-Met-NH(2) yields a 2:1 preference for sulfoxide formation at the C-terminal Met versus the N-terminal Nor residue, respectively, while incubation of the peptide with H(2)O(2) alone results in a 1:1 ratio. These results are rationalized by the better access of the thioether side chain of the flexible C-terminal Met residue to the peptide-bound iron compared with the conformationally restricted Nor residue. It is commonly believed that Fenton oxidation reactions involve hydroxyl radicals, and that Met oxidation in proteins is predominantly controlled by the surface-accessibility of the respective Met residues. However, occasionally protein oxidation in formulations shows selectivities, which are not consistent with these paradigms. Our results demonstrate additional features of the Fenton reaction such as the formation of a metal-bound oxidant specific for Met (and not Tyr or His), which may assist formulation scientists in the rationalization of unexpected oxidation selectivities.