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Various gluten-related diseases (celiac disease, wheat allergy, gluten sensitivity) are known and their incidence is growing. Gluten is a specific type of plant storage protein that can impair the health of gluten-prone persons following consumption, depending on the origin. The most severe effects are induced by wheat, barley, and rye. The only treatment is based on the absolute avoidance of those foods, as even traces might have severe effects on human well-being. With the goal of binding gluten impurities after ingestion, an in vitro setting was created. A special processed kind of zeolite, purified clinoptilolite-tuff (PCT), was implemented as an adsorber of gluten derived from different origins. Zeolites are known for their excellent sorption capacities and their applications in humans and animals have been studied for a long time. Tests were also performed in artificial gastric and intestinal fluids, and the adsorption capacity was determined via a certified validated method (ELISA). Depending on the kind of gluten source, 80–130 µg/mg of gluten were bound onto PCT. Hence, purified clinoptilolite-tuff, which was successfully tested for wheat, barley, and rye, proved to be suitable for the adsorption of gluten originating from different kinds of crops. This result might form the basis for an expedient human study in the future.

Clostridioides difficile (C. difficile) infection is a major public health problem worldwide. The current treatment of C. difficile-associated diarrhea relies on the use of antibacterial agents. However, recurrences are frequent. The main virulence factors of C. difficile are two secreted cytotoxic proteins toxin A and toxin B. Alternative research exploring toxin binding by resins found a reduced rate of recurrence by administration of tolevamer. Hence, binding of exotoxins may be useful in preventing a relapse provided that the adsorbent is innocuous. Here, we examined the toxin binding capacity of G-PUR®, a purified version of natural clinoptilolite-tuff. Our observations showed that the purified clinoptilolite-tuff adsorbed clinically relevant amounts of C. difficile toxins A and B in vitro and neutralized their action in a Caco-2 intestinal model. This conclusion is based on four independent sets of findings: G-PUR® abrogated toxin-induced (i) RAC1 glucosylation, (ii) redistribution of occludin, (iii) rarefaction of the brush border as visualized by scanning electron microscopy and (iv) breakdown of the epithelial barrier recorded by transepithelial electrical resistance monitoring. Finally, we confirmed that the epithelial monolayer tolerated G-PUR® over a wide range of particle densities. Our findings justify the further exploration of purified clinoptilolite-tuff as a safe agent in the treatment and/or prevention of C. difficile-associated diarrhea.

Summary Horizontal gene transfer (HGT) was observed by incubation of an amino acid-deficient strain of Escherichia coli (AB1157) with particles gained from an oligotrophic environment, when all deficiencies were restored with frequencies up to 1.94 × 10−5 and no preference for a single marker. Hence, the DNA transfer to the revertant cells was carried out by generalized transduction. Those particles display structural features of outer membrane vesicles (OMVs) but contain high amounts of DNA. Due to a process called serial transduction, the revertant’s particles were likewise transferring genetic information to deficient E. coli AB1157 cells. These results indicate a new way of HGT, in which mobilized DNA is transferred in particles from the donor to the recipient. Extracted OMV-associated DNA of known alpha-, and gamma-proteobacterials, Ahrensia kielensis and Pseudoalteromonas marina, respectively, was larger than 30 kbp with all sequences in single copy and identified as prokaryotic sequences. Inserted viral sequences were not found.

