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Results for “multiplexed perturbation”

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PubMed2003

Frequent p16INK4A/CDKN2A alterations in chemically induced Syrian golden hamster pancreatic tumors.

The p16INK4A/CDKN2A (p16) tumor suppressor gene is known to be inactivated in up to 98% of human pancreatic cancer specimens. Chemically induced pancreatic tumors in Syrian golden hamsters have been demonstrated to share many morphological and biological similarities with human pancreatic tumors and represent a potentially suitable model for the evaluation of therapies targeting p16. The purpose of this study was to evaluate primary hamster pancreatic tumor specimens for potentially inactivating p16 alterations. Tumors were induced with N-nitroso-bis-(2-oxopropyl) amine, followed by two cycles of augmentation pressure, and were harvested on day 100. Foci of tumor cells were identified by light microscopy after staining with hematoxylin and eosin, and corresponding tumor tissues were excised for DNA extraction. The techniques of multiplex real-time PCR, direct sequencing and methylation-specific PCR were used to evaluate 30 tumor specimens for homozygous deletions, mutations and aberrant methylation of 5' CpG islands, respectively. Homozygous deletions were identified in 11 of 30 (36.7%) specimens, mutations were identified in four of 30 (13.3%) specimens, and aberrant methylation of 5' CpG islands was found in 14 of 30 (46.7%) specimens. The overall frequency of p16 alterations was 93.3% (28 of 30 specimens) and the majority of changes (83.3%) were noted to be secondary to methylation or homozygous deletion. The four mutations significantly impaired cyclin-dependent kinase 4 inhibitory activity, and two resulted in perturbation of the global structure of P16 protein. These findings indicate that p16 inactivation is a common event in chemically induced hamster tumors, and that this animal model is appropriate for comparative studies evaluating pancreatic cancer therapeutic strategies targeting p16.

Animals↗

PubMed2006

A perspective on the use of iTRAQ reagent technology for protein complex and profiling studies.

Proteomic research includes the characterization of protein mixtures in order to understand complex biological systems and determine relationships between proteins, their function, and protein-protein interactions. Often the goal of such research is to monitor changes of proteins in perturbed systems, a type of study referred to as differential expression analysis. To perform these studies requires the ability to execute some type of differential comparison of a given protein state in reference to some type of a control. The iTRAQ reagents are a set of isobaric reagents which are amine specific and allow for the identification and quantitation of up to four different samples simultaneously. The amine specificity of these reagents makes most peptides in a sample amenable to this labeling strategy with no loss of information from samples involving post-translational modifications, such as the scrutiny of signal transduction pathways that often involve phosphorylation phenomena. In addition, the multiplexing capacity of these reagents allows for information replication within certain LC-MS/MS experimental regimes, providing additional statistical validation within any given experiment. The results presented herein demonstrate a few examples of the wide variety of quantitative information that can be realized when undertaking such experimental approaches. These include temporal analysis of drug-induced-protein expression, discovery and elucidation of disease markers, and protein-protein interactions in multi-protein complexes.

Animals↗

PubMed2006

Symmetry properties of vibrational modes in mesoporphyrin IX dimethyl ester investigated by polarization-sensitive resonance Raman and CARS spectroscopy.

The symmetry properties of selected vibrational modes of mesoporphyrin IX dimethyl ester (MP-IX-DME) in solution are investigated under different electronic resonance conditions. The Raman band parameters of the macrocycle modes nu(2), nu(10), nu(11), and nu(19) are determined from a quantitative analysis of polarized spontaneous resonance Raman (RR) and polarization-sensitive (PS) multiplex coherent anti-Stokes Raman scattering (CARS) spectra obtained with pre-resonant B band and resonant Qx band excitation, respectively. Additionally, the molecular geometry and the vibrational modes of MP-IX-DME are calculated by employing density functional theory (DFT) on the B3LYP/6-31G(d) level. Both the DFT-derived structure and the Raman spectroscopic parameters of MP-IX-DME indicate minor deviations from an ideal D2h macrocycle symmetry. To assess the influence of the beta substitution pattern on the in-plane symmetry, calculated normal-mode vectors and several experimentally detected parameters, such as peak positions, depolarization ratios, and coherent phases, are analyzed. The effects of the macrocycle substitution pattern are different for the selected vibrational modes: nu(2) in particular is very sensitive to subtle perturbations of the in-plane symmetry. The considerable activity of totally symmetric vibrations observed in the PS CARS spectra of MP-IX-DME and the correlation of mode symmetries with coherent phases confirm earlier PS CARS results on octaethylporphine (OEP) acquired under the same electronic resonance conditions.

