An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue

MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H₂O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 μg/mm². Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.     

Determination of retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection

A method for separating and detecting retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection is described. Packed columns with an inner diameter of 180 μm were employed for the separation using a C₁₈ stationary phase and a mobile phase containing acetonitrile-water-methanol (65:32.5:2.5, v/v/v) with 1% tetrabutylammonium perchlorate and 0.174 M acetate buffered at pH 5. The detection cell consisted of a carbon fiber barrel electrode held at 0.9 V versus an Ag/AgCl reference. Injection volumes of 2 μl produced detection limits of 2.73, 0.472, 0.428, and 0.267 fmol (or 410, 64.1, 60.9, and 38.2 pg ml⁻¹) for 13-cis-retinoic acid, all-trans-retinoic acid, retinaldehyde, and retinol, respectively. This represents an improvement in detection limits of at least three orders of magnitude for similar analyses using liquid chromatography and UV absorbance detection. The detector signal was linear over two orders of magnitude of analyte concentration. Retinoid concentrations in bovine serum were determined and found to be in good agreement with previously reported values.     

The effect of substrate, dihydrobiopterin, and dopamine on the EPR spectroscopic properties and the midpoint potential of the catalytic iron in recombinant human phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH(4)) and non-heme iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic ferric iron at the active site of recombinant human PAH (hPAH) and its midpoint potential at pH 7.25 (E(m)(Fe(III)/Fe(II))) were studied by EPR spectroscopy. Similar EPR spectra were obtained for the tetrameric wild-type (wt-hPAH) and the dimeric truncated hPAH(Gly(103)-Gln(428)) corresponding to the "catalytic domain." A rhombic high spin Fe(III) signal with a g value of 4.3 dominates the EPR spectra at 3.6 K of both enzyme forms. An E(m) = +207 +/- 10 mV was measured for the iron in wt-hPAH, which seems to be adequate for a thermodynamically feasible electron transfer from BH(4) (E(m) (quinonoid-BH(2)/BH(4)) = +174 mV). The broad EPR features from g = 9.7-4.3 in the spectra of the ligand-free enzyme decreased in intensity upon the addition of L-Phe, whereas more axial type signals were observed upon binding of 7,8-dihydrobiopterin (BH(2)), the stable oxidized form of BH(4), and of dopamine. All three ligands induced a decrease in the E(m) value of the iron to +123 +/- 4 mV (L-Phe), +110 +/- 20 mV (BH(2)), and -8 +/- 9 mV (dopamine). On the basis of these data we have calculated that the binding affinities of L-Phe, BH(2), and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme, with respect to the ferric form. Interestingly, an E(m) value comparable with that of the ligand-free, resting form of wt-hPAH, i.e. +191 +/- 11 mV, was measured upon the simultaneous binding of both L-Phe and BH(2), representing an inactive model for the iron environment under turnover conditions. Our findings provide new information on the redox properties of the active site iron relevant for the understanding of the reductive activation of the enzyme and the catalytic mechanism.     

The unique cyanobacterial protein OpcA is an allosteric effector of glucose-6-phosphate dehydrogenase in Nostoc punctiforme ATCC 29133

Glucose-6-phosphate dehydrogenase (G6PD), encoded by zwf, is essential for nitrogen fixation and dark heterotrophic growth of the cyanobacterium Nostoc punctiforme ATCC 29133. In N. punctiforme, zwf is part of a four-gene operon transcribed in the order fbp-tal-zwf-opcA. Genetic analyses indicated that opcA is required for G6PD activity. To define the role of opcA, the synthesis, aggregation state, and activity of G6PD in N. punctiforme strains expressing different amounts of G6PD and/or OpcA were examined. A single tetrameric form of G6PD was consistently observed for all strains, as well as for recombinant N. punctiforme His-G6PD purified from Escherichia coli, regardless of the quantity of OpcA present. However, His-G6PD and the G6PD of strain UCD 351, which lacks OpcA, had low affinities for glucose 6-phosphate (G6P) substrate (K(m)(app) = 65 and 85 mm, respectively) relative to wild-type N. punctiforme G6PD (K(m)(app) = 0.5 mm). Near wild-type affinities for G6P were observed for these enzymes when saturating amounts of His-OpcA- or OpcA-containing extract were added. Kinetic studies were consistent with OpcA acting as an allosteric activator of G6PD. A role in redox modulation of G6PD activity was also indicated, because thioredoxin-mediated inactivation and reactivation of His-G6PD occurred only when His-OpcA was present.     

Trans-Atlantic dispersal and phylogeography of Cerastium arcticum (Caryophyllaceae) inferred from RAPD and SCAR markers

