Structural reorganization of CuO/Cu2[Fe(CN)6] nanocomposite: characterization and electrocatalytic effect for the hydrogen peroxide reduction.

We report the study on the formation of the Cu2[Fe(CN)6] nanocomposite, which was obtained from copper oxide nanoparticles (CuO NPs) and Prussian Blue precursors. UV-vis analysis indicated that Cu2+ ions are released from CuO NPs, while Fe3+ ions are adsorbed onto the structure of CuO due to a sharp increase in zeta potential (from -30 to 0 mV) after the formation of the Cu2[Fe(CN)6]. Moreover, energy dispersive spectroscopy confirmed that Fe3+ ions are trapped in the CuO NPs structure. The CuO/Cu2[Fe(CN)6] nanocomposite exhibited the monoclinic and face-centered cubic phases that correspond to the CuO and Cu2[Fe(CN)6] components. Cyclic voltammetry (CV) for the Nanocomposite modified electrode revealed two well-defined redox couples at -0.073 ((E1/2)1) and 0.665 mV ((E1/2)2), attributed to the conversion of Cu2+ to Cu+ and CuFe2+ CuFe3+ pairs, respectively, which is similar to those in the CuO and Cu2[Fe(CN)6]components. Furthermore, the catalytic activity of the nanocomposite towards hydrogen was investigated through CV, where the reduction of H2O2 led to increased currents for the electrochemical process associated with the first redox pair. In contrast, for isolated materials (CuO NPs and Cu2[Fe(CN)6]), there was no significant increase in the current associated with either redox pair.

Molecular interactions and structure of a supramolecular arrangement of glucose oxidase and palladium nanoparticles.

This paper presents studies about the molecular interactions and redox processes involved in the formation of palladium nanoparticles associated to glucose oxidase (GOx-PdNPs) in a supramolecular arrangement. The synthesis occurs in two steps, the Pd reduction and the formation of the 80 nm sized supramolecular aggregates containing multiples units of GOx associated to 3.5 nm sized PdNPs. During synthesis, GOx molecules interact with Pd salt leading to metal ion and FAD reduction probably via the thiol group of the cysteine 521 residue. For the growing of PdNPs, formic acid was necessary as a co-adjuvant reducing agent. Besides the contribution for the redox processes, GOx is also necessary for the NP stability preventing the formation of precipitates resulted from uncontrolled growing of NPs Cyclic voltammetry of the GOx-PdNPs demonstrated electroactivity of the bionanocomposite immobilized on ITO (indium-tin oxide) electrode surface and also the NP is partially blocked due to strong interaction GOx and the surface of PdNPs. Vibrational spectroscopy (FTIR) showed that significant structural changes occurred in GOx after the association to PdNP. These mechanistics and structural studies can contribute for modulation of bionanocomposites properties.

Presence of regulatory sequences within intron 4 of human and murine c-myb genes.

The molecular mechanisms that modulate c-myb mRNA transcription in hematopoietic cells appear to involve intron regulatory sequences. We have characterized the fourth of ten introns from both human and murine c-myb genes in regard to nucleotide sequence and specific protein binding. For this approach complete genomic c-myb intron 4 fragments were isolated from mouse and human DNA using PCR amplification with flanking exon-primers derived from the mouse gene. Comparison of the obtained sequences revealed strong homology between the two species. Using crude nuclear protein extracts from mouse and human myb expressing cells (70Z/3B; Molt4) and gel shift experiments we found specific protein interaction for both introns and to determine the protein binding site in detail, we performed DNase I footprinting. Our results indicate that the binding factor is absent in control cell lines without c-myb transcriptional activity, suggesting a possible positive regulatory function of the DNA-protein complex. To confirm these findings we introduced the human c-myb intron 4 DNA sequence into the EcoRI site of the pCAT-Promoter plasmid and transfected Molt4 cells with this chimeric construct. The transient expression studies revealed that intron 4 sequences possess enhancer activity. Thus, we have demonstrated that intron 4 sequences can be important for the regulation of c-myb proto-oncogene expression.

A rapid protocol for the purification of mitochondrial DNA suitable for studying restriction fragment length polymorphisms.

When analyzing mitochondrial DNA (mtDNA) from various normal and malignant human tissues, it became necessary to enhance mtDNA isolation for improved yields and quality. The method described here consists of rapid and simple-to-perform steps, avoiding complicated instrumentation. It was designed for preparation of undegraded mtDNA and is highly useful when limited amounts of tissues, cells and unique biopsies of tumors (fresh or frozen) are available. The resulting mtDNA is sufficiently pure for restriction analysis, subcloning, labeling and various types of hybridization. Using Sau3A and MspI, restriction analysis revealed new restriction-fragment length polymorphisms for Caucasians, independent of the DNA source, and hence excluding tissue-specific DNA modifications.

