Expression of Cathepsin D and pS2 in imprint smears of breast carcinoma

athanassiadou p.p., athanassiades p. h., daffaris p., petrakakou e. i., fflerffa ch. i. and kffirkou k. a. (1998) Cytopathology 9, 240–247
Expression of Cathepsin D and pS2 in imprint smears of breast carcinoma
The aim of this study was to add to existing information on the effects of certain tumour markers expressed by breast cancers on tumour malignancy as evidenced by size of primary and occurrence of lymph node invasion. One hundred freshly resected breast cancers were examined by immunocytochemical staining of imprint smears for Cathepsin D and pS2. Oestrogen receptor (ER) and progesterone receptor (PR) were tested for by dextrose-coated charcoal (DCC) assay and the results correlated with tumour size, histology and presence or absence of lymph node metastases at the time of surgery using χ² analysis. A significant positive correlation was demonstrated between Cathepsin D positivity and ER, PR and pS2 positivity. In tumours < 2 cm in diameter at surgery a positive correlation was observed between Cathepsin D positivity and the presence of lymph node metastases. The findings support the hypothesis that Cathepsin D may promote early metastasis, possibly by its proteolytic activity.     

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine

The reported cDNA structrre, of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olsonet al. Proc. Natl. Acad. Sci USA, 87: 2284–2288, 1990). The calculated Mr is 107, 534 whereas the estimate by SDS-PAGE is approximately 130, 000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors, in the cDNA sequence for non-muscle MLCK and that the NH₂-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH₂-terminally blocked. A CNBr peptide derived from smMLCK contains the NH₂-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating, that Met-1 is present. Using a limited thermolysin digest we isolated an NH₂-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH₂-terminal Met being blocked by actylation. The results demonstrate that the NH₂-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating, Met is not removed, but modified by -NH₂ acetylation of the translation product.     

Structural and biosensor analyses of a synthetic biotinylated peptide probe for the isolation of adenomatous polyposis coli tumor suppressor protein complexes.

Large numbers of colon tumors stem from mutations in the gene coding for the production of the adenomatous polyposis coli (APC) tumor suppressor protein. This protein contains a coiled-coil N-terminal domain that is known to be responsible for homodimerization. Previous work by others has led to the design of a specific 54-residue anti-APC peptide (anti-APCp1) that dimerizes preferentially with this domain. We have undertaken the chemical synthesis of a modified form of this peptide (anti-APCp2) that bears a biotin moiety at its N-terminus for use in subsequent ligand-binding analysis studies. The peptide was subjected to comprehensive chemical characterization to confirm its purity. Secondary structural analysis by circular dichroism spectroscopy and Fourier transform infrared spectroscopy indicated that the peptide could assume a wide range of potential conformations, depending upon the precise microenvironment. Significantly, a stable alpha-helical structure was generated when the solvent conditions supported intramolecular salt-bridge formation along the helix barrel. The biotinylated anti-APCp2 was immobilized onto a streptavidin sensor surface, in a specific orientation leaving all amino acids available to form a coiled structure. In one experiment, injection of colonic cell lysate extracts (LIM1215) onto a size-exclusion column resulted in the isolation of a high molecular mass protein peak (> 600 kDa) that reacted specifically with the immobilized anti-APCp2 on the biosensor surface. In another experiment, a high molecular mass protein (M(r) > 250 kDa on SDS-PAGE) could be specifically immunoprecipitated from this peak using either the anti-APCp2 peptide or an anti-APC polyclonal antibody. This demonstrates the specific interaction between the anti-APCp2 peptide and native APC and highlights the potential use of the former peptide in a multidimensional micropreparative chromatographic/biosensor/proteomic protocol for the purification of APC alone and APC complexed with different biopolymers in various cell lines, and stages of tumor development.     

Budgerigar-fancier's lung: the commonest variety of allergic alveolitis in Britain.