Absorption↗

PubMed1999

Intracellular signaling of the TGF-beta superfamily by Smad proteins.

TGF-beta is a potent inhibitor of cell growth, and accumulating evidence suggests that perturbation of the TGF-beta signaling pathway leads to tumorigenesis. Smads are recently identified proteins that mediate intracellular signaling of the TGF-beta superfamily. Smads 2 and 3 are phosphorylated by the TGF-beta type I receptor. Smad4 was originally identified as a candidate tumor suppressor gene in pancreatic cancers. Smads 2 and 3 form complexes with Smad4 upon TGF-beta stimulation. The heteromeric Smad complexes translocate into the nucleus, where they activate expression of target genes. Our recent study demonstrated that Smads exist as monomers in the absence of TGF-beta. Smads 2 and 3 form homo- as well as hetero-oligomers with Smad4 upon ligand stimulation. Both homo-oligomers and hetero-oligomers directly bind to DNA, suggesting that the signaling pathway of Smads may be multiplex. Smads 2 and 3 associate with transcriptional coactivators such as p300 in a ligand-dependent manner, p300 enhances transactivation by TGF-beta, suggesting that coactivators link Smads to the basal transcriptional machinery. A missense mutation of Smad2 identified in colorectal and lung cancers was introduced to Smad3. The mutant, Smad3(DE), blocked the activation of wild-type Smad2 and Smad3. Thus, the missense mutation not only disrupts the function of the wild-type Smad but also creates a dominant-negative Smad, which could actively contribute to oncogenesis.

Animals↗

PubMed1992

Correlated, simultaneous, multiple-wavelength optical monitoring in vivo of localized cerebrocortical NADH and brain microvessel hemoglobin oxygen saturation.

Current forms of brain monitoring, such as electroencephalography (EEG), have had limited clinical utility. The EEG records spontaneous cerebrocortical activity and thus is an indirect indicator of metabolic demand and, to a lesser extent, an indicator of mismatch of supply versus demand. Ischemia modulates EEG activity in ways that can usually be detected, but EEG patterns can be similarly modulated by many other factors, including temperature and pharmacologic manipulation. This in vivo study in physiologically monitored animals evaluated the use of correlated optical spectroscopy, performed with an instrument having a fiberoptic light-guide bundle in contact with the cerebral cortex, for the simultaneous monitoring of cerebrovascular oxygen availability and intracellular oxygen delivery. A highly specific monitor of cerebral intracellular oxygen supply, the cerebrocortical intramitochondrial NADH redox state, was monitored in vivo with a fluorescence technique. Absorption spectroscopy was used concurrently to monitor hemoglobin content (blood volume) and oxygen saturation in the microcirculation. Correlated changes in optical signals from cerebrocortical NADH and hemoglobin were studied in a swine model (n = 7) of nitrogen hypoxia. Measurements were made at four wavelengths with a time-division, multiplexed fluorometer/reflectometer. Because the NADH fluorescence signal at 450 nm is affected by local changes in blood volume, a "corrected" fluorescence signal is usually calculated. In previous studies, where only two wave lengths have been measured, attempts at correction were based on reflectance at the excitation wavelength (366 nm). We compared estimators of changes in microcirculatory blood volume using reflection at two wavelengths: 366 nm and 585 nm, the wavelengths for maximum and isobestic absorption. The results of the studies were as follows: (1) during transient hypoxia, NADH and local hemoglobin saturation signals changed in concert with arterial pulse oximetry, with changes in NADH lagging behind changes in saturation by an average of 5.3 seconds; (2) after hypocapnic ventilation to a mean PaCO2 of 20.2 +/- 0.8 mm Hg, NADH increased by 11.5 +/- 8.7% (as compared with maximal change during anoxia), local hemoglobin saturation decreased by 7.7 +/- 6.4%, and local blood volume decreased by 12.5 +/- 13%, while arterial SpO2 was unchanged; (3) our two measures of local blood volume were closely correlated during carbon dioxide perturbations, but poorly correlated during hypoxic perturbation; and (4) NADH fluorescence provided a more rapid, sensitive indicator of oxygen deprivation than did the EEG. During transient hypoxia, EEG changes occurred 57.4 +/- 10.4 seconds after the onset of decline in local hemoglobin saturation, after NADH had completed 50% of its maximal increase.