Cross-hybridization between the avian myeloblastosis oncogene and eukaryotic 28S ribosomal RNA.

In Northern blots, avian myeloblastosis (myb) oncogene probes (genomic or cDNA) cross-hybridize to the 28S rRNA band mimicking a myb-specific transcript. A misinterpretation of the hybridization data can be avoided by using an oligodeoxyribonucleotide probe.

Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture.

Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated on histidine residues, however, only the B isoform appeared to be serine phosphorylated.

Expression of the breast cancer associated gene pS2 and the pancreatic spasmolytic polypeptide gene (hSP) in diffuse type of stomach carcinoma.

Expression of the pancreatic spasmolytic peptide (hSP) gene and pS2 (a gene isolated from oestrogen-induced breast carcinoma cells) were analysed in 36 samples of human stomach carcinoma. 17 tumours were investigated at the RNA level (by northern blots) as well as at the gene product level (by immunochemistry). Since pS2 had been shown to be expressed in normal stomach mucosa its activity in carcinoma samples was expected. Surprisingly, strong pS2 immunoreactivity was noted in the diffuse carcinoma type, whereas the intestinal type displayed weak reactivity. The tumour samples showing strong immunostaining expressed the regular 0.6 kb pS2 RNA band and weak staining was paralleled by aberrant transcripts. Additionally, only in tumour samples with regular pS2 transcription was the typical 0.7 kb hSP RNA band seen; samples with aberrant pS2 bands did not express hSP at all. This is the first demonstration of hSP gene activity in a human tumour.

Nonradioactive southwestern analysis using chemiluminescent detection.

Replacing radioactively labeled probes by nonradioactive ones and detection by chemiluminescence instead of colorimetry allows a nonhazardous handling and offers the possibility of easily reprobing filters in Southwestern analysis. Using the described procedure, we were able to determine the molecular weight of DNA-binding proteins and achieve a high signal-to-noise ratio.

The cellular myb oncogene is amplified, rearranged and activated in human glioblastoma cell lines.

The human c-myb gene which encodes a DNA binding protein and which is rarely amplified in neoplastic cells was found to be altered in four human glioblastoma cell lines. It exists in multiple copies in 2 out of 4 cases studied. The degree of amplification as determined by densitometry was about 10-fold, a rearrangement within the coding region and an enhanced gene activity of c-myb were noted. The observation of c-myb oncogene amplification and activity in glioblastoma cell lines presents the first report of this effect in human brain tumor cells.

c-myb intron I protein binding and association with transcriptional activity in leukemic cells.

Specific binding of nuclear proteins to the region of transcriptional attenuation has been shown to modulate the expression of c-myb, a nuclear proto-oncogene preferentially expressed in lympho-hematopoietic cells. Here, it plays an important role in processes of differentiation and proliferation. The mechanism that regulates c-myb expression is not yet fully understood. The block of transcriptional elongation which has been mapped to a 1 kb region within murine intron 1 may represent one regulatory pathway. The DNA sequences containing the transcriptional pause site are well conserved between murine and human species, thus Implying similar transcription-control strategies. We compared the binding potential of nuclear extracts (from human fibroblasts and MOLT4 as well as murine NIH3T3- and 70Z/3B- cell lines) to oligonucleotide sequences previously shown to be target binding sites in the murine system. One complex containing a 70 D protein was found to be associated specifically with transcriptionally active leukemia cells. We performed transient expression studies with a CAT reporter construct containing this putative enhancer sequence and yielded significant CAT activity. We identified further a putative 20 kD repressor protein in transcriptionally silent cells and demonstrated that c-Jun is part of an ubiquitously present complex. Our results confirm the participation of intron 1 in transcriptional regulation of the c-myb gene (in mouse and human) and implicate multiple and complex regulatory mechanisms of activation during myelomonocytic differentiation and leukemic cell growth control.

Constitutive C-myc overexpression and p1/p2 promoter shift in a small-cell lung-cancer cell-line.

A number of genes display altered activity in human small cell lung cancer (SCLC), in particular members of the myc family (c-myc, L-myc, N-myc) that frequently are expressed at high levels. GLC4, a cell line established at the State University of Groningen from a SCLC, contains a 30 fold amplified c-myc gene and the cells show a high steady state c-myc transcription rate. The amplified c-myc copies of this cell line are not grossly rearranged and contain the gene's known major EcoRI (12 kb) fragment. To investigate in detail the mechanism(s) responsible for transcriptional activation we analyzed the c-myc promoter region. Whereas point mutations, which often occur in a mutational hot spot region in Burkitt's lymphoma, were not detected, c-myc transcription in GLC4 initiates mainly from P1 instead of the P2 promoter. This promoter shift seems to overcome transcript termination at a transcriptional pause site described for Burkitt's lymphoma c-myc sequence. Our data suggest that the shift of promoter usage - together with gene amplification - plays a likely role in c-myc transcriptional activation in GLC4 SCLC cells.

