Correction: A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

Correction for 'A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags' by B. Söveges, et al., Org. Biomol. Chem., 2016, 14, 6071-6078.

A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

The linker region of breast cancer resistance protein ABCG2 is critical for coupling of ATP-dependent drug transport.

The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an α-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux.

Parallel functional and immunological detection of human multidrug resistance proteins, P-glycoprotein and MRP1.

The proper assessment of the expression and drug extrusion activity of multidrug resistance proteins in various tumor cells is a challenging clinical laboratory problem. Recently, we have introduced a fluorescent dye (calcein) accumulation assay for the estimation of the functional expression of both P-glycoprotein (MDR1) and the multidrug resistance-associated protein (MRP1). Since both MDR1 and MRP1 decrease the intracellular accumulation of the fluorescent free calcein, by applying appropriate inhibitors of MDR1 and MRP1, the transport activity of these proteins could be quantitatively and selectively estimated in fluorometry or flow-cytometry assays. In the present work single-cell fluorescence digital imaging has been applied to characterize the kinetics and inhibitor-sensitivity of calcein accumulation in a mixture of HL60 MRP1 and NIH 3T3 MDR1 cells. Subsequent immunofluorescence labeling was performed by the anti-MDR1 monoclonal antibody (mAb) UIC2 in the same cell population. We report that the double labeling approach, based on the single cell calcein accumulation assay and an immunofluorescence detection, provides good sensitivity and selectivity for the simultaneous functional and immunological detection of cellular MDR1 and MRP1.

Membrane topology and glycosylation of the human multidrug resistance-associated protein.

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.

Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells.

In this paper we demonstrate that the expression of the multidrug resistance-associated protein (MRP) in a variety of intact human tumour cells results in the ATP-dependent, mutually exclusive extrusion of both the acetoxymethyl ester and the free anion forms of the fluorescent dye calcein, as well as that of a fluorescent pyrenemaleimide-glutathione conjugate. The MRP-dependent transport of all these three model compounds closely correlates with the expression level of MRP and is cross-inhibited by hydrophobic anticancer drugs, by reversing agents for MDR1, and also by compounds not influencing MDR1, such as hydrophobic anions, alkylating agents, and inhibitors of organic anion transporters. Cellular glutathione depletion affects neither the MRP-dependent extrusion of calcein AM or free calcein, nor its modulation by most hydrophobic or anionic compounds, although eliminating the cross-inhibitory effect of glutathione conjugates. These results suggest that the outward pumping of both hydrophobic uncharged and water-soluble anionic compounds, including glutathione conjugates, is an inherent property of MRP, and offer sensitive methods for the functional diagnostics of this transport protein as well as for the rapid screening of drug-resistance modulating agents.

[Relationship between the management of the placental phase and the frequency of indication for uterine intervention in the puerperium]. / Osszefüggés a lepényi szak vezetésének módja és a gyermekágyban szükséges méhmütétek gyakorisága között.

Authors examined the frequency of operative intervention in the uterine cavity, becoming inevitably necessary in late puerperium, in 28,835 mothers divided in 3 groups. In group A the controlling of the placental phase was realised in conservative, exspective way; manual or instrumental intervention in the uterine cavity was applied in the case of suspicion of placental remains. In group B the control of placental phase was performed by active procedure. In this group, as well in group A the uterine cavity was controlled only in the case of suspicion of placental remains. In group C the placental phase was effectuated by active procedure. Immediately in postplacentar period the uterine cavity was controlled with curettage. The numerical data of inevitably necessary operative intervention touching the uterus in late puerperium, were in group A the most favourable.

[Cytological analysis of peritoneal lavage fluid in patients during operation for cancer of the myometrium]. / Kismedencei mosófolyadék cytologiai vizsgálata méhtestrák mütéti kezelése során.

In this study the authors present their findings after the cytological examination of the peritoneal wash at the primary operation of 115 patients suffering from I-IV stages of carcinoma corporis uteri. They found tumor cells in 14 cases and suspicious ones in 2 cases out of 93 cases of stage I. Besides post-operative vaginal radiotherapy they applied percutan radiotherapy and a long-term gestogen and cytostatic treatment in case of the patients with tumor cells in the peritoneal wash.

[Current problems and complications of extensive uterine and pelvic lymph node excision for cancer of the uterus based on a 25-year case load]. / Méhrák miatt végzett kiterjesztett méh- és kismedencei nyirokcsomó eltávolítás egyes aktuális kérdései és szövödményei osztályunk 25 éves anyagában.

The authors studied the complications and other current problems of surgery of 212 cases subjected to Wertheim-Okabayashi operation. Of them 118 were cervix cancer in stage I and 90 in stage II and 4 were cancer of the uterus body in stage II. Two cases of operation death, 1 case of ureter injury and 1 case of large vessel injury occurred. Four ureteral and 1 late rectal-vaginal fistula developed. Difficulty of urination was observed in 49 cases. Those who consider surgical monotherapy best, hold the postoperative percutaneous irradiation necessary only in cases of metastases of the lymph nodes.

