Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.     

Cytokinin oxidase/cytokinin dehydrogenase assay: optimized procedures and applications

1H NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)3 at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M(r) metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz 1H NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO>3)3 was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results.     

Identifying metameric variation in extant hominoid and fossil hominid mandibular molars

Landmark data were collected from cross sections and occlusal images of mandibular molar crowns, and Euclidean distance matrix analysis (EDMA) was used to identify metameric morphological variation between the first and second mandibular molars of living taxa: Gorilla gorilla (n = 30), Pan troglodytes (n = 34), and Homo sapiens (n = 26). Two patterns of metameric variation were identified, one unique to humans and the other shared by chimpanzees and gorillas. In order to assess the utility of this type of analysis for the interpretation of the hominid fossil record, 19 mandibular molars from Sterkfontein Member 4, South Africa, were examined. The pattern of metameric variation of the Sterkfontein molars resembled that of the African great apes, and differed from the modern human pattern. These results demonstrate that data on metameric variation may provide information regarding function or developmental processes previously indiscernible from fossil material.     

Immunoprecipitation of DNA-protein complexes cross-linked by cis-diamminedichloroplatinum

For the study of in vitro and in vivo DNA-protein interactions, cross-linking reactions driven by UV or formaldehyde have been frequently used, followed by standard protocols of immunoprecipitation and analysis of the DNA isolated from the complexes. Here we present a basically modified method to analyze the DNA-protein cross-linked complexes obtained by an alternative cross-linking reagent. The innovations presented here include cross-linking by cis-diamminedichloroplatinum II, a fast method to isolate DNA-protein complexes using gel-filtration chromatography, and a modified procedure to obtain specific immunocomplexes that can be analyzed either for DNA or for protein content. The application of this method to two nuclear proteins from chicken liver nuclei is described.     

Development of reconstituted embryos derived from transgenic embryonic stem cell nuclei

This study was carried out to transform embryonic stem (ES) cells and to produce the reconstituted embryos derived from transgenic ES cell nuclei. Then, in vitro/vivo developmental potency of transgenic ES cells were compared to that of control ES cells (non-transgenic ES cells) in the reconstituted embryos. Unfertilized B6D2F1 ooplasm at metaphase II (M II) and two kinds of ES cell lines, 129SV and transgenic (tg) 129SV transformed by EGFP gene, were used as nuclear recipients and nuclear donors, respectively. The M II chromosome-spindle complex was aspirated into the pipette with a minimal volume of ooplasm. After enucleation, the ES cell nuclei was injected into the enucleated ooplasm directly. Then, reconstituted embryos were activated in SrCl2, and they were cultured in HTF medium. There was no difference of developmental rate between reconstituted embryos derived from the control (non-transgenic) and the tg ES cells. From this result, we indicated that transgenic ES cells might not change the property of peculiarity of the ES cell by gene transfer. The expression of GFP gene in the embryos was observed by fluorescence microscope at the 4-cell and more stage. As comparison with development of the embryos derived from the control and tg ES cells, the difference of the development could not be confirmed between the two cell groups. When the reconstituted embryos derived from the control and tg ES cells were transferred into oviduct or uterus of pseudopregnant females, fetuses were observed 13.5 days post coitum. However, in all fetuses, developmental arrest and regression were seen 19.5 days post coitum.     

Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS

Novel algorithms are presented for automated NOESY peak picking and NOE signal identification in homonuclear 2D and heteronuclear-resolved 3D [¹H,¹H]-NOESY spectra during de novoprotein structure determination by NMR, which have been implemented in the new software ATNOS (automated NOESY peak picking). The input for ATNOS consists of the amino acid sequence of the protein, chemical shift lists from the sequence-specific resonance assignment, and one or several 2D or 3D NOESY spectra. In the present implementation, ATNOS performs multiple cycles of NOE peak identification in concert with automated NOE assignment with the software CANDID and protein structure calculation with the program DYANA. In the second and subsequent cycles, the intermediate protein structures are used as an additional guide for the interpretation of the NOESY spectra. By incorporating the analysis of the raw NMR data into the process of automated de novoprotein NMR structure determination, ATNOS enables direct feedback between the protein structure, the NOE assignments and the experimental NOESY spectra. The main elements of the algorithms for NOESY spectral analysis are techniques for local baseline correction and evaluation of local noise level amplitudes, automated determination of spectrum-specific threshold parameters, the use of symmetry relations, and the inclusion of the chemical shift information and the intermediate protein structures in the process of distinguishing between NOE peaks and artifacts. The ATNOS procedure has been validated with experimental NMR data sets of three proteins, for which high-quality NMR structures had previously been obtained by interactive interpretation of the NOESY spectra. The ATNOS-based structures coincide closely with those obtained with interactive peak picking. Overall, we present the algorithms used in this paper as a further important step towards objective and efficient de novoprotein structure determination by NMR.     

Exact solutions for chemical bond orientations from residual dipolar couplings

New methods for determining chemical structures from residual dipolar couplings are presented. The fundamental dipolar coupling equation is converted to an elliptical equation in the principal alignment frame. This elliptical equation is then combined with other angular or dipolar coupling constraints to form simple polynomial equations that define discrete solutions for the unit vector(s). The methods are illustrated with residual dipolar coupling data on ubiquitin taken in a single anisotropic medium. The protein backbone is divided into its rigid groups (namely, its peptide planes and C frames), which may be solved for independently. A simple procedure for recombining these independent solutions results in backbone dihedral angles and that resemble those of the known native structure. Subsequent refinement of these - angles by the ROSETTA program produces a structure of ubiquitin that agrees with the known native structure to 1.1 Å C rmsd.     

PROSITE: a documented database using patterns and profiles as motif descriptors

Among the various databases dedicated to the identification of protein families and domains, PROSITE is the first one created and has continuously evolved since. PROSITE currently consists of a large collection of biologically meaningful motifs that are described as patterns or profiles, and linked to documentation briefly describing the protein family or domain they are designed to detect. The close relationship of PROSITE with the SWISS-PROT protein database allows the evaluation of the sensitivity and specificity of the PROSITE motifs and their periodic reviewing. In return, PROSITE is used to help annotate SWISS-PROT entries. The main characteristics and the techniques of family and domain identification used by PROSITE are reviewed in this paper.