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Biomedical subjects

D Prochnow

Publications and source records attributed to D Prochnow.

11 recordsLinked to original sources
PubMed2005

Differentiation of prostate cancer from normal prostate tissue in an animal model: conventional MRI and dynamic contrast-enhanced MRI.

PURPOSE: To differentiate orthotopically implanted prostate cancer from normal prostate tissue using magnetic resonance imaging (MRI) and Gd-DTPA-BMA-enhanced dynamic MRI in the rat model. MATERIAL AND METHODS: Tumors were induced in 15 rats by orthotopic implantation of G subline Dunning rat prostatic tumor cells. MRI was performed 56 to 60 days after tumor cell implantation using T1-weighted spin-echo, T2-weighted turbo SE sequences, and a 2D FLASH sequence for the contrast medium based dynamic study. The interstitial leakage volume, normalized permeability and the permeability surface area product of tumor and healthy prostate were determined quantitatively using a pharmacokinetic model. The results were confirmed by histologic examination. RESULTS: Axial T2-weighted TSE images depicted low-intensity areas suspicious for tumor in all 15 animals. The mean tumor volume was 46.5 mm(3). In the dynamic study, the suspicious areas in all animals displayed faster and more pronounced signal enhancement than surrounding prostate tissue. The interstitial volume and the permeability surface area product of the tumors increased significantly by 420 % (p < 0.001) and 424 % (p < 0.001), respectively, compared to normal prostate tissue, while no significant difference was seen for normalized permeability alone. CONCLUSION: The results of the present study demonstrate that quantitative analysis of contrast-enhanced dynamic MRI data enables differentiation of small, slowly growing orthotopic prostate cancer from normal prostate tissue in the rat model.

Animals↗
PubMed1995

Histone-poly(A) hybrid molecules as tools to block nuclear pores.

Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histones, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 1:1 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histones covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins--presumably in the nuclear pore--with molecular weights of 110, 80, and 71.4 kDa.

Animals↗
PubMed1994

Efflux of RNA from resealed nuclear envelope ghosts.

mRNA translocation across the nuclear envelope and the appropriate signal-receptor interactions have been studied using resealed rat liver nuclear envelope ghosts (RNEG). We compared export kinetics of nonadenylated (tRNAs, histone-2 poly(A)- mRNA), and adenylated RNAs (poly(A)+ tRNAs, synthetic histone-2 poly(A) +mRNA, albumin mRNA, beta-globin poly(A) +mRNA and a total poly(A) + mRNA extract from rat liver cells). ATP-dependent export of mRNAs and of total poly(A)+ RNA was prevented by inhibitors of a nuclear envelope NTPase. All adenylated RNA species competed with each other for export, but nonadenylated RNAs did not. This indicates the existence of different translocation mechanisms for different RNA species with their appropriate nuclear envelope associated RNA receptors involved in export. The attachment of a poly(A)250 sequence at the 3'-end of tRNA or histone messenger masks the intrinsic RNA export signal of nonadenylated RNAs and results in efflux comparable to that of beta-globin poly(A)+ mRNA. The attachment on oligo(A)5 does not have any comparable effect of nonadenylated RNA translocation. Export of all polyadenylated RNAs from RNEGs is blocked by a monoclonal antibody, which is directed against an intranuclear envelope poly(A) binding protein. The results suggest that the pore complexes do not select RNAs for export to the cytoplasm and are therefore not responsible for nuclear restriction of mRNA precursors.

Adenosine Triphosphate↗
PubMed1994

Cross-linking of nucleic acids to proteins. Modified poly(A) as mRNA for Escherichia coli ribosomes.

Poly(adenylic acid) was modified by methylchlorotetrolic ester in a reproducible and defined content of the derivatized bases. The nucleic acid derivative is protein reactive and was coupled to 70S ribosomes from Escherichia coli, in order to identify proteins along the mRNA pathway. The binding of the label becomes specific under the direction of tRNA(Lys) and is then almost exclusively located on the small subunit. The proteins S1, S12, S18 and S21 were labeled, as shown by an antibody assay. The yield of the affinity label was 5.4%, as calculated from the labeled nucleic acid. This compares favourably with the yields from photolabile compounds.

Affinity Labels↗
PubMed1993

Immunological evidence for the localization of a 110 kDa poly(A) binding protein from rat liver in nuclear envelopes and its phosphorylation by protein kinase C.