Effects of endogenous and tobacco-related amines and nitrosamines on cell growth and morphology of a cell line derived from a human neuroendocrine lung cancer.

This report is part of a comprehensive research programme to elucidate the role of physiological functions and pathways of pulmonary neuroendocrine cells in the cascade of events that lead to the development of neuroendocrine lung cancer. In this study, a well differentiated neuroendocrine cell line (NCI-H727) derived from a human lung carcinoid was used to investigate the ability of endogenous and tobacco-related amines to stimulate cell proliferation in neuroendocrine tumour cells. Cell line NCI-H727 was exposed in vitro to the endogenous amine serotonin and its precursor 5-hydroxytryptophan (5-HTP), to the tobacco-related amine nicotine, and the tobacco-related nitrosamines N-nitrosodiethylamine (DEN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The experiments were conducted in both 8% CO(2)-92% air and in 5% CO(2)-95% air. The effect on growth kinetics and ultrastructure of these conditions were studied. All amines had a dose-dependent stimulating effect on cell growth when tested in 8% CO(2)-92% air with nicotine exerting the strongest effect. No such effect was observed in 5% CO(2)-95% air and the control cells did not grow under these conditions. DEN, 5-HTP, serotonin and NNK all caused dedifferentiation of cytoplasmic organelles. Nicotine caused a change in size and ultrastructure of dense-cored granules suggestive of a change in the nature of stored neuroendocrine material.

Inhibition of N-diethylnitrosamine metabolism by human lung cancer cell lines with features of well differentiated pulmonary endocrine cells.

Cell lines derived from a human pulmonary carcinoid tumor (NCI-H727) and from a human pulmonary large cell carcinoma (NCI-H460) were investigated by transmission electron microscopy. Both cell lines, at early in vitro passage, demonstrated ultrastructural features of well differentiated pulmonary endocrine cells. Line NCI-H727 had more endoplasmic reticulum than line NCI-H460 and demonstrated L-dopa decarboxylase activity as well as production of calcitonin and bombesin. Because of their ultrastructural resemblance with normal pulmonary endocrine cells, these cell lines were used to test the theory derived from experiments in hamsters that human pulmonary endocrine cells can metabolize N-nitrosodiethylamine (DEN). The cells were incubated in vitro with [14C]DEN. Metabolism was assessed by 14CO2 production. Both cell lines metabolized DEN to a much greater extent than previously investigated human lung cancer cell lines of Clara cell and alveolar type II cell morphology. In keeping with its abundant endoplasmic reticulum, line NCI-H727 yielded 14CO2 in the 300 nM range, whereas NCI-H460 was less active. Metabolism was time dependent. Preincubation with various enzyme inhibitors yielded a highly significant inhibition of DEN metabolism with the two inhibitors of the fatty acid cyclooxygenase component of prostaglandin endoperoxide synthetase, aspirin and indomethacin. Inhibitors of cytochrome P-450 (CO, piperoxylbutoxide) did not inhibit DEN metabolism. Preincubation with sinigrin yielded similar negative results as CO and piperonylbutoxide. Our data are in support of the theory that human pulmonary endocrine cells can metabolize nitrosamines. Moreover, the experiments with enzyme inhibitors suggest that in this cell type such metabolism is largely dependent on prostaglandin endoperoxide synthetase.

Cell type-specific differences in metabolic activation of N-nitrosodiethylamine by human lung cancer cell lines.

The metabolism of N-nitrosodiethylamine (NDEA) and its modulation by inhibitors of cytochrome P450 and prostaglandin H synthetase enzymes was investigated in seven well-differentiated early-passage human lung cancer cell lines. NDEA metabolism was assessed by covalent binding and evolution of carbon dioxide. Morphological diagnosis of cell lines was done by light and electron microscopy. Two cell lines (NCI-H69, NCI-H128) with characteristics of small-cell cancer did not metabolize NDEA. Two cells lines (NCI-H322) with features of adenocarcinoma, comprised of Clara cells, and (NCI-H727), with features of pulmonary endocrine cells, were more potent than all other cell lines in metabolizing NDEA. A cell line divided from an adenocarcinoma but comprised of alveolar type-II cells (NCI-H358) metabolized NDEA predominantly via prostaglandin H synthetase. Similarly, several cell lines with features of well-differentiated pulmonary endocrine cells (NCI-H727, NCI-H460) metabolized NDEA via prostaglandin H synthetase, while the cell line comprised of Clara cells (NCI-H322) activated the nitrosamine by cytochrome P450 but not by prostaglandin H synthetase. Although cancer cells may react differently from normal cells to xenobiotics, our data provide substantial evidence for the hypothesis that--as in the hamster--Clara cells and pulmonary endocrine cells are potential major targets of NDEA carcinogenesis in human lung. It is of particular interest that different cell types activate the nitrosamine via different enzyme systems.