Regional loss of the y-chromosome in urothelial carcinoma.

Specific chromosomal losses have been reported for various human tumors. We have now investigated ten cases of urothelial carcinoma and observed genomic alterations by a new method allowing detection of chromosomal losses directly in the tissue section. In 6 out of 8 male carcinomas, the Y-chromosome was lost either in single cells and isolated areas or in extended regions of the tumor sample. Presence of chromosome 1 served as an internal control. This new in situ method allows studies of chromosomal alterations in relation to their tumor topology and the observations constitute the first report on such localised tumor-specific genomic changes.

PCR expression analysis of the estrogen-inducible gene BCEI in gastrointestinal and other human tumors.

A polymerase chain reaction (PCR) assay was developed to test for tumor cell specific expression of the BCEI gene. This new marker gene, reported at first for human breast cancer, was found specifically active in various gastrointestinal carcinomas by previously applying immunohistochemistry and RNA (Northern blot) analysis. Presently, by using reverse transcription-PCR analysis, a series of primary tumor tissues and established tumor cell lines were tested for BCEI transcription. This approach was compared to immunostaining achieved by an antibody directed against the BCEI gene's product. The result demonstrate the superior sensitivity of PCR by indicating the gene's expression in cases where immunohistochemical testing remained negative.

Differential expression of the pS2 protein in the human prostate and prostate cancer: association with premalignant changes and neuroendocrine differentiation.

The distribution of the estrogen inducible pS2 protein was investigated in benign and malignant prostate tissue by the avidin-biotin complex method. Prostate tissue obtained from 20 patients without clinical and histological evidence of malignant disease consistently lacked pS2 immunoreactivity. Conversely, nonneoplastic tissue from 36 total prostatectomies with locally advanced prostate cancer showed a variable degree of pS2 reactivity in normal or hyperplastic glands and in prostatic intraepithelial neoplasia (PIN) adjacent to the cancerous lesions. This suggests that the pS2 gene expression detected in nonmalignant tissue may be related to early premalignant changes of prostate glands harboring significant carcinomas. In prostate cancer the pS2 protein was detected in close association with neuroendocrine (NE) differentiation as assessed by Chromogranin A (Chr A) immunoreactivity. Double labeling techniques showed that pS2 immunoreactivity recognizes both endocrine (Chr A-positive) and adjacent exocrine (Chr A-negative) cell types within NE foci. Whereas pS2 expression was consistently confined to NE differentiation in untreated tumors, carcinomas that relapsed after hormonal therapy showed increased pS2 immunoreactivity, even in the absence of NE features. The differential expression of the pS2 peptide in nonneoplastic tissue from patients with and without malignant disease indicates that pS2 immunohistochemistry may be useful in the diagnostic evaluation of negative biopsy specimens. Furthermore, the results suggest that the immunohistochemical spectrum of pS2 in prostate cancer may include endocrine differentiated and presumably related cell populations.

Increased amount of a mitochondria-associated 21 x 10(3) dalton protein in human gastrointestinal tumors.

Differences in the structure and number of mitochondria in tumor cells were found. Using isoelectrofocusing two-dimensional polyacrylamide gel electrophoresis which allows detection of alterations in the protein pattern of tumor mitochondria, we studied both quantitative and qualitative changes in the mitochondrial protein pattern of human gastrointestinal tumors and corresponding normal matrix tissues. One low molecular protein spot was found to be quantitatively changed in the tumors. The approximate molecular weight was 21 x 10(3) daltons and the pI value 5.7.

NM23-H1 and NM23-H2 gene expression in human renal tumors.

The two nm23 genes, nm23-H1 and nm23-H2, are implicated in the metastatic process and tumor progression in some human tumors. Until now no data exist about nm23 expression in the different types of human renal tumors. To investigate if the nm23 genes play a central role in the progression of renal tumors, we have examined nm23-H1 and nm23-H2 gene expression using Northern-blot analysis and immunohistochemistry. We analysed clear cell type RCC, chromophilic RCC, chromophobic RCC, collecting duct type RCC and renal oncocytomas. Our results indicate that the nm23 genes do not play a central role in the prognosis of renal cell carcinoma in the analysed tumors.