We have purified a 110 kDa poly(A) binding protein (P110) from rat liver which is thought to be involved in mRNA translocation through the nuclear pores and have demonstrated its localisation in the nuclear envelope using polyclonal antibodies and confocal laser scanning microscopy. Although P110 was prepared from highly purified nuclear envelopes, the polyclonal antibodies raised against them bind to nucleo- and cytoplasmic structures to a minor extent, but not to nucleolar structures. P110 decays spontaneously into several fragments which are also recognized by the polyclonal antibodies. The 110 kDa polypeptide and its fragments were phosphorylated by a nuclear envelope kinase and this phosphorylation was inhibited by a monoclonal antibody against protein kinase C and by a specific protein kinase C inhibitor obtained from bovine brain. Scatchard analysis was used to determine the influence of protein kinase C activators and inhibitors on nuclear envelope protein phosphorylation and RNA binding. The data indicate a close association between the RNA translocation machinery (the 110 kDa protein) and protein kinase C within the nuclear envelope. We suggest that the fragmentation of P110 is triggered before or during mRNA export and is not due to nonspecific proteolysis.

Animals↗
PubMed1992

Covalent attachment of ribonucleic acids to proteins.

As a prerequisite for the synthesis of affinity labels, we describe methods to couple histones to ribonucleic acids. For the synthesis of these covalent hybrid molecules, we used a population of histones H1, H2A, H2B, H3, and H4 from calf thymus and polyadenylic acid with an average chain length of up to 260-280 bases, representing the size of poly(A)-tails from mature mRNAs. Three methods were investigated. (a) Poly(A) containing an 8-N3-A residue was cross-linked to histones by ultraviolet irradiation. (b) The 3'-end of the polynucleotide was connected to a mononucleotide containing an aliphatic amino group, and the resulting poly(A)-derivative was coupled to histones via derivation with a bromoacetyl group. (c) The 3'-end of the polynucleotide was oxidized with sodium periodate and bound covalently to an amino group of the polypeptide. To demonstrate the RNA content of the hybrid molecule, the poly(A) was removed with RNase T2.

Animals↗
PubMed1990

Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).

Animals↗
PubMed1987

Permeability measurements with closed vesicles from rat liver nuclear envelopes.

Closed nuclear envelope ghosts in the physiological orientation were prepared from rat liver and nuclei as previously described. Here we report transport measurements of various proteins and ribonucleic acids across the envelope of these vesicles. Histones were accumulated rapidly in the ghosts, in contrast to other, nonnuclear, proteins. Triton X-100 removal of the external nuclear membrane from loaded vesicles, as well as comparative studies with open vesicles, excluded the effects of external adsorption. The exchange rate of histones across the nuclear envelope is strongly depressed in the presence of GTP and GDP. The vesicles contain the translocation mechanism for poly(A)-containing RNA. The translocation of poly(A), messenger RNA, and ribosomal RNA was investigated after entrapment of these nucleic acids during the preparation of vesicles. Our data show that the complete export of only poly(A)-containing RNA from the vesicles is enhanced in the presence of 2 mM ATP. This RNA, as well as poly(A), is transported unidirectionally.

Animals↗
PubMedDate not supplied

17O NMR studies of low silicate zeolites.

Multiple-quantum magic-angle spinning and double-rotation NMR techniques were applied in the high field of 17.6 T to the study of oxygen-17-enriched zeolites A and LSX with the ratio Si/Al = 1. A monotonic correlation between the isotropic value of the chemical shift and the Si-O-Al bond angle alpha (taken from X-ray data) could be found. Hydration of the zeolites causes a downfield 17O NMR chemical shift of about 8 ppm with respect to the dehydrated zeolites. Ion exchange of the hydrated zeolites generates stronger chemical shift effects. The increase of the basicity of the oxygen framework of the zeolite LSX is reflected by a downfield shift of approx. 10 ppm going from the lithium to the cesium form, and the substitution of sodium by thallium in the zeolite A causes a shift of 34 ppm for the O3 signal. 17O DOR NMR spectra are superior to 17O 3QMAS NMR spectra, featuring a resolution increase by a factor of 2 and are about equal with respect to the sensitivity. The residual linewidths of the signals in the 17O DOR and 17O 5QMAS NMR spectra can be explained by a distribution of the Si-O-Al angles in the zeolites.

Magnetic Resonance Spectroscopy↗