Molecular cloning and characterization of human kinectin.

We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted alpha-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain.

Translational proteomics: developing a predictive capacity -- a review.

Over the past decade, proteomics has undergone a rapid development and radiation, diversifying across the biochemical landscape. While no single technique yet delivers complete proteomic coverage, application-specific adaptations afford significant opportunity for discovery and the development of predictive capacity (e.g. surrogate biomarker and clinical diagnostics). Targeted proteomic approaches, protein profiling strategies using affinity capture mass spectrometry and solution array represent realistic opportunities to deliver predictive capacity. The aim of this review is to provide an overview of proteomic technologies and how the outcomes delivered by such platforms may be translated into applications of predictive utility in clinical and basic science. In particular, recent applications in protein/peptide profiling (solid-phase affinity capture mass spectrometry and the targeted approach of antibody arrays) and the opportunities they afford researchers within the discipline of reproductive biology to develop new diagnostic and prognostic tests and surrogate biomarkers to improve the delivery of women's health care are considered.

Synthesis and antitumor and antiviral activities of a series of 1-beta-D-ribofuranosyl-5-halocytosine (5-halocytidine) cyclic 3',5'-monophosphates.

A series of 1-beta-ribofuranosyl-5-halocytosine cyclic 3',5'-monophosphates (1-4) has been prepared. Direct halogenation of cytidine 3',5'-monophosphate (cCMP) yielded the Cl, Br, and I compounds while 5-F-cCMP (1) was obtained on cyclization of the 5'-monophosphate. On in vitro testing of 1-4 against L1210 and P388 leukemias, only 1 showed significant low-level activity (ID50 = 3.1 X 10(-4) mmol/L). Derivatives 2-4 were inactive at 10(-1) mmol/L and also proved to have low viral ratings against a series of RNA and DNA virus strains in vitro. By contrast the 5-F-cCMP showed moderate activity against VV, HSV-1, and HSV-2 strains (VR = 0.6-0.9). Both 5-fluorocytidine and 5-fluorocytidine 5'-monophosphate had marked antiviral activity (VR = 1.0-2.1) with the above viruses as well as with parainfluenza virus type 3. The nucleoside and nucleotide also were more active than 5-F-cCMP against L1210 and P388 cells. However, comparison of the cytotoxicities and antiviral ED50 values of 5-F-cCMP, 5-fluorocytidine 5'-monophosphate, and 5-fluorocytidine suggests a potential therapeutic advantage for 5-F-cCMP. Possible rationales for these activities are discussed in terms of 5-F-cCMP and the corresponding 5'-monophosphate as potential prodrugs and as sources, following enzymatic deamination, of cytotoxic 5-fluorouridine or its 5'-monophosphate.

Stable expression of a selectable myeloproliferative sarcoma virus in murine T lymphocyte and monocyte cell lines.

We have investigated whether a retroviral vector based on the myeloproliferative sarcoma virus (MPSV) can be expressed in murine T cells and macrophages. This vector (neoR MPSV) carries the dominant selection marker for neomycin resistance (neoR) and the mos oncogene. The murine T cell line BW5147 and the monocytic cell line P388D1 were either transfected with neoR MPSV DNA or infected with neoR MPSV virus. From both lines, neoR cell clones could be established by retroviral infection, but not by calcium-phosphate precipitation-mediated DNA transfection. The efficiency of infection could be increased 60- to 200-fold upon cocultivation of target cells with irradiated neoR MPSV virus-producing cells. All neoR clones showed neoR MPSV specific sequences as revealed by dot blot and Southern blot analysis. The integration and expression of neoR MPSV was stable over a period of now more than 4 months, even in the absence of selection for neomycin resistance. Northern blot analysis showed that neoR clones express full length neoR MPSV. Further, clones of both T cell and monocyte origin were capable to produce infectious virus particles as revealed by focus formation on fibroblasts and conversion of neomycin sensitive fibroblasts to a neomycin resistant phenotype.

Rapid characterization of combinatorial libraries using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The relatively new field of combinatorial chemistry has enabled researchers to create large mixtures of compounds that can be screened for leads in developing potential drug candidates. The new synthetic method has also created a need for better procedures to analyze the complex mixtures that are generated. The immediate goal in most cases is to verify the synthetic procedure and to determine the purity and completeness of the library sample before binding studies are initiated. We report here a method to rapidly characterize small-molecule combinatorial libraries in solution. All combinatorial library samples were synthesized by combining a core molecule bearing two acid chloride functionalities with various amino acids to generate libraries of 36, 78 and 120 components. Using electrospray ionization fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) we were able to identify 70-80% of the library components. All samples were analyzed as mixtures by direct infusion without chromatographic separation. Furthermore, nominally isobaric components could be resolved and identified through exact mass assignments without tandem mass spectrometery. ESI-FTICR-MS is a rapid and convenient tool for the characterization of small-molecule libraries. The method is especially useful for the analysis of larger libraries that contain many nominally isobaric components and impurities.

Fluorinated psychopharmacological agents: noninvasive observation by fluorine-19 nuclear magnetic resonance.