Overexpression of nm23-H4 RNA in colorectal and renal tumours.

A new member of the nm23 gene family, designated nm23-H4, was cloned recently. Nm23-H4 is a mitochondrial protein. To determine whether nm23-H4 could have a function in tumour progression like nm23-H1 or nm23-H2, we analysed nm23-H4 expression in 18 renal tumours and 42 colorectal carcinomas obtained from patients who underwent curative resection. As compared to the corresponding healthy tissue, 14 renal tumours and 41 colorectal carcinomas showed increased nm23-H4 mRNA expression. While the increase was only moderate in renal cell carcinoma, a strong overexpression of nm23-H4 was noted in most colorectal carcinomas, possibly indicating a role of this gene in tumour genesis. The nm23-H4 transcript levels did not correlate with tumour stage, grade of differentiation or lymph node involvement.

Tumor-specific methylation patterns of erbB2 (HER2/neu) sequences in gastro-intestinal cancer.

To determine whether the sporadically occurring amplification of the oncogene erbB2/HER2 in gastrointestinal carcinomas is associated with additional changes of this sequence, DNA from 17 colorectal and 5 stomach carcinomas was analyzed for copy number, sequence rearrangement and DNA methylation by Southern blot hybridization. Amplification was detected in two cases. By applying the isochizomers Hpall and Mspl we tested for alterations in the DNA methylation status. Whereas in colon tumors with non-amplified erbB2 this status was unchanged, one case with erbB2 amplification showed additional MspI bands indicating a methylation of the amplified gene sequences. In stomach carcinoma, however, we detected differences between tumor and mucosa samples but not between amplified and non-amplified tumor samples. Independent of the DNA methylation status, significant amounts of the erbB2 oncoprotein were detected in the cases with gene amplification; weaker immunostaining of erbB2 was also seen in a few additional tumors.

Evaluation of killed and modified live porcine rotavirus vaccines in cesarean derived colostrum deprived pigs.

Twenty-eight cesarean derived, colostrum deprived (CDCD) piglets were used to evaluate the efficacy of killed and modified live rotavirus (MLV) vaccines against challenge with virulent A-1 and A-2 rotaviruses. Two killed rotavirus vaccines were evaluated: an experimental vaccine and a commercially available vaccine. Efficacy parameters included: average daily weight gains, rotavirus shedding in feces, morbidity incidence and duration, and rotavirus serum antibody conversion post-vaccination and post-challenge. Piglets vaccinated orally/intramuscularly with the modified live vaccine were completely protected from A-1 and A-2 virulent rotavirus challenge. Nonvaccinated control piglets and piglets receiving killed rotavirus vaccines developed diarrhea, shed virus and exhibited reduced weight gains post-challenge. Only the MLV rotavirus vaccine was able to prevent virus shedding in feces after virulent challenge. Both controls and pigs which received killed vaccines intraperitoneally, orally or intramuscularly shed virus in the feces for 7 days post-challenge and virus peak titers approached 10(7) fluorescent antibody infectious dose (FAID)50/g feces. These studies clearly reflected the inability of killed rotavirus vaccines to induce active local immunity to rotaviral diarrhea in piglets.

Expression of the candidate tumor suppressor gene nm23 in the bronchial system of patients with squamous cell lung cancer.

OBJECTIVE: A number of oncogenes and tumor suppressor genes participating in tumorigenesis have been identified, one of them being nm23. The expression of the candidate tumor suppressor gene nm23 depends on the cell type of tumors. Both, reduced expression as well as overexpression of nm23 is associated with a high potential of malignancy. In a variety of tumor cell lines secretion of the nm23 protein can be detected. In an earlier investigation we showed, that the nm23 expression in squamous cell lung carcinoma is considerably elevated. In order to establish the potential diagnostic value of this finding we investigated the nm23 expression in healthy and diseased lungs in patients with squamous cell lung cancers. METHODS: We examined bronchial lavage samples of 20 patients with bronchogenic squamous cell carcinoma. The lavage was separately performed in the bronchus of the tumor bearing lobe and in the corresponding bronchus of the unaffected contralateral lung. RESULTS: Using Western blot analysis we found 2-7 fold elevated amount of nm23 protein in bronchial lavage of the tumor bearing lung in comparison to the healthy side. This finding was neither related to tumor stage nor to tumor location. Thus we have a strong hint that the nm23 protein is secreted by the bronchogenic squamous cell carcinoma. CONCLUSIONS: With respect to these results the proof of nm23 protein in bronchial lavage fluid might be of relevance to establish the diagnosis when pulmonary nodules of unknown etiology are found.