A questionnaire survey of 1005 consecutive attenders at four outpatient clinics yielded 117 (12%) budgerigar fanciers (exposed to budgerigars- known in North America as parakeets-for at least three months) and 296 (29%) former fanciers. Twnety had precipitins to budgerigar serum or droppings or both, and 10 of these together with 39 precipitin-negative patients reported undue breathlessness on exertion during exposure to buderigars. These 59 patients were investigated further, seven completing a series of inhalation provocation tests with budgerigar antigens designed to confirm or exclude budgerigar-fancier''s lung (BrFL). Typical positive responses were obtained from four current and one former fancier. The prevalence of confirmed BrFL among the 11n current budgerigar fanciers was 3.4% (four cases). This was biased, however, by the inclusion of one patient whose attendance at the surveyed clinic was attributable to the disease. With the exclusion of this patient, confidence limits suggested that the true prevalence of BrFL among current budgerigar fanciers in the general population lies between 0.5% and 7.5%, which is similar to the prevalence of farmer''s lung in farm workers. In view of the enormous population at risk, however, this implies that BrFL rather than farmer''s lung is by far the commonest type of allergic alveolitis in Britain.     

Green synthesis and characterization of selenium nanoparticles and its augmented cytotoxicity with doxorubicin on cancer cells

Green synthesis of selenium nanoparticles (SeNPs) was achieved by a simple biological procedure using the reducing power of fenugreek seed extract. This method is capable of producing SeNPs in a size range of about 50–150 nm, under ambient conditions. The synthesized nanoparticles can be separated easily from the aqueous sols by a high-speed centrifuge. These selenium nanoparticles were characterized by UV–Vis spectroscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and elemental analysis by X-ray fluorescence spectrometer (XRF). Nanocrystalline SeNPs were obtained without post-annealing treatment. FTIR spectrum confirms the presence of various functional groups in the plant extract, which may possibly influence the reduction process and stabilization of nanoparticles. The cytotoxicity of SeNPs was assayed against human breast-cancer cells (MCF-7). It was found that SeNPs are able to inhibit the cell growth by dose-dependent manner. In addition, combination of SeNPs and doxorubicin shows better anticancer effect than individual treatments.     

Late Emsian Rutoceratoidea (Nautiloidea) from the Prague Basin,Czech Republic: morphology,diversity and palaeoecology

Abstract: Nautiloids of the superfamily Rutoceratoidea from the late Emsian (late Early Devonian) of the Prague Basin (Czech Republic) are commented upon. Species recognized include the hercoceratids Hercoceras mirum, H.? transiens, Ptenoceras proximum, P. nudum, P. minusculum and Anomaloceras anomalum, as well as the rutoceratids Adelphoceras bohemicum, Homoadelphoceras devonicans, Pseudorutoceras bolli and Goldringia? devonicans. In addition, four new species are described: Parauloceras regulare sp. nov., Roussanoffoceras chlupaci sp. nov., Otomaroceras sp. nov. and Goldringia sp. nov. Morphology and distribution patterns of Pragian and late Emsian rutoceratoid faunas from the Prague Basin are compared. They show that an increased diversity was accompanied by a higher level of specialization of rutoceratoids, which manifested itself in low abundance, increased facies dependence and greater variation in shell size during the Early Devonian. The evolution of sculpture and a contracted aperture, both regarded as protective adaptive features, was also examined, but no adaptive trend towards more pronounced sculpture and constriction of the aperture was found to have occurred in the Early Devonian. A more distinctive sculpture was, however, observed in shallow‐water assemblages of P. proximum in comparison with deeper‐water faunules, and two additional cephalopod species were examined in order to obtain comparative data. The presence of distinct sculpture patterns in coeval shallow‐ and deeper‐water assemblages suggests limited migration between them and consequently reflects some degree of territoriality in Devonian nautiloids. New data on early shell development in P. proximum are presented. During the Chote? Event, rutoceratoid generic diversity dropped dramatically, one family became extinct and the Early Devonian diversification of the group came to an end. The recovery of nautiloid faunas was slower than that of other cephalopods and associated, unrelated invertebrates. The absence of change in abundance patterns between Pragian and late Emsian rutoceratoid faunas, i.e. prior to and subsequent to ammonoid radiation, suggests that the appearance and radiation of the latter group in the early Emsian did not affect the structure of nautiloid assemblages, i.e. these two clades did not occupy the same niches.