Fluorinated psychopharmacological agents were measured with fluorine-19 nuclear magnetic resonance spectroscopy in the brain of intact rats that had been treated with fluphenazine. These in vivo experiments were compared to in vitro measurements of fluphenazine-treated rats. A high-field shift was observed in both in vivo and in vitro measurements. On the basis of the in vitro measurements, fluphenazine concentration in the brains of treated rats was estimated. Our observations demonstrate the feasibility of this technique for determining fluorinated neuroleptics in live mammals.

[Low frequency electro-stimulation and ultrasonic therapy (author's transl)]. / Niederfrequenz-und Ultraschallbehandlung. Prospektive Studie über 1200 Behandlungssequenzen.

In a prospective study 1200 sequences of low frequency electrostimulation and ultrasonic therapy have been examined. The basics of the type of currents applied, the therapy scheme and the indication routine are presented. These parameters were kept constant in the course of the 2 years' study. For the treatment 8 different apparatuses were available. The actual current shapes of the generators were measured, the influence of constant-current and constant-voltage output circuits were tested and were discussed in relation to the electrode types.--Advantages and disadvantages of disposable-type, sponge-type, lead-type and vacuum-type electrodes are reported. Treatments were carried out with the current types DF and CP of the diadynamic currents alone, as combined therapy together with ultrasound, as mere ultrasound treatment, as ultrastimulation current, as iontophoresis and galvanic current. The results are compared with comparable examinations by other authors and they are discussed with respect to different influencing factors.

Murine gamma interferon inhibits v-mos-induced fibroblast transformation via down regulation of retroviral gene expression.

Expression of the retroviral vector Neor myeloproliferative sarcoma virus (MPSV), which contains the v-mos oncogene and the neomycin resistance gene, leads to neoplastic transformation of mouse fibroblasts. Murine recombinant gamma interferon (IFN-gamma) could revert the neoplastic properties of established Neor MPSV-transformed cell lines to an apparently untransformed phenotype. In the presence of IFN-gamma, the Neor MPSV transformants showed a greater than 97% reduction of cloning efficiency in soft agar, strongly reduced proliferative capacity, and morphological changes. The IFN-gamma-induced phenotypic reversion was preceded by a rapid and selective reduction of all retroviral RNA species, apparently due to IFN-gamma action on the long terminal repeat of Neor MPSV. The mRNA levels of cellular genes either remained unaffected (beta-actin) or were even enhanced (H-2) in IFN-gamma-treated Neor MPSV-transformed cells. Upon removal of IFN-gamma, retroviral gene expression was fully recovered and a gradual reappearance of the transformed phenotype of these cells within 3 weeks was noted. These data show that IFN-gamma can cause a virtually complete, but reversible, inhibition of v-mos-induced neoplastic properties in transformed fibroblasts by selective down regulation of retroviral RNA levels.

In vivo metabolism of [4-13C]phenacetin in an isolated perfused rat liver measured by continuous flow 13C NMR spectroscopy.

Continuous flow 13C NMR spectroscopy has been used for the first time to monitor the metabolism of a 13C labeled drug in an isolated liver. Continuous and almost immediate information on the metabolite formation could be obtained using 13C labeled phenacetin without alteration of the biological system. The data are consistent with those observed by conventional techniques (HPLC, aliquot 13C NMR measurements). From the biological point of view the sensitivity of continuous flow 13C NMR spectroscopy is still low (10(-3) M). The results presented demonstrate however that non-invasive and non-radioactive real time monitoring of drug metabolism in intact organs is possible.

Inhibition of tumor necrosis factor (TNF)-mediated NF-kappa B activation by selective blockade of the human 55-kDa TNF receptor.

The numerous biologic activities of TNF appear mediated by two types of specific cell surface receptors of 55 to 60 kDa (TR55) and 75 to 80 kDa (TR75) molecular mass, respectively. The role of TR55 in the activation of the nuclear transcription factor kappa B (NF-kappa B) was investigated using an antagonistic, mAb, H398, specific for the human TR55. The human leukemic T cell line, Jurkat, which expresses both types of receptors at comparable levels, was used to test for NF-kappa B activation by electrophoretic mobility shift assays using as a probe an oligonucleotide encompassing the two tandemly arranged kappa B sites of the HIV-1 LTR enhancer. mAb H398 is shown to efficiently block not only TNF- but also lymphotoxin-mediated activation of NF-kappa B. Furthermore mAb H398 also impeded TNF- or lymphotoxin-mediated activation of chloramphenicol acetyl transferase gene expression from the HIV-1-LTR as determined by transient transfection assays. These findings indicate that both, induction of NF-kappa B binding to DNA, and transcriptional activity can be efficiently inhibited by selective blockade of TR55. Finally it is shown, that human TR55 confers NF-kappa B inducibility when expressed in the mouse pre-B cell line 70Z/3, which does not respond to TNF in its parental state. Together, the results of this study indicate that TR55 is both necessary and sufficient for mediating TNF activation of NF-kappa B.