We studied Golgi apparatus disorganizations and reorganizations in human HepG2 hepatoblastoma cells by using the nonmetabolizable glucose analogue 2-deoxy-D-glucose (2DG) and analyzing the changes in Golgi stack architectures by 3D-electron tomography. Golgi stacks remodel in response to 2DG-treatment and are replaced by tubulo-glomerular Golgi bodies, from which mini-Golgi stacks emerge again after removal of 2DG. The Golgi stack changes correlate with the measured ATP-values. Our findings indicate that the classic Golgi stack architecture is impeded, while cells are under the influence of 2DG at constantly low ATP-levels, but the Golgi apparatus is maintained in forms of the Golgi bodies and Golgi stacks can be rebuilt as soon as 2DG is removed. The 3D-electron microscopic results highlight connecting regions that interlink membrane compartments in all phases of Golgi stack reorganizations and show that the compact Golgi bodies mainly consist of continuous intertwined tubules. Connections and continuities point to possible new transport pathways that could substitute for other modes of traffic. The changing architectures visualized in this work reflect Golgi stack dynamics that may be essential for basic cell physiologic and pathologic processes and help to learn, how cells respond to conditions of stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Antibodies against nuclear components (ANAs) occur in sera of approximately 50% of patients with primary biliary cirrhosis (PBC). By indirect immunofluorescence (IIF) ANA-positive PBC sera generate most frequently, homogeneous, speckled, centromere, and rim-like staining patterns. A perinuclear staining pattern is indicative for the reactivity of the sera with the components of the nuclear envelope. A substantial subset of PBC patients develops antibodies against constituents of the nuclear pore complexes (NPCs). These autoantibodies target two major autoantigens: gp210 glycoprotein and p62 kDa nucleoporin. Originally, a strong reaction of PBC with a 60 kDa protein of NPCs that was affinity purified on wheat-germ agglutinin (WGA)-Sepharose was described. Recently, using human recombinant p62 nucleoporin the identity of the reactivity was confirmed. In this work we compared by immunoprecipitation the reactivity of 20 PBC sera with the two recombinant autoantigens of the NPCs. Two out of 20 (10%) PBC sera precipitated recombinant gp210 glycoprotein and 11 out of 20 (55%) PBC sera reacted with p62 nucleoporin. These results evidence that anti-p62 antibodies occur more frequently than the autoantibodies against gp210 glycoprotein. Considering the recently reported clinical significance of ANAs in PBC, the prognostic value of the anti-NPC antibodies and their correlation with severity and progression of the disease is under evaluation.

Roscovitine (ROSC), a potent cyclin-dependent kinase inhibitor (CDI), inactivates cyclin-dependent kinase (CDK)2 resulting in the arrest of human MCF-7 breast cancer cells in G2 phase of the cell cycle. We have recently observed a strong activation of wild-type (wt) p53 protein in human MCF-7 breast cancer cells upon treatment with ROSC implicating that upregulated p53 might additionally modulate the primary action of ROSC. ROSC stabilized wt p53 protein resulting in a marked extension of its half-life. Since ROSC exhibits low cytotoxicity, it seems to upregulate p53 protein in a way different from DNA damage. ROSC induced phosphorylation of p53 protein at serine 46. Therefore, we decided to examine whether other anticancer drugs are also able to induce phosphorylation of wt p53 protein at serine 46. Exposure of MCF-7 cells to doxorubicin (DOX) at doses inducing a strong G2 arrest resulted in a weak upregulation of p53. No site-specific phosphorylation of p53 at serine 46 was detected. These results indicate that p53 activation is dispensable for DOX-induced G2 arrest. Moreover, the pattern of p53 phosphorylation strongly depends on the type of the stimulating factor.

We have recently observed activation of wild-type (wt) p53 protein in human MCF-7 breast cancer cells upon treatment with roscovitine (ROSC), a potent cyclin-dependent kinase inhibitor. It has been previously suggested that ROSC repressed transcription of Mdm-2, a negative p53 regulator, and that the lack of Mdm-2 contributes to the ROSC-induced upregulation of p53 protein. Therefore, we decided to see whether the prevention of p53 degradation by proteasome inhibitors will mimic the effects generated by ROSC. Exposure of human MCF-7 cells to different proteasome inhibitors resulted in a time-dependent increase of p53. However, unlike ROSC, they failed to modify p53 protein at Ser46 and to induce p53AIP1 protein. Moreover, whereas ROSC arrested MCF-7 cells in the G2-phase of the cell cycle, proteasome inhibitors blocked cells primarily in the S-phase, presumably because of the prevention of cyclin degradation. Our results indicate that prevention of p53 degradation by proteasome inhibitors does not mimic the action of ROSC.