Cerastium arcticum is an autogamous pioneer species with a distribution limited to the North Atlantic region. It has been suggested that such species must have survived in ice-free refugia on both sides of the Atlantic throughout the last, or even several, of the Pleistocene glaciations, because they lack special adaptations for long-distance dispersal. To address the possibility for recent trans-Atlantic dispersal of C. arcticum, we analyzed random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) differentiation among 26 populations of this high-polyploid species. Three SCAR markers were obtained that verified the main patterns identified in the RAPD analysis. Eighty-four multilocus RAPD phenotypes were observed in the 126 plants analyzed, based on 35 polymorphic markers. Multivariate analyses and analyses of molecular variance (AMOVAs) identified two highly divergent groups of populations: one arctic group (western and eastern Greenland, and the archipelagos of Svalbard and Franz Josef Land) and one nonarctic group (southern and northern Norway, and Iceland), indicating that C. arcticum is composed of two lineages with different evolutionary histories. However, there was little geographic structuring within each lineage, in spite of the fact that both lineages are disjunctly distributed across the Atlantic. Occurrence of very similar, in some cases even identical RAPD multilocus phenotypes on both sides of the Atlantic in this autogamous allopolyploid is most probably caused by postglacial dispersal. The present geographic distribution of C. arcticum may thus have been established after trans-Atlantic expansion from two Weichselian refugia, one for each evolutionary lineage. Unexpectedly, the level of intrapopulational variation increased towards the north. This may reflect that interpopulational migration is most extensive in the treeless arctic environment, where the species has a more continuous distribution than in the more southerly areas.     

Rate of intrachain contact formation in an unfolded protein: temperature and denaturant effects

We have measured the effect of temperature and denaturant concentration on the rate of intrachain diffusion in an unfolded protein. After photodissociating a ligand from the heme iron of unfolded horse cytochrome c, we use transient optical absorption spectroscopy to measure the time scale of the diffusive motions that bring the heme, located at His18, into contact with its native ligand, Met80. Measuring the rate at which this 62 residue intrachain loop forms under both folding and unfolding conditions, we find a significant effect of denaturant on the chain dynamics. The diffusion of the chain accelerates as denaturant concentration decreases, with the contact formation rate approaching a value near approximately 6x10(5) s(-1) in the absence of denaturant. This result agrees well with an extrapolation from recent loop formation measurements in short synthetic peptides. The temperature dependence of the rate of contact formation indicates an Arrhenius activation barrier, Ea approximately 20 kJ/mol, at high denaturant concentrations, comparable to what is expected from solvent viscosity effects alone. Although Ea increases by several kBT as denaturant concentration decreases, the overall rate of diffusion nevertheless increases. These results indicate that inter-residue energetic interactions do not control conformational diffusion in unfolded states, even under folding conditions.     

Beta 1-subunits are required for regulation of coupling between Ca2+ transients and Ca2+-activated K+ (BK) channels by protein kinase C

Colonic myocytes have spontaneous, localized, Ins (1,4,5) trisphosphate (IP3) receptor-dependent Ca2+ transients that couple to the activation of Ca2+-dependent K+ channels and spontaneous transient outward currents (STOCs). We previously reported that the coupling strength between spontaneous Ca2+ transients and large conductance Ca2+ activated K+ (BK) channels is regulated by Ca2+ influx through nonselective cation channels and activation of protein kinase C (PKC). Here, we used confocal microscopy and the patch-clamp technique to further investigate the coupling between localized Ca2+ transients and STOCs in colonic myocytes from animals lacking the regulatory beta1-subunit of BK channels. Myocytes from beta1-knockout (beta1-/-) animals loaded with fluo 4 showed typical localized Ca2+ transients, but the STOCs coupled to these events were of abnormally low amplitude. Reduction in external Ca2+ or application of inhibitors of nonselective cation channels (SKF-96365) caused no significant change in the amplitude or frequency of STOCs. Likewise, an inhibitor of PKC, GF 109203X, had no significant effect on STOCs. Single-channel recording from BK channels showed that application of an activator (PMA) and an inhibitor (GF 109203X) of PKC did not affect BK channel openings in myocytes of beta1-/- mice. These data show that PKC-dependent regulation of coupling strength between Ca2+ transients and STOCs in colonic myocytes depends upon the interaction between alpha- and beta1-subunits.     

IL-2 intratumoral immunotherapy enhances CD8+ T cells that mediate destruction of tumor cells and tumor-associated vasculature: a novel mechanism for IL-2

Therapeutic use of IL-2 can generate antitumor immunity; however, a variety of different mechanisms have been reported. We injected IL-2 intratumorally (i.t.) at different stages of growth, using our unique murine model of mesothelioma (AE17; and AE17 transfected with secretory OVA (AE17-sOVA)), and systematically analyzed real-time events as they occurred in vivo. The majority of mice with small tumors when treatment commenced displayed complete tumor regression, remained tumor free for >2 mo, and survived rechallenge with AE17 tumor cells. However, mice with large tumors at the start of treatment failed to respond. Timing experiments showed that IL-2-mediated responses were dependent upon tumor size, not on the duration of disease. Although i.t. IL-2 did not alter tumor Ag presentation in draining lymph nodes, it did enhance a previously primed, endogenous, tumor-specific in vivo CTL response that coincided with regressing tumors. Both CD4(+) and CD8(+) cells were required for IL-2-mediated tumor eradication, because IL-2 therapy failed in CD4(+)-depleted, CD8(+)-depleted, and both CD4(+)- and CD8(+)-depleted C57BL/6J animals. Tumor-infiltrating CD8(+) T cells, but not CD4(+) T cells, increased in association with a marked reduction in tumor-associated vascularity. Destruction of blood vessels required CD8(+) T cells, because this did not occur in nude mice or in CD8(+)-depleted C57BL/6J mice. These results show that repeated doses of i.t. (but not systemic) IL-2 mediates tumor regression via an enhanced endogenous tumor-specific CTL response concomitant with reduced vasculature, thereby demonstrating a novel mechanism for IL-2 activity.