Gamma interferon regulates long terminal repeat-controlled oncogene expression in transformed mouse fibroblasts at the level of mRNA transcription.

In transformed NIH 3T3 cells, murine gamma interferon reduces the expression of the long terminal repeat-controlled oncogenes v-mos, c-myc, and v-Ha-ras by a direct effect on the activity of retroviral promoters, as revealed by analyses of RNA half-life and transcriptional activity of retroviral genes as well as by analyses of chloramphenicol acetyltransferase activity in cells transformed with the cat gene under the control of long terminal repeats.

Deprotonation reactions of multiply protonated ubiquitin ions.

The gas-phase deprotonation reactions of multiply protonated ubiquitin ions have been studied in a Fourier-transform ion cyclotron resonance mass spectrometer. Electrospray ionization was used to generate ubiquitin ions with attachment of 7-13 protons. Rate constants were measured for the reactions of these protein ions with four amines: n-propylamine, di-n-propylamine, tri-n-propylamine, and N,N,N',N'-tetramethyl-1,4-diaminobutane. The gas-phase basicities of the amines ranged from 210.1 kcal/mol to 232.6 kcal/mol. The rate constants were found to increase as the charge state of the ion increased and as the basicity of the amine increased. Several reactions proceed at near the collision rate and have rate constants in excess of 10(-8) cm3 molecule-1 s-1. With the more basic reactants, multiple protons could be stripped sequentially from ubiquitin ions at roughly equivalent rates, suggesting that these protons are attached to sites with similar basicities. In general, deprotonation occurs if the gas-phase basicity of the amine is within 10 kcal/mol of the intrinsic gas-phase basicity of the amino acid residue being deprotonated. For [M+nH]n+, n = 4-6, nonlinear pseudo-first-order kinetic behavior indicated the presence of multiple ion structures. Kinetic, structural and thermodynamic aspects of these reactions are discussed.

In vivo 19F nuclear magnetic resonance spectroscopy of trifluorinated neuroleptics in the rat.

In vivo 19F NMR measurements of trifluorinated neuroleptics in the rat brain were made using a 13 x 18 mm surface coil at 4.7 T. The signal of fluphenazine was obtained within 8-15 min from brains of living rats treated chronically with drug doses of 5-30 mg/kg. Following the intravenous injection of a single dose of trifluoperazine (30 mg/kg), brain levels could be monitored with a time resolution of 30 min. The data demonstrate the possibility of obtaining in vivo pharmacokinetics of fluorinated agents in the rat brain. 19F NMR is expected to become an important tool in neurochemical research.

Multipole storage assisted dissociation, a novel in-source dissociation technique for electrospray ionization generated ions.

In this work we present a novel in-source dissociation scheme referred to as multipole storage assisted dissociation (MSAD) for electrospray ionization (ESI) generated ions in which dissociation is effected by employing extended ion accumulation intervals in a high pressure rf-only hexapole assembly prior to mass analysis. Following an extended ion accumulation interval in which ions are confined in the rf-only hexapole, ions are gated out of the hexapole, trapped, and mass analyzed in the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The accumulation region is comprised of an rf-only hexapole ion guide which separates two electrodes, a biased skimmer cone, and an auxiliary 'gate' electrode at the low pressure end of the hexapole. This technique should be applicable to other mass spectrometry platforms compatible with pulsed ionization sources including quadrupole ion traps, and time-of-flight mass analyzers. This concept is demonstrated with the dissociation of a small protein in which selective fragmentation is observed at labile amino acid linkages producing primarily y-type fragment ions.

The significance of monoisotopic and carbon-13 isobars for the identification of a 19-component dodecapeptide library by positive ion electrospray Fourier transform ion cyclotron resonance mass spectrometry.

Harnessing the ultra high resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and positive ion electrospray, we have demonstrated the significance and utility of cumulative mass defect high resolution mass separation stable isotope distribution, exact mass measurement and elemental formula as a means of simultaneously identifying 19 components of the dodecapeptide library Ac-ANKISYQS[X]STE-NH(2). With an instrument resolution of 275 000 (average), isobaric multiplets attributed to monoisotopic and carbon-13 components of peptides: Ac approximately SLS approximately NH(2); Ac approximately SNS approximately NH(2); Ac approximately SOS approximately NH(2); Ac approximately SDS approximately NH(2); within the mass window of 1380-1385 Da, and Ac approximately SQS approximately NH(2); Ac approximately SKS approximately NH(2); Ac approximately SES approximately NH(2); Ac approximately SMS approximately NH(2), within the mass window 1395-1400 Da, were mass resolved, accurately mass measured and identified from the computed molecular formulas. This experimental procedure enabled the separation of monoisotopic and carbon-13 isobars yielding enhanced selectivity and specificity and serves to illustrate the significance of monoisotopic and carbon-13 isobars in final product analysis. Chromatographic separation (HPLC) was of limited utility except for monitoring the overall extent of reaction and apparent product distribution. Positive ion electrospray-FTICR-MS and fast atom bombardment (FAB) MS were used to assess final product quality and apparent component distribution.