Diagnostic efficacy of immunocytochemistry on fine needle aspiration biopsies processed by thin-layer cytology

OBJECTIVE: To evaluate the efficacy of immunocytochemistry (ICC) performed on smears processed by thin-layer cytology (TLC). STUDY DESIGN: During the period January 2001-September 2003, 3,573 consecutive fine needle aspiration biopsies were processed with both conventional smears (CSs) and TLC diagnosed by a single pathologist; 113 required immunocytochemical study. CSs were fixed in ethanol whereas TLC slides were processed with the ThinPrep 2000 method (Cytyc Co., Marlborough, Massachusetts, U.S.A); both were stained with Papanicolaou stain. ICC staining was carried out on only TLC slides. RESULTS: The 113 cytologic cases were grouped as follows: 32 thyroid nodules with 16 histologic controls (HCs), 24 lymph nodes (regardless of location) with 15 HCs, 18 liver and pancreatic lesions (3 HCs), 11 lung nodules (6 HCs), 5 kidney and adrenal gland lesions (1 HC), 6 abdominal (2 HCs) and 4 mediastinal masses (1 HC), 6 salivary gland tumors (3 HCs), 4 bone masses (2 HCs) and 3 subcutaneous lesions (1 HC). ICC contributed to the diagnosis in 104 cases (92%), whereas it was inconclusive in 9. The cytologic diagnoses were histologically confirmed in 46 of 50 cases (92%). CONCLUSION: ICC can be successfully applied on TLC slides with better results than on CSs, and its yield can be useful in making the correct diagnosis on fine needle aspiration biopsy.     

5-Aza-2'-deoxycytidine and depsipeptide synergistically induce expression of BIK (BCL2-interacting killer)

DNA methylation and histone acetylation are main epigenetic events regulating gene expression, serving as anticancer drug targets. A combination of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine with the histone deacetylase inhibitor depsipeptide synergistically induces apoptosis. To characterize genes involved in this process, we measured expression of 376 apoptosis-related genes with microarrays after treatment with the two inhibitors alone or in combination. The pro-apoptotic BIK (Bcl2-interacting killer) was the only gene synergistically upregulated in all four cancer cell lines tested (A549, PC-3, TK-10, and UO-31). BIK induction was confirmed by RT-PCR and Western blots. Histone acetylation of the BIK promoter region increased with depsipeptide treatment but was not further affected by 5-aza-2'-deoxycytidine. In summary, synergistic upregulation of pro-apoptotic BIK-previously shown to suppress tumor growth-appears to play a critical role in anticancer effects of 5-aza-2'-deoxycytidine plus depsipeptide.     

Salmonella enterica SpvB ADP-ribosylates actin at position arginine-177-characterization of the catalytic domain within the SpvB protein and a comparison to binary clostridial actin-ADP-ribosylating toxins

The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila.     

Microbial endoxylanases: effective weapons to breach the plant cell-wall barrier or, rather, triggers of plant defense systems?

Endo-beta-1,4-xylanases (EC 3.2.1.8) are key enzymes in the degradation of xylan, the predominant hemicellulose in the cell walls of plants and the second most abundant polysaccharide on earth. A number of endoxylanases are produced by microbial phytopathogens responsible for severe crop losses. These enzymes are considered to play an important role in phytopathogenesis, as they provide essential means to the attacking organism to break through the plant cell wall. Plants have evolved numerous defense mechanisms to protect themselves against invading pathogens, amongst which are proteinaceous inhibitors of cell wall-degrading enzymes. These defense mechanisms are triggered when a pathogen-derived elicitor is recognized by the plant. In this review, the diverse aspects of endoxylanases in promoting virulence and in eliciting plant defense systems are highlighted. Furthermore, the role of the relatively recently discovered cereal endoxylanase inhibitor families TAXI (Triticum aestivum xylanase inhibitor) and XIP (xylanase inhibitor protein) in plant defense is discussed.     

Failure to maintain eye-specific segregation in nob, a mutant with abnormally patterned retinal activity

Axon terminals from the two eyes initially overlap in the dorsal-lateral geniculate nucleus (dLGN) but subsequently refine to occupy nonoverlapping territories. Retinal activity is required to establish and maintain this segregation. We show that despite the presence of retinal activity, segregated projections desegregate when the structure of activity is altered. Early in development, spontaneous retinal activity in the no b-wave (nob) mouse is indistinguishable from that of wild-type mice, and eye-specific segregation proceeds normally. But, around eye-opening, spontaneous and visually evoked activity in nob retinas become abnormal, coincident with a failure to preserve precise eye-specific territories. Dark-rearing studies suggest that altered visual experience is not responsible. Transgenic rescue of the mutated protein (nyctalopin) within nob retinal interneurons, without rescuing expression in either retinal projection neurons or their postsynaptic targets in the dLGN, restores spontaneous retinal activity patterns and prevents desegregation. Thus, normally structured spontaneous retinal activity stabilizes newly refined retinogeniculate circuitry.     

