Automated selection of DAB-labeled tissue for immunohistochemical quantification.

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.     

Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of L-propargylglycine.

L-Propargylglycine, a naturally occurring gamma, delta-acetylenic alpha-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine gamma-synthase and of 8:1 for methionine gamma-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [alpha-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [alpha-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, only one alpha and one beta subunit may be labeled in an inactive alpha 2 beta 2 tetramer of methionine gamma-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine gamma-synthase may reprotonate C2 of the enzyme--inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine gamma-lyase has lambda max values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.     

Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets

NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRalphabeta NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Valpha14i-positive and approximately 50% of Valpha14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Valpha14i-negative than Valpha14i-positive NKT cells. In spleen only, subsets of Valpha14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-gamma/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4(-) Valpha14i-negative NKT cells that were L-selectin(high) and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Valpha14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1(+) Valpha14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Valpha14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4(+) or CD4(-)CD8(-) and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Valpha14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.     

Contrasting distributions of bacteriophages and eukaryotic viruses from contaminated coastal sediments

Viruses are ubiquitous, abundant and play an important role in all ecosystems. Here, we advance understanding of coastal sediment viruses by exploring links in the composition and abundance of sediment viromes to environmental stressors and sediment bacterial communities. We collected sediment from contaminated and reference sites in Sydney Harbour and used metagenomics to analyse viral community composition. The proportion of phages at contaminated sites was significantly greater than phages at reference sites, whereas eukaryotic viruses were relatively more abundant at reference sites. We observed shifts in viral and bacterial composition between contaminated and reference sites of a similar magnitude. Models based on sediment characteristics revealed that total organic carbon in the sediments explained most of the environmental stress-related variation in the viral dataset. Our results suggest that the presence of anthropogenic contaminants in coastal sediments could be influencing viral community composition with potential consequences for associated hosts and the environment.     

Pattern formation in single cells

Single-cell patterning begins with an asymmetric cue that orients the axis of polarity. Despite great diversity in the types of cues, common mechanisms appear to mediate the polarizing response. Rho-family GTPases initially process and reinforce polarity cues by remodelling cortical actin, and these local asymmetries are subsequently propagated to the microtubules, membrane and secretory pathway to generate the final pattern. Homologues of the yeast polarity genes fulfil similar functions in higher eukaryotes, revealing a fundamental conservation in how polarity arises. Unlike yeast, however, more complex eukaryotic cells can manifest multiple axes of polarity, suggesting that additional mechanisms have evolved to generate more elaborate patterns.     

Sustained activation of the raf-MEK-ERK pathway elicits cytokine unresponsiveness in T cells

Activation of T cells via the TCR and other costimulatory receptors triggers a number of signaling cascades. Among them, the Ras-activated Raf-mitogen-activated protein/extracellular signal-related kinase (ERK) kinase (MEK)-ERK signaling cascade has been demonstrated to be crucial for both T cell development and activation. It has previously been demonstrated that high doses of Ag or anti-CD3 mAb are able to induce in T cells a nonresponsive state to subsequent treatment with cytokines such as IL-2. The precise biochemical mechanisms underlying this effect are not fully characterized. In this study, we demonstrate that cytokine nonresponsiveness is accompanied by the induction of the cyclin-dependent kinase inhibitor p21Cip1 that is mediated, at least in part, by the activation of the Raf-MEK-ERK pathway. Furthermore, we demonstrate that selective activation of the Raf-MEK-ERK signaling pathway in T cells is sufficient to induce cytokine nonresponsiveness in both a T cell clone and naive primary T cells. In this case, nonresponsiveness is accompanied by the induction of p21Cip1 and the prevention of p27Kip1 down-regulation, leading to inhibition of cyclin E/cyclin-dependent kinase 2 activity. These data suggest that anti-CD3 mAb-induced cytokine nonresponsiveness may be a consequence of hyperactivation of the Raf-MEK-ERK pathway, leading to alterations in the expression of key cell cycle regulators. These observations may provide a novel insight into the mechanisms of induction of peripheral tolerance.     

Patterns of neural activation associated with exposure to odors from a familiar winner in male golden hamsters

The neural mechanisms underlying recognition of familiar individuals and responses appropriate to them are not well known. Previous studies with male golden hamsters have shown that, after a series of brief aggressive encounters, a loser selectively avoids his own, familiar winner but does not avoid other males. Using this paradigm, we investigated activity in 20 areas of the brain using immunohistochemistry for c-Fos and Egr-1 during exposure to a familiar winner compared to control groups not exposed to another male. Behavioral data showed that 1 day after fights males that lost avoided the familiar winner, suggesting that they recognized this individual. The c-Fos and Egr-1 immunohistochemistry showed that the losers exposed to familiar winners had a greater density of stained cells in the basolateral amygdala, the CA1 region of anterior dorsal hippocampus and the dorsal subiculum than control groups had in these areas. These results suggest that these brain areas may be involved in the memory for other males, the learned fear of familiar winners, or related processes.