Animals↗

PubMedDate not supplied

Profiling lymphocyte subpopulations in peripheral blood under efalizumab treatment of psoriasis by multi epitope ligand cartography (MELC) robot microscopy.

CD11a-blocking efalizumab has recently been approved as a systemic treatment of moderate to severe chronic plaque psoriasis. When treating 6 psoriasis patients with efalizumab over 12 weeks in the present study, we observed an overall good tolerability and 5 treatment responders characterized by a decrease of PASI from 21.3 +/- 5.4 to 3.9 +/- 0.6. The accompanying significant increase of peripheral blood lymphocytes from 1.9 +/- 0.7 to 4.3 +/- 1.0 x 10(9)/L (p < 0.05) was analyzed by multi epitope ligand cartography (MELC) robot microscopy. Thereby a high-dimension simultaneous multiplex immunophenotyping was pursued using 39 fluorophore-labeled antibodies including labeled efalizumab and 3 other affinity reagents such as lectins. Due to efalizumab treatment there was a substantial decrease of the cellular expression of CD11a (detected by mab clone 25.3.1) and efalizumab binding sites (EfaBSs). This was paralleled by an increase of the number of EfaBS- and EfaBS+ lymphocytes by a factor of 2.4x and 2.2x, respectively. The latter effect was mainly derived from a subpopulation showing a low degree of EfaBS expression. Efalizumab treatment led furthermore to an increase of the numbers of CD3+, CD4+, CD8+, CD44+, CD45+, CD45R0+, CD45 RA+, CD52+, CD58+, CD247+, HLA-DR+ and Sambucus nigra lectin-reactive lymphocytes (by factors from 2.0 to 3.3x). In terms of a combinatorial molecular phenotype we identified a CD3+/CD4+/CD44+/CD52+ lymphocyte subpopulation which accumulated most predominantly from 0.824 +/- 0.270 x 10(9)/L up to 1.616 +/- 0.152 x 10(9)/L under efalizumab treatment (p < 0.01). Thus, the current study extends the knowledge of efalizumab-dependent perturbations of recirculating blood lymphocyte subpopulations in psoriasis patients.

Adult↗

PubMed2005

A locus on chromosome 9p predisposes to a specific disease manifestation, acute anterior uveitis, in ankylosing spondylitis, a genetically complex, multisystem, inflammatory disease.

OBJECTIVE: Uveitis or intraocular inflammation is a major cause of visual loss. Acute anterior uveitis (AAU) affects approximately 40% of patients with ankylosing spondylitis (AS) but also affects patients with no evidence of spondylarthritis. We sought to determine whether a unique genetic region could be implicated in a specific manifestation-AAU-of a multisystem, inflammatory, genetically complex disease, AS. METHODS: Individuals from families multiplex for AAU were genotyped at 400 markers representing the ABI PRISM linkage map MD-10, and at the HLA-B, DRB1, DQA1, DQB1, and DPB1 alleles. Among the family members with AAU, 76 affected sibpairs were analyzed (6 without concomitant AS, 12 discordant for AS, and 58 concordant for AS). Two-point and multipoint nonparametric linkage analyses were performed, and 1-parameter allele-sharing model logarithm of odds (LOD) scores were determined. RESULTS: As previously reported for AS, linkage at the major histocompatibility complex region (chromosome 6p21) was evident, exhibiting the highest multipoint LOD score (4.96 at marker HLA-B). Strong linkage was seen at a region on chromosome 9p21-9p24, with a LOD score of 3.72 at marker D9S157. When compared with a companion cohort of AS families, the linkage at this region was found in association with AAU but not with AS. A third region on chromosome 1q23-1q31 was observed to have suggestive linkage (LOD 2.05 at marker D1S238), which overlaps with a region associated with AS. CONCLUSION: This is the first study in which a genetic region for AAU has been identified by genome-wide scan. Even though AS was highly prevalent in this cohort of families, a locus at chromosome 9p21-9p24 was identified that uniquely associates with AAU. Identifying the genetic perturbation at this region may advance our understanding of the mechanisms involved in tissue-specific pathology of complex inflammatory diseases.

Acute Disease↗