Patients with primary biliary cirrhosis (PBC) generate a variety of autoantibodies, which are primarily directed against mitochondrial antigens (AMA). However, a subgroup of patient sera are also positive for antibodies to nuclear components (ANAs). At indirect immunofluorescence (IIF), PBC sera mostly produce homogeneous, nuclear dot, speckled, centromere, or rim-like patterns. During the last two decades, a number of nuclear structures have been recognized as specific targets of ANA in PBC. These include Sp100 and promyelocytic leukemia proteins, which generate a nuclear dot IIF pattern, and two components of the nuclear pore complex specifically associated with a perinuclear pattern (i.e., gp210 and p62). In recent years, the clinical significance of ANA in PBC has been widely investigated and data indicate that, unlike AMAs, PBC-specific ANAs correlate with disease severity and may therefore be a marker of poor prognosis.

The classic Golgi apparatus organization, an arrangement of highly ordered cisternal stacks with tubular-vesicular membrane specializations on both sides, is the functional image of a continuous flow of contents and membranes with input, metabolization, and output in a dynamic steady state. In response to treatment with 2-deoxy-D-glucose (2-DG), which lowers the cellular ATP level by about 70% within minutes, this organization is rapidly replaced by tubular-glomerular membrane convolutes described as Golgi networks and bodies. 2-DG is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis, which has become attractive in the context of therapeutic approaches for several kinds of tumors specifically targeting glycolysis in cancer. With the question of whether the functions of the Golgi apparatus in lipid synthesis would be influenced by the 2-DG-induced Golgi apparatus reorganization, we focused on lipid metabolism within the Golgi bodies. For this, we applied a fluorophore-labeled short-chain ceramide (BODIPY-Cer) in various combinations with 2-DG treatment to HepG2 cell cultures and followed uptake, enrichment and metabolization to higher ordered lipids. The cellular ATP status in each experiment was controlled with a bioluminescence assay, and the response of the Golgi apparatus was tracked by immunostaining of the trans-Golgi network protein TGN46. For electron microscopy, the fluorescent BODIPY-Cer signals were converted into electron-dense precipitates by photooxidation of diaminobenzidine (DAB); DAB precipitates labeled trans-Golgi areas in control cultures but also compartments at the periphery of the Golgi bodies formed in response to 2-DG treatment, thus indicating that concentration of ceramide takes place in spite of the Golgi apparatus reorganization. Lipid analyses by thin-layer chromatography (TLC) performed in parallel showed that BODIPY-Cer is not only concentrated in compartments of the 2-DG-induced Golgi bodies but is partly metabolized to BODIPY-sphingomyelin. Both, uptake and condensation of BODIPY-Cer and its conversion to complex lipids indicate that functions of the Golgi apparatus in the cellular lipid metabolism persist although the classic Golgi apparatus organization is abolished.

Detailed insight into the fine structure and 3D-architecture of the complex and dynamic compartments of the endocytic system is essential for a morpho-functional analysis of retrograde traffic from the cell surface to different intracellular destinations. Here, we describe a cytochemical approach for electron microscopic exploration of endocytic pathways with the use of wheat germ agglutinin (WGA) in combination with either conventional chemical fixation or ultrafast physical fixation of the cells by high pressure-freezing. Horseradish peroxidase-labeled WGA endocytozed by human hepatoma cells for various periods of time served as a marker. Its intracellular routes were visualized by means of diaminobenzidine oxidation either done conventionally after chemical fixation or in living cells prior to physical fixation. The latter protocol permits the combination of peroxidase-catalyzed cytochemistry with high pressure-freezing (HPF), which is state of the art for ultrastructural studies of complex and dynamic organelles at high spatial and temporal resolutions. The technique yields distinct cytochemical reactions and excellently preserved fine structures well qualified for detailed electron microscopic and 3D-studies of the complex endocytic architectures.

