Expression and characterization of a novel CD6 ligand in cells derived from joint and epithelial tissues

CD6 is a T cell surface glycoprotein that plays an important role in interactions of thymocytes with thymic epithelial cells and in mature T cell interactions with selected nonprofessional tissue APCs. We describe a novel CD6 ligand (CD6L) 3A11 Ag that is distinct from the known CD6L (CD166). The 3A11 protein is expressed on cells derived from human thymus, skin, synovium, and cartilage, and its expression is enhanced by IFN-gamma. mAbs directed against the 3A11 Ag and CD166 exhibit distinct patterns of binding to a panel of cell lines. Confocal microscopy shows that both CD166 and the 3A11 Ag are expressed at the cell surface, and that these proteins colocalize. The 3A11 Ag has a molecular mass of 130 kDa and is immunoprecipitated using either mAb 3A11 or soluble CD6-Ig fusion protein. mAbs directed against individual CD6L were less potent than was soluble CD6-Ig fusion protein in reducing adhesion of T cells to adherent 3A11-positive epithelial cells in vitro, suggesting that these Abs recognize epitopes on the 3A11 Ag and CD166 that are distinct from CD6 binding sites. Finally, transfection of epithelial cells with CD166-specific small interfering RNAs significantly decreased CD166 expression without alteration in 3A11 Ag levels, and thus confirmed that these two CD6L are distinct. Taken together, our data identifies a novel 130-kDa CD6L that may mediate interactions of synovial and epithelial cells with T lymphocytes.     

Myoepithelial carcinoma arising in a pleomorphic adenoma of the parotid gland: report of a case with cytopathologic findings

BACKGROUND: Carcinoma arising in a mixed tumor, or carcinoma ex pleomorphic adenoma (CEPA), is an uncommon primary salivary gland neoplasm. Among the various types of carcinomas that can be seen histologically in a CEPA, myoepithelial carcinoma is one of the rarest forms. CASE: A 76-year-old woman presented with an incidental parotid/parapharyngeal mass. Computed tomography-guide fine needle aspiration (FNA) showed a biphasic neoplasm with epithelial and stromal components consistent with pleomorphic adenoma (PA). However, in addition, a distinct population of discohesive atypical and pleomorphic cells with high nuclear/cytoplasmic ratio was noted in the background. In the cytopathologic diagnosis a suspicion was raised about a possible CEPA. Subsequent resection of the parotid mass confirmed the presence of low grade myoepithelial carcinoma arising in a PA. CONCLUSION: Although uncommon, CEPA should be suspected on FNA when atypical cytomorphologic characteristics are observed. In rare cases a myoepithelial carcinoma also arises in a preexisting PA, necessitating an accurate interpretation for more definitive therapy.     

Analysis of glioma cell platinum response by metacomparison of two-dimensional chromatographic proteome profiles

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response.     

Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions Rather than Electrostatic Repulsion

RNA folding occurs via a series of transitions between metastable intermediate states for Mg²⁺ concentrations below those needed to fold the native structure. In general, these folding intermediates are considerably less compact than their respective native states. Our previous work demonstrates that the major equilibrium intermediate of the 154-residue specificity domain (S-domain) of the Bacillus subtilis RNase P RNA is more extended than its native structure. We now investigate two models with falsifiable predictions regarding the origins of the extended intermediate structures in the S-domains of the B. subtilis and the Escherichia coli RNase P RNA that belong to different classes of P RNA and have distinct native structures. The first model explores the contribution of electrostatic repulsion, while the second model probes specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering and Langevin dynamics simulations, we show that electrostatics plays only a minor role, whereas specific interactions largely account for the extended nature of the intermediate. Structural contacts in the core, including a nonnative base pair, help to stabilize the intermediate conformation. We conclude that RNA folding intermediates adopt extended conformations due to short-range, nonnative interactions rather than generic electrostatic repulsion of helical domains. These principles apply to other ribozymes and riboswitches that undergo functionally relevant conformational changes.     

Thiol Peroxidase Protects Salmonella enterica from Hydrogen Peroxide Stress In Vitro and Facilitates Intracellular Growth

