Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

Synthesis and antinematodal activity of 3-n-alkylphenols

Several 3-alkylphenols including 3-undecylphenol, which was isolated from a Sumatran rainforest plant, were synthesized to investigate their antinematodal activity against the phytopathogenic nematodes, Bursapherencus xylophilus. A three-step synthesis involving the treatment of 2-cyclohexen-1-one with the Grignard reagent, oxidation of the resulting 1-alkyl-2-cyclohexen-1-ol and subsequent aromatization of 3-alkyl-2-cyclohexen-1-one successfully afforded such phenols. Among the 3-alkylphenols, 3-nonylphenol showed the highest activity, while 3-decylphenol and 3-undecylphenol also showed high activity.     

Protective effects of green tea catechins on alveolar macrophages against bacterial infections

Bacterial pneumonia in immunocompromised patients as well as elderly persons often becomes a life threatening disease, even when effective antibiotics are used extensively. In addition, the appearance of antibiotic-resistant bacteria in medical facilities as well as in patients requires another approach to treat such patients besides treatment with antibiotics. In this regard, green tea catechins, such as epigallocatechin gallate (EGCg), may be one of the potential agents for such purpose due to its possible potential immunomodulatory as well as antimicrobial activity. The studies by us showed that EGCg enhanced the in vitro resistance of alveolar macrophages to Legionella pneumophila infection by selective immunomodulatory effects on cytokine formation. Furthermore, the tobacco smoking-induced impairment of alveolar macrophages regarding antibacterial as well as immune activity was also recovered by EGCg treatment. These results indicate that EGCg may be a possible potential immunotherapeutic agent against respiratory infections in immunocompromised patients, such as heavy smokers.     

The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein

Intermedilysin is a pore-forming cytolysin belonging to the streptolysin O gene family known as the 'Cholesterol-binding/dependent cytolysins' and is unique within the family in that it is highly humanspecific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome-embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome-binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human-specific step of intermedilysin action, that the host cell membrane-binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human-specific action step and the cell-binding site of domain 4 lies within the 56 amino acids of the C-terminal, excluding the undecapeptide region.     

Chromosomal analysis of mouse spermatozoa following physical and chemical treatments that are effective in inactivating HIV

Human immunodeficiency virus (HIV) can be inactivated by heating at 56 degrees C for 30 min, treating with 50% ethanol at room temperature for 10 min, or treating with 2% sodium hypochlorite solution (NaClO) at room temperature for 60 min. Using a mouse model, we evaluated the risk of generating chromosome damage in spermatozoa following these treatments. The spermatozoa were all dead after the treatments. Although 41.3% of oocytes injected with ethanol-treated spermatozoa successfully activated, none of the oocytes injected with heated or NaClO-treated spermatozoa activated. When artificial stimulation with strontium was used, the fertilization of oocytes with heated or ethanol-treated spermatozoa was completely rescued. Sperm nuclei treated with NaClO neither decondensed nor developed to a male pronucleus. The incidences of structural chromosome aberrations in 1-cell zygotes derived from the heated spermatozoa (45.6%) and ethanol-treated spermatozoa (91.2%) were significantly higher than those in the matched controls (5.5% and 10.5%, respectively). Further study is needed to develop a methodology for the protection of spermatozoa against chromosome damage or the separation of damaged spermatozoa before intracytoplasmic sperm injection.     

Involvement of syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.     

Cystatin 10, a novel chondrocyte-specific protein,may promote the last steps of the chondrocyte differentiation pathway

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.