Depicting the Spatial Distribution of Proteins in Human Tumor Tissue Combining SELDI and MALDI Imaging and Immunohistochemistry

Carcinoma tissue consists of not only tumor cells but also fibroblasts, endothelial cells or vascular structures, and inflammatory cells forming the supportive tumor stroma. Therefore, the spatial distribution of proteins that promote growth and proliferation in these complex functional units is of high interest. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a newly developed technique that generates spatially resolved profiles of protein signals directly from thin tissue sections. Surface-enhanced laser desorption/ionization mass spectrometry (MS)combined with tissue microdissection allows analysis of defined parts of the tissue with a higher sensitivity and a broader mass range. Nevertheless, both MS-based techniques have a limited spatial resolution. IHC is a technique that allows a resolution down to the subcellular level. However, the detection and measurement of a specific protein expression level is possible only by semiquantitative methods. Moreover, prior knowledge about the identity of the proteins of interest is necessary. In this study, we combined all three techniques to gain highest spatial resolution, sensitivity, and quantitative information. We used frozen tissue from head and neck tumors and chose two exemplary proteins (HNP1–3 and S100A8) to highlight the advantages and disadvantages of each technique. It could be shown that the combination of these three techniques results in congruent but also synergetic data. (J Histochem Cytochem 58:929–937, 2010)     

The effects of temperature and salinity on vital biological functions of the Antarctic crustacean Serolis polita

Effects of temperature rise (from 0 to +5°C) and salinity decline (from 34 to 30 psu) on vital biological functions of the Antarctic isopod Serolis polita were studied in laboratory experiments. Behavioural reactions to food odour, as well as righting responses and reburying in the sediments, were measured. Both temperature increase and salinity decline impaired the ability of S. polita to perform these biological functions critical for their long-term survival, by lowering the number of isopods able to right and rebury in the sediment, increasing time-to-right, reducing locomotory activity and weakening isopod reaction to food odour. Significant interactive effects between temperature and salinity on time-to-right and time spent swimming were observed, with isopods being more vulnerable to lower salinities when exposed to higher temperatures. Some biological functions (righting, reburying) were more sensitive to temperature and salinity changes than others (swimming). In conclusion, our findings strongly suggest that Antarctic isopods are vulnerable to environmental changes, and their ability to cope with them is limited.     

Spoiling the Whole Bunch: Quality Control Aimed at Preserving the Integrity of High-Throughput Genotyping

False-positive or false-negative results attributable to undetected genotyping errors and confounding factors present a constant challenge for genome-wide association studies (GWAS) given the low signals associated with complex phenotypes and the noise associated with high-throughput genotyping. In the context of the genetics of kidneys in diabetes (GoKinD) study, we identify a source of error in genotype calling and demonstrate that a standard battery of quality-control (QC) measures is not sufficient to detect and/or correct it. We show that, if genotyping and calling are done by plate (batch), even a few DNA samples of marginally acceptable quality can profoundly alter the allele calls for other samples on the plate. In turn, this leads to significant differential bias in estimates of allele frequency between plates and, potentially, to false-positive associations, particularly when case and control samples are not sufficiently randomized to plates. This problem may become widespread as investigators tap into existing public databases for GWAS control samples. We describe how to detect and correct this bias by utilizing additional sources of information, including raw signal-intensity data.     

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A_2A receptor (A_2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α_olf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ₇ subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ₇ protein controls the hierarchial assembly of a specific G-protein α_olfβ₂γ₇ heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A_2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ₇ subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A_2AR G-protein α_olfβ₂γ₇ interface as a possible therapeutic target for Parkinson disease.     

Nitric oxide stimulates PC12 cell proliferation via cGMP and inhibits at higher concentrations mainly via energy depletion.

We investigated the mechanisms by which nitric oxide (NO) from an NO donor (DETA/NO) regulates proliferation of pheochromocytoma PC12 cells. The NO donor stimulated proliferation at low concentrations, but reversibly and completely inhibited proliferation at higher concentrations. The stimulation (but not the inhibition) of proliferation was apparently due to NO stimulation of soluble guanylate cyclase to produce cGMP, as it was prevented by a specific cyclase inhibitor (ODQ), and replicated by a cell-permeable form of cGMP. The NO-induced cytostasis was not reversed by inhibitors of MEK kinase or poly(ADP-ribose)polymerase, or by treatments that bypass inhibition of ribonucleotide reductase or ornithine decarboxylase. Cytostatic concentrations of DETA/NO strongly inhibited respiration of PC12 cells, and specific respiratory inhibitors (rotenone, myxothiazol, or azide) caused complete cytostasis. Uridine and pyruvate reversed the cytostasis induced by the specific respiratory inhibitors, but not that induced by DETA/NO. However, the combination of uridine, pyruvate, and N-acetyl-cysteine did reverse DETA/NO-induced cytostasis. DETA/NO strongly and progressively inhibited glycolysis measured by glucose consumption, lactate production, and ATP level, and a specific glycolytic inhibitor (5 mM 2-deoxy-d-glucose) caused complete cytostasis. Our results indicate that NO at low concentrations increases cell proliferation via cGMP, while high concentrations of NO block proliferation via inhibition of both glycolysis and respiration, causing energy depletion.     

pH-sensitive pHLIP® coated niosomes

Nanomedicine is becoming very popular over conventional methods due to the ability to tune physico-chemical properties of nano vectors, which are used for encapsulation of therapeutic and diagnostic agents. However, the success of nanomedicine primarily relies on how specifically and efficiently nanocarriers can target pathological sites to minimize undesirable side effects and enhance therapeutic efficacy. Here, we introduce a novel class of targeted nano drug delivery system, which can be used as an effective nano-theranostic for cancer. We formulated pH-sensitive niosomes (80–90?nm in diameter) using nonionic surfactants Span20 (43–45?mol%), cholesterol (50?mol%) and 5?mol% of pH (Low) insertion peptide (pHLIP) conjugated with DSPE lipids (DSPE-pHLIP) or hydrophobic fluorescent dye, pyrene, (Pyr-pHLIP). In coating of niosomes, pHLIP was used as an acidity sensitive targeting moiety. We have demonstrated that pHLIP coated niosomes sense the extracellular acidity of cancerous cells. Intravenous injection of fluorescently labeled (R18) pHLIP-coated niosomes into mice bearing tumors showed significant accumulation in tumors with minimal targeting of kidney, liver and muscles. Tumor-targeting niosomes coated with pHLIP exhibited 2–3 times higher tumor uptake compared to the non-targeted niosomes coated with PEG polymer. Long circulation time and uniform bio-distribution throughout the entire tumor make pHLIP-coated niosomes to be an attractive novel delivery system.