Cross-Study Projections of Genomic Biomarkers: An Evaluation in Cancer Genomics

Human disease studies using DNA microarrays in both clinical/observational and experimental/controlled studies are having increasing impact on our understanding of the complexity of human diseases. A fundamental concept is the use of gene expression as a “common currency” that links the results of in vitro controlled experiments to in vivo observational human studies. Many studies – in cancer and other diseases – have shown promise in using in vitro cell manipulations to improve understanding of in vivo biology, but experiments often simply fail to reflect the enormous phenotypic variation seen in human diseases. We address this with a framework and methods to dissect, enhance and extend the in vivo utility of in vitro derived gene expression signatures. From an experimentally defined gene expression signature we use statistical factor analysis to generate multiple quantitative factors in human cancer gene expression data. These factors retain their relationship to the original, one-dimensional in vitro signature but better describe the diversity of in vivo biology. In a breast cancer analysis, we show that factors can reflect fundamentally different biological processes linked to molecular and clinical features of human cancers, and that in combination they can improve prediction of clinical outcomes.     

Adenosine amine congener mitigates noise-induced cochlear injury

Hearing loss from noise exposure is a leading occupational disease, with up to 5% of the population at risk world-wide. Here, we present a novel purine-based pharmacological intervention that can ameliorate noise-induced cochlear injury. Wistar rats were exposed to narrow-band noise (8–12 kHz, 110 dB SPL, 2–24 h) to induce cochlear damage and permanent hearing loss. The selective adenosine A₁ receptor agonist, adenosine amine congener (ADAC), was administered intraperitoneally (100 μg/kg/day) at time intervals after noise exposure. Hearing thresholds were assessed using auditory brainstem responses and the hair cell loss was evaluated by quantitative histology. Free radical damage in the organ of Corti was assessed using nitrotyrosine immunohistochemistry. The treatment with ADAC after noise exposure led to a significantly greater recovery of hearing thresholds compared with controls. These results were upheld by increased survival of sensory hair cells and reduced nitrotyrosine immunoreactivity in ADAC-treated cochlea. We propose that ADAC could be a valuable treatment for noise-induced cochlear injury in instances of both acute and extended noise exposures.     

A Parameterized Model of Amylopectin Synthesis Provides Key Insights into the Synthesis of Granular Starch

A core set of genes involved in starch synthesis has been defined by genetic studies, but the complexity of starch biosynthesis has frustrated attempts to elucidate the precise functional roles of the enzymes encoded. The chain-length distribution (CLD) of amylopectin in cereal endosperm is modeled here on the basis that the CLD is produced by concerted actions of three enzyme types: starch synthases, branching and debranching enzymes, including their respective isoforms. The model, together with fitting to experiment, provides four key insights. (1) To generate crystalline starch, defined restrictions on particular ratios of enzymatic activities apply. (2) An independent confirmation of the conclusion, previously reached solely from genetic studies, of the absolute requirement for debranching enzyme in crystalline amylopectin synthesis. (3) The model provides a mechanistic basis for understanding how successive arrays of crystalline lamellae are formed, based on the identification of two independent types of long amylopectin chains, one type remaining in the amorphous lamella, while the other propagates into, and is integral to the formation of, an adjacent crystalline lamella. (4) The model provides a means by which a small number of key parameters defining the core enzymatic activities can be derived from the amylopectin CLD, providing the basis for focusing studies on the enzymatic requirements for generating starches of a particular structure. The modeling approach provides both a new tool to accelerate efforts to understand granular starch biosynthesis and a basis for focusing efforts to manipulate starch structure and functionality using a series of testable predictions based on a robust mechanistic framework.     

Primary structure of the Drosophila laminin B2 chain and comparison with human, mouse, and Drosophila laminin B1 and B2 chains

Laminin, a major component of basement membranes, is a large glycoprotein consisting of three disulfide-bonded subunits, A, B1, and B2. We have isolated and sequenced a Drosophila laminin B2 chain cDNA clone that spans 5737 nucleotides. The deduced amino acid sequence predicts that the mature and nonglycosylated polypeptide has a chain length of 1606 residues (Mr = 178,665). This B2 chain contains 100 half-cystine residues, most of which are located in two cysteine-rich domains, and 11 N-X-S or N-X-T sequences which are potential sites of N-linked glycosylation. The predicted secondary structure reveals the presence of six structurally distinct domains, of which two are mainly alpha-helical, two are cysteine-rich with homologous repeats, and two are globular regions. The Drosophila B2 chain is 40.3 and 41.1% identical to the human and mouse B2 chains, respectively, and 29.6, 30.0, and 29.4% identical to the Drosophila, human, and mouse B1 chains, respectively.     

Characterization of pneumolysin from Streptococcus pneumoniae,interacting with carbohydrate moiety and cholesterol as a component of cell membrane

The cytolytic mechanism of cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target cell membrane. Membrane cholesterol was thought to serve as the common receptor for these toxins, but not all CDCs require cholesterol for binding. One member of this toxin family, pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae, and the mechanism via which PLY binds to its putative receptor or cholesterol on the cell membrane is still poorly understood. Here, we demonstrated that PLY interacted with carbohydrate moiety and cholesterol as a component of the cell membrane, using the inhibitory effect of hemolytic activity. The hemolytic activity of PLY was inhibited by cholesterol-MβCD, which is in a 3β configuration at the C3-hydroxy group, but is not in a 3α-configuration. In the interaction between PLY and carbohydrate moiety, the mannose showed a dose-dependent increase in the inhibition of PLY hemolytic activity. The binding ability of mannose with truncated PLYs, as determined by the pull-down assay, showed that mannose might favor binding to domain 4 rather than domains 1–3. These studies provide a new model for the mechanism of cellular recognition by PLY, as well as a foundation for future investigations into whether non-sterol molecules can serve as receptors for other members of the CDC family of toxins.     

Neutrophil depletion does not prevent lung edema after endotoxin infusion in goats

Neutropenia was produced in goats by injection of either nitrogen mustard, (1.5 mg/kg) or hydroxyurea (200 mg X kg-1 X day-1). A nitrogen mustard (M + E) group (n = 6), a hydroxyurea (H + E) group (n = 5), and a control (E) group (n = 7) were given 1-h infusions of endotoxin (5 micrograms/kg total dose), then monitored for up to 5 h. Postmortem extravascular lung water (EVLW) was significantly higher in the M + E group (14.2 +/- 4.4 ml/kg) and the E group (11.9 +/- 3.9 ml/kg) when compared with a normal control (6.6 +/- 1.3 ml/kg) group that did not receive endotoxin. EVLW in a group made neutropenic with nitrogen mustard (6.7 +/- 1.3 ml/kg) and the H + E (7.9 +/- 1.5 ml/kg) groups were not statistically different from each other or from normal controls. Circulating neutrophil counts averaged 32 +/- 42 cells/microliter in the M + E group and 180 +/- 210 cells/microliter in the H + E group. Only minimal histological changes were seen in the H + E group, but the E and M + E lungs had severe pulmonary edema. We conclude that neutrophils are not required for increased EVLW and decreased arterial O2 partial pressure after endotoxin infusion, and hydroxyurea prevents at least part of the pulmonary edema after endotoxin by a mechanism that is not neutrophil dependent.