Fine needle aspiration appearance of extragastrointestinal stromal tumor. A case report

BACKGROUND: Gastrointestinal stromal tumors (GISTs) rarely develop outside the digestive tract and in the soft tissues of abdomen and retroperitoneum. Such tumors are designated extra-GISTs (EGISTs). Cytologic and immunocytochemical features of a case of EGIST are reported. CASE: A 54-year-old woman presented with a peritoneal mass, diameter 22 cm, adherent to the omentum and without a connection to the digestive tract. Fine needle aspiration biopsy (FNAB) of the excised tumor showed high cellularity in two patterns: monotonous spindle cells were intermingled with a mildly atypical epithelioid component. Immunocytochemistry performed on cytospins revealed reactivity for c-kit (CD117), CD34 and smooth muscle actin and negativity for S-100. The findings were concordant with a histologic diagnosis of EGIST. CONCLUSION: EGISTs are infrequent neoplasms and can be diagnosed in FNAB samples. The clinical/radiologic setting must be considered together with the cytologic features. Immunocytochemistry is a clue to the diagnosis when it detects c-kit reactivity.     

Characterization of the specificity of arginine-specific gingipains from Porphyromonas gingivalis reveals active site differences between different forms of the enzymes

Porphyromonas gingivalis is a pathogen associated with periodontal disease, and arginine-specific proteases (gingipains-R) from the bacterium are important virulence factors. The specificity of two forms of gingipain-R, HRgpA and RgpB, for substrate positions C-terminal to the cleavage site was analyzed, and notable differences were observed between the enzymes. Molecular modeling of the HRgpA catalytic domain, based on the structure of RgpB, revealed that there are four amino acid substitutions around the active site of HRgpA relative to RgpB that may explain their different specificity. Previously, differences in the ability of these two gingipain-R forms to cleave a number of proteins were attributed to additional adhesins on HRgpA mediating increased interaction with the substrates. Here, purified RgpA(cat), the catalytic domain of HRgpA, which like RgpB also lacks adhesin subunits, was used to show that the differences between HRgpA and RgpB are probably due to the amino acid substitutions at the active site. The kinetics of cleavage of fibrinogen, a typical protein substrate for the gingipain-R enzymes, which is bound by HRgpA but not RgpA(cat) or RgpB, were evaluated, and it was shown that there was no difference in the cleavage of the fibrinogen Aalpha-chain between the different enzyme forms. HRgpA degraded the fibrinogen Bbeta-chain more efficiently, generating distinct cleavage products. This indicates that while the adhesin domain(s) play(s) a minor role in the cleavage of protein substrates, the major effect is still provided by the amino acid substitutions at the active site of rgpA gene products versus those of the rgpB gene.     

Identification of UNC119 as a novel activator of SRC-type tyrosine kinases

Lyn, an Src-type tyrosine kinase, is associated with the interleukin (IL)-5 receptor in eosinophils. The mechanism of its activation is unknown. Through yeast two-hybrid screening we have cloned and characterized a new signaling molecule, Unc119, that associates with IL-5Ralpha and Src family tyrosine kinases. Unc119 induces the catalytic activity of these kinases through interaction with Src homology 2 and 3 domains. IL-5 stimulation of eosinophils increases Unc119 association with Lyn and induces its catalytic activity. Lyn is important for eosinophil survival. Eosinophils that are transduced with Unc119 have increased Lyn activity and demonstrate prolonged survival in the absence of IL-5. Inhibition of Unc119 down-regulates eosinophil survival. To our knowledge Unc119 is the first receptor-associated activator of Src family tyrosine kinases.     

Heterodimerization of substance P and mu-opioid receptors regulates receptor trafficking and resensitization

The micro-opioid receptor (MOR1) and the substance P receptor (NK1) coexist and functionally interact in nociceptive brain regions; however, a molecular basis for this interaction has not been established. Using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET), we show that MOR1 and NK1 can form heterodimers in HEK 293 cells coexpressing the two receptors. Although NK1-MOR1 heterodimerization did not substantially change the ligand binding and signaling properties of these receptors, it dramatically altered their internalization and resensitization profile. Exposure of the NK1-MOR1 heterodimer to the MOR1-selective ligand [D-Ala2,Me-Phe4,Gly5-ol]enkephalin (DAMGO) promoted cross-phosphorylation and cointernalization of the NK1 receptor. Conversely, exposure of the NK1-MOR1 heterodimer to the NK1-selective ligand substance P (SP) promoted cross-phosphorylation and cointernalization of the MOR1 receptor. In cells expressing MOR1 alone, beta-arrestin directs the receptors to clathrin-coated pits, but does not internalize with the receptor. In cells expressing NK1 alone, beta-arrestin internalizes with the receptor into endosomes. Interestingly, in cells coexpressing MOR1 and NK1 both DAMGO and SP induced the recruitment of beta-arrestin to the plasma membrane and cointernalization of NK1-MOR1 heterodimers with beta-arrestin into the same endosomal compartment. Consequently, resensitization of MOR1-dependent receptor functions was severely delayed in coexpressing cells as compared with cells expressing MOR1 alone. Together, our findings indicate that MOR1 by virtue of its physical interaction with NK1 is sequestered via an endocytotic pathway with delayed recycling and resensitization kinetics.     