At present, Salmonella is considered to express two peroxiredoxin-type peroxidases, TsaA and AhpC. Here we describe an additional peroxiredoxin, Tpx, in Salmonella enterica and show that a single tpx mutant is susceptible to exogenous hydrogen peroxide (H₂O₂), that it has a reduced capacity to degrade H₂O₂ compared to the ahpCF and tsaA mutants, and that its growth is affected in activated macrophages. These results suggest that Tpx contributes significantly to the sophisticated defense system that the pathogen has evolved to survive oxidative stress.Salmonella is an important human pathogen which causes a variety of diseases, including gastroenteritis, septicemia, and typhoid fever. In the host, salmonellae reside inside phagocytic cells and are exposed to various host defense mechanisms, including oxidative stress (13). The production of superoxide anion (O₂⁻) is crucial, as individuals with chronic granulomatous disease, which is due to a defective phagocyte NADPH oxidase, are more susceptible to infections with Salmonella (10). Likewise, diminished NADPH oxidase activity leads to increased susceptibility to Salmonella in murine macrophages (20-22, 25). Superoxide anion (O₂⁻) is weakly reactive and fails to pass through the bacterial cell wall. After conversion to H₂O₂ by either spontaneous or enzymatic dismutation by superoxide dismutases, it readily diffuses into the bacterial cell and forms reactive hydroxyl radicals (OH) that damage macromolecules such as DNA, proteins, and lipids (12, 17).In principle, Salmonella possesses two classes of enzymes to degrade H₂O₂. Catalases degrade H₂O₂ to water and molecular oxygen independent of an additional reductant. Peroxiredoxin-type peroxidases (peroxiredoxins) reduce organic hydroperoxides to alcohols and hydrogen peroxide to water at the expense of NADH or NADPH. In a recent study by Hébrard et al., three members of the catalase family, KatG, KatE, and KatN, and two members of the peroxiredoxin family, AhpC and TsaA, were characterized in Salmonella (14). Previously it had been shown that single katE, katG, and katN Salmonella mutants did not show increased susceptibility to exogenous H₂O₂ (3, 24). In macrophages a katG katE katN triple mutant had no growth defect, whereas an ahpCF tsaA double mutant showed a reduced growth rate in macrophages (14). These observations point out the multiple routes that have evolved in Salmonella to protect the pathogen against oxidative stress and suggest that peroxiredoxins play a dominant role in the antioxidant defense during infection. In this study we characterized a third peroxiredoxin-type peroxidase, Tpx. Surprisingly, a simple tpx mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) was more susceptible to exogenous H₂O₂ than the wild type (WT). The mutant grew less well in activated macrophages and showed a reduced peroxidase activity toward H₂O₂.     

Uev1A, a ubiquitin conjugating enzyme variant, inhibits stress-induced apoptosis through NF-κB activation

We have previously shown that UEV1 is up-regulated in all tumor cell lines examined and when SV40-transformed human embryonic kidney cells undergo immortalization; however, it is unclear whether and how UEV1 plays a critical role in this process. UEV1A encodes a ubiquitin conjugating enzyme variant, which is required for Ubc13 (ubiquitin conjugating enzyme) catalyzed poly-ubiquitination of target proteins through Lys63-linked chains. One of the target proteins is NEMO/IKKγ (nuclear factor-κB essential modulator/inhibitor of κB protein kinase), a regulatory subunit of IκB kinase in the NF-κB signaling pathway. In this report, we show that constitutive high-level expression of UEV1A alone in cultured human cells was sufficient to cause a significant increase in NF-κB activity as well as the expression of its target anti-apoptotic protein, Bcl-2 (B-cell leukemia/lymphoma 2). Overexpression of UEV1A also conferred prolonged cell survival under serum-deprived conditions, and protected cells against apoptosis induced by diverse stressing agents. All of the effects of Uev1A were reversible upon suppression of UEV1 expression by RNA interference. Our observations presented in this report provide evidence that Uev1A is a critical regulatory component in the NF-κB signaling pathway in response to environmental stresses and identify UEV1A as a potential proto-oncogene.     

Characterization of oil palm MADS box genes in relation to the mantled flower abnormality

In vitro propagation of oil palm (Elaeis guineensis Jacq.) frequently induces a somaclonal variant called ‘mantled’ abnormality, in which the stamens of both male and female flowers are transformed into carpels. This leads to a reduced yield or complete loss of the harvest of palm oil. The high frequency of the abnormality in independent lines and the high reversal rate suggest that it is due to an epigenetic change. The type of morphological changes suggest that it involves homeotic MADS box genes that regulate the identity of the flower whorls. We have isolated a number of MADS box genes from oil palm inflorescences by a MADS box-directed mRNA display approach. The isolated partial cDNAs included genes that were likely to function at the initial stages of flowering as well as genes that may function in determination of the inflorescence and the identity of the flower whorls. For four genes that were homologous to genes known to affect the reproductive parts of the flower, full length cDNAs were isolated. These were a B-type MADS box gene which may function in the determination of stamen formation, a C-type gene expected to be involved in stamen and carpel formation, and two putative SEP genes which act in concert with the A-, B- and C-type MADS box gene in determining flower whorl formation. The B-type gene EgMADS16 was functionally characterized as a PISTILLATA orthologue; it was able to complement an Arabidopsis thaliana pi mutant. Whether EgMADS16, or any of the other EgMADS genes, are functionally involved in the mantled condition remains to be established.