Novel putative targets of N-ethylmaleimide sensitive fusion protein (NSF) and alpha/beta soluble NSF attachment proteins (SNAPs) include the Pak-binding nucleotide exchange factor betaPIX

N-ethylmaleimide sensitive fusion protein (NSF) is a chaperone that plays a crucial role in the fusion of vesicles with target membranes. NSF mediates the ATP-consuming dissociation of a core protein complex that assembles during vesicle fusion and it thereby recharges the fusion machinery to perform multiple rounds of fusion. The binding of NSF to the core complex is mediated by co-chaperones named soluble NSF attachment proteins (SNAPs), for which three isoforms (alpha, beta and gamma) are known. Here, we sought to identify novel targets of the NSF-SNAP complex. A yeast two-hybrid screen using the brain specific betaSNAP isoform as bait revealed, as expected, NSF and several isoforms of the SNARE protein syntaxin as interactors. In addition, three isoforms of the reticulon protein family and two isoforms of BNIP3 interacted with betaSNAP. A yeast two-hybrid screen using NSF as bait identified Rab11-FIP3 and the Pak-binding nucleotide exchange factor betaPIX as putative binding partners. betaPIX interacts with recombinant NSF in co-sedimentation assays and the two proteins may be co-immunoprecipitated. A leucine zipper (LZ) motif within the C-terminus of betaPIX mediates binding to NSF; however, this fragment of betaPIX does not exhibit dominant negative effects in a cellular assay. In summary, our results support the evolving view that NSF has numerous targets in addition to conventional SNARE complexes.     

Evidence for a novel domain of bacterial outer membrane ushers

Many pathogenic bacteria possess adhesive surface organelles (called pili), anchored to their outer membrane, which mediate the first step of infection by binding to host tissue. Pilus biogenesis occurs via the "chaperone-usher" pathway: the usher, a large outer membrane protein, binds complexes of a periplasmic chaperone with pilus subunits, unloads the subunits from the chaperone, and assembles them into the pilus, which is extruded into the extracellular space. Ushers comprise an N-terminal periplasmic domain, a large transmembrane beta-barrel central domain, and a C-terminal periplasmic domain. Since structural data are available only for the N-terminal domain, we performed an in-depth bioinformatic analysis of bacterial ushers. Our analysis led us to the conclusion that the transmembrane beta-barrel region of ushers contains a so far unrecognized soluble domain, the "middle domain", which possesses a beta-sandwich fold. Two other bacterial beta-sandwich domains, the TT0351 protein from Thermus thermophilus and the carbohydrate binding module CBM36 from Paenibacillus polymyxa, are possible distant relatives of the usher "middle domain". Several mutations reported to abolish in vivo pilus formation cluster in this region, underlining its functional importance.     

Effects of three AM fungi on growth, distribution of glandular hairs, and essential oil production in Ocimum basilicum L. var. Genovese

The essential oils of basil are widely used in the cosmetic, pharmaceutical, food, and flavoring industries. Little is known about the potential of arbuscular mycorrhizal (AM) fungi to affect their production in this aromatic plant. The effects of colonization by three AM fungi, Glomus mosseae BEG 12, Gigaspora margarita BEG 34, and Gigaspora rosea BEG 9 on shoot and root biomass, abundance of glandular hairs, and essential oil yield of Ocimum basilicum L. var. Genovese were studied. Plant P content was analyzed in the various treatments and no differences were observed. The AM fungi induced various modifications in the considered parameters, but only Gi. rosea significantly affected all of them in comparison to control plants or the other fungal treatments. It significantly increased biomass, root branching and length, and the total amount of essential oil (especially α-terpineol). Increased oil yield was associated to a significantly larger number of peltate glandular trichomes (main sites of essential oil synthesis) in the basal and central leaf zones. Furthermore, Gi. margarita and Gi. rosea increased the percentage of eugenol and reduced linalool yield. Results showed that different fungi can induce different effects in the same plant and that the essential oil yield can be modulated according to the colonizing AM fungus.