Role of N-methyl-D-aspartate receptors in the neuroprotective activation of extracellular signal-regulated kinase 1/2 by cisplatin

Neurons are exposed to damaging stimuli that can trigger cell death and subsequently cause serious neurological disorders. Therefore, it is important to define defense mechanisms that can be activated in response to damage to reduce neuronal loss. Here we report that cisplatin (CPDD), a neurotoxic anticancer drug that damages DNA, triggered apoptosis and activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in cultured rat cortical neurons. Inhibition of ERK1/2 activation using either pharmacological inhibitors or a dominant-negative mutant of the ERK1/2 activator, mitogen-activated protein kinase kinase 1, increased the toxicity of CPDD. Interestingly, N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonists reduced the ERK1/2 activation and exacerbated apoptosis in CPDD-treated neurons. Pre-treatment with CPDD increased ERK1/2 activation triggered by exogenous NMDA, suggesting that CPDD augmented NMDAR responsiveness. CPDD-enhanced response of NMDAR and CPDD-mediated ERK1/2 activation were both decreased by inhibition of poly(ADP-ribose) polymerase (PARP). Interestingly, PARP activation did not produce ATP depletion, suggesting involvement of a non-energetic mechanism in NMDAR regulation by PARP. Finally, CPDD toxicity was reduced by brain-derived neurotrophic factor, and this protection required ERK1/2. In summary, our data identify a novel compensatory circuit in central nervous system neurons that couples the DNA injury, through PARP and NMDAR, to the defensive ERK1/2 activation.     

Insight into functional aspects of Stt3p, a subunit of the oligosaccharyl transferase. Evidence for interaction of the N-terminal domain of Stt3p with the protein kinase C cascade

Over a decade ago, the gene STT3 was identified in a staurosporine and temperature sensitivity screen of yeast. Subsequently the product of this gene was shown to be a subunit of the endoplasmic reticulum-localized oligosaccharyl transferase (OT) complex. Although stt3 mutants are known to be staurosporine-sensitive, we found that mutants of other OT subunits (except ost4 Delta) are staurosporine-resistant, which indicates that this phenotype of stt3 mutants is not simply a consequence of their defect in glycosylation, as previously speculated. Staurosporine sensitivity was found to be an allele-specific phenotype restricted to cells harboring mutations in highly conserved residues in the N-terminal domain of the STT3 protein. Cells bearing mutations in one of the cytosolic-oriented loops (amino acids 158-168) in the N terminus of Stt3p were found to be specifically susceptible to staurosporine. Staurosporine is a specific inhibitor of Pkc1p, and a genetic link had previously been suggested between PKC1 and STT3. It is known that overexpression of PKC1 suppresses the staurosporine sensitivity of the stt3 mutants in an allele-specific manner, which is typical of mutants of Pkc1p cascade. It has been shown that the pkc1 null mutant exhibits lowered OT activity. Our results combined with these previous observations indicate that the N-terminal domain of Stt3p may interact with members of the Pkc1p cascade and consequently mutations in this domain result in staurosporine sensitivity. We further speculate that the Pkc1p regulates OT activity through the N-terminal domain of Stt3p, the C-terminal domain of which possesses the recognition and/or catalytic site of the OT complex.     

Intratumor CpG-oligodeoxynucleotide injection induces protective antitumor T cell immunity

Tumor cells are typically poorly immunogenic. The same mechanisms that evolved to avoid the induction of immune responses against self tissues, and, hence, autoimmune disease, also have to be overcome for immune therapy of cancer. Toll-like receptor-activating microbial products such as CpG motif containing DNA are among the primary stimuli that the immune system uses to distinguish between infectious nonself (that is to be attacked) and noninfectious self (that must not be attacked). We tested in a murine RMA lymphoma/C57BL/6 model whether providing the infectious nonself context in a tumor-by injecting CpG-oligodeoxynucleotides directly into the tumor-would elicit a protective antitumor response. Complete remission of established solid tumors was achieved in immune competent mice, but not in T cell/B cell-deficient RAG-1 knockout mice. Intratumor injection of CpG-oligodeoxynucleotides was shown to induce a tumor-specific CD4(+) and CD8(+) T cell response of the type 1 effector class, and T cells adoptively transferred the protection to RAG-1 knockout mice. The data show that intratumor injection of CpG-oligodeoxynucleotides is a promising strategy for rendering tumors immunogenic.     

Regulation of membrane transport by rab GTPases

Membrane flow through the cell is a highly dynamic process in which intracellular compartments communicate via tubulo-vesicular structures shuttling cargo molecules to their destinations. Transport carriers are formed at a donor compartment and navigate through the cytoplasm to the target organelle, on which they subsequently dock and fuse. Many of these events are regulated by the cooperative action of monomeric rab GTPases and their effector proteins. Research in recent years resulted in the identification of many rab effectors, providing first glimpses how the GTPase switch of individual rab proteins is utilized in discrete transport steps.