Correlative microscopic approaches combine the advantages of both light and electron microscopy. Here we show a correlative approach that uses the photooxidation capacity of fluorescent dyes. Through illumination with high energetic light, the chromogen diaminobenzidine is oxidized and stable deposits are formed at the sites of the former fluorescent signals, which after osmification are then visible in the electron microscope. The potential of the method is illustrated by tracing the endocytic pathway of three different ligands: the lipid ceramide, high density lipoproteins, and the lectin wheat germ agglutinin. The ligands were labeled either with BODIPY or Alexa dyes. Following cell surface binding, uptake, and time-dependent intracellular progression, the route taken by these molecules together with the organelles that have been visited is characterized. Correlative microscopic data are recorded at various levels. First, by fluorescence and phase contrast illumination with the light microscope, followed by the analysis of semithin sections after photooxidation, and finally of thin sections at the ultrastructural level.

Detailed insight into the fine structure and 3D-architecture of the complex and dynamic compartments of the endocytic system is essential for a morpho-functional analysis of retrograde traffic from the cell surface to different intracellular destinations. Here, we describe a cytochemical approach for electron microscopic exploration of endocytic pathways with the use of wheat germ agglutinin (WGA) in combination with either conventional chemical fixation or ultrafast physical fixation of the cells by high pressure-freezing. Horseradish peroxidase-labeled WGA endocytozed by human hepatoma cells for various periods of time served as a marker. Its intracellular routes were visualized by means of diaminobenzidine oxidation either done conventionally after chemical fixation or in living cells prior to physical fixation. The latter protocol permits the combination of peroxidase-catalyzed cytochemistry with high pressure-freezing (HPF), which is state of the art for ultrastructural studies of complex and dynamic organelles at high spatial and temporal resolutions. The technique yields distinct cytochemical reactions and excellently preserved fine structures well qualified for detailed electron microscopic and 3D-studies of the complex endocytic architectures.

Correlative microscopic approaches combine the advantages of both light and electron microscopy. Here we show a correlative approach that uses the photooxidation capacity of fluorescent dyes. Through illumination with high energetic light, the chromogen diaminobenzidine is oxidized and stable deposits are formed at the sites of the former fluorescent signals, which after osmification are then visible in the electron microscope. The potential of the method is illustrated by tracing the endocytic pathway of three different ligands: the lipid ceramide, high density lipoproteins, and the lectin wheat germ agglutinin. The ligands were labeled either with BODIPY or Alexa dyes. Following cell surface binding, uptake, and time-dependent intracellular progression, the route taken by these molecules together with the organelles that have been visited is characterized. Correlative microscopic data are recorded at various levels. First, by fluorescence and phase contrast illumination with the light microscope, followed by the analysis of semithin sections after photooxidation, and finally of thin sections at the ultrastructural level.

In this study, the ceramide-enriched trans-Golgi compartments representing sites of synthesis of sphingomyelin and higher organized lipids were visualized in control and ATP-depleted hepatoma and endothelial cells using internalization of BODIPY-ceramide and the diaminobenzidine photooxidation method for combined light-electron microscopical exploration. Metabolic stress induced by lowering the cellular ATP-levels leads to reorganizations of the Golgi apparatus and the appearance of tubulo-glomerular bodies and networks. The results obtained with three different protocols, in which BODIPY-ceramide either was applied prior to, concomitantly with, or after ATP-depletion, revealed that the ceramide-enriched compartments reorganize together with other parts of the Golgi apparatus under these conditions. They were found closely associated with and integrated in the tubulo-glomerular bodies formed in response to ATP-depletion. This is in line with the changes of the staining patterns obtained with the Helix pomatia lectin and the GM130 and TGN46 immuno-reactions occurring in response to ATP-depletion and is confirmed by 3D electron tomography. The 3D reconstructions underlined the glomerular character of the reorganized Golgi apparatus and demonstrated continuities of ceramide positive and negative parts. Most interestingly, BODIPY-ceramide becomes concentrated in compartments of the tubulo-glomerular Golgi bodies, even though the reorganization took place before BODIPY-ceramide administration. This indicates maintained functionalities although the regular Golgi stack organization is abolished; the results provide novel insights into Golgi structure–function relationships, which might be relevant for cells affected by metabolic stress.

Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclear pore complexes (NPCs), purified by affinity chromatography on wheat-germ agglutinin (WGA)-Sepharose, was previously detected. Recently, clinical significance of the anti-NPC antibodies in PBC became evident. In the light of recent reports, indicating the correlation of the anti-NPC antibodies with severity and progression of the disease, the characterization of the reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibody detection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinant p62 protein and validated an immunoprecipitation assay for the detection of anti-p62. We also demonstrated that the generated human recombinant p62 nucleoporin was modified by N-acetylglucosamine residues. More than 50% of tested PBC sera precipitated 35S-radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinant antigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence of anti-p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer of autoantibodies in p62-positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant protein completely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodies reacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigen source for future evaluations of the clinical role of anti-p62 in PBC. J. Cell. Biochem. 104: 27–37, 2008. © 2007 Wiley-Liss, Inc.

Human MCF-7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase-3 activity. We reported recently that roscovitine (ROSC), a potent cyclin-dependent kinase 2 inhibitor, arrests human MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF-7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time- and dose-dependent manner. The p53 level increased despite upregulation of Hdm-2 protein and was attributable to the site-specific phosphorylation at Ser-46. The p53 protein phosphorylated at serine 46 causes the up-regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC-induced p53 activation. Exposure of MCF-7 cells to ROSC activated homeodomain-intereacting protein kinase-2 (HIPK2). The overexpression of wild-type but not kinase inactive HIPK2 increased the basal and ROSC-induced level of p53 phosphorylation at Ser-46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC-induced P-Ser-46-p53, thereby stabilizing wt p53 and increasing the efficacy of drug-induced apoptosis in MCF-7 cells. These results identify HIPK2 as a component of the ROSC-induced signaling pathway leading to the stabilization and activation of wt p53 protein.

Estrogens play an important role in the growth and terminal differentiation of the mammary gland. Prolonged exposure to estrogens seems to predispose women to breast cancer. It recently became evident that not only the intrinsic hormonal status but also external factors such as the occurrence of pharmaceuticals and chemicals with hormone activity in the environment may put women at greater risk of developing breast cancer. We focused on the interference of endocrine disruptors in breast cancer therapy. We observed that phenol red added to the culture medium strongly promoted the cell proliferation and cell cycle progression of human cells expressing the estrogen receptor, and affected their susceptibility to chemotherapy.

We reported recently that roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrested human MCF-7 breast cancer cells in G2 phase of the cell cycle and concomitantly induced apoptosis. On the other hand, ROSC-induced G1 arrest observed by another group has not been accompanied by apoptosis. Therefore, we decided to prove to which extent components of tissue culture media could affect the primary action of ROSC. For this purpose we compared the efficacy of the ROSC treatment on MCF-7 cells cultivated in medium with and without phenol red. The kinetics of MCF-7 cell proliferation strongly depended on the presence of phenol red that has been recognized previously as a weak estrogen. Exposure of MCF-7 cells cultivated in phenol red-deprived medium to ROSC resulted in a strong G2 arrest and apoptosis. However, the anti-proliferative and pro-apoptotic action of ROSC was strongly diminished in cells maintained in medium containing phenol red. The ratio of the G2 cell population after 12 h ROSC was reduced by approximately 20% in the latter and correlated with the lack of CDK2 inactivation. Moreover, the kinetics of ROSC-induced apoptosis was delayed in the presence of phenol red. These results clearly evidence that the efficacy of the therapy of ER-positive breast cancers by CDK inhibitors is diminished in the presence of estrogen-mimicking compounds and indicate that phytoestrogens and xenoestrogens could interfere with the therapy. Therefore, the exposure of cancer patients to the estrogen mimics should be avoided at least during chemotherapy by CDK inhibitors.

The efficacy of distinct anti-cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti-proliferative and pro-apoptotic action of a number of anti-cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti-proliferative and pro-apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose- and time-dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases-3/7 in two separate microtiter plates using the CellTiter-Glo Luminescent Cell Viability Assay and the Caspase-Glo 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter-Blue Cell Viability Assay and Caspase-Glo 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases-3/7. The new multiplexing assay offers a range of benefits over standard assays.