Yeast proteins that recognize nuclear localization sequences.

A variety of peptides can mediate the localization of proteins to the nucleus. We have identified yeast proteins of 70 and 59 kD that bind to nuclear localization peptides of SV-40 T antigen, Xenopus nucleoplasmin, and the yeast proteins Ga14 and histone H2B. These proteins are assayed by the binding of peptide-albumin conjugates to proteins immobilized on nitrocellulose filters. These binding proteins fractionate with nuclei and are extractable with salt but not detergent. Radiolabeled peptide-albumin conjugates also bind to isolated nuclei; the binding is saturable and can be extracted with salt. Different nuclear localization peptides compete with each other, implying that a single class of proteins is responsible for their recognition. The 70- and 59-kD proteins have the properties expected for a receptor that would act to direct proteins to the nucleus.

Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton.

Interaction with extracellular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847-5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393). We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2-terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine-rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene expression.

A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein.

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.

The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes.

The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 10(8) porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P < 0.01), and significantly smaller pyruvate (day 1 vs day 14 P < 0.03) and threonine consumption (day 1 vs day 10 P < 0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P < 0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P < 0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies.

Solution conformation of an ET(B) selective agonist, ET-1[Cys(Acm)1,15,Ala3,Leu7,Aib11], in CD3OH/H2O by 1H NMR and molecular modelling.

To understand the basic structural requirements for the biological activity of endothelin peptides, the solution structure of an ETB selective agonist, ET-1[Cys-(Acm)1,15, Ala3,Leu7,Aib11, was investigated by 1H NMR spectroscopy and molecular modelling. The structure is characterised by an alpha-helical conformation between residues Ser5-His16 but is undefined at both the N and C termini. To date, neither the solution structures of linear modified peptides nor the effects of a methanol/water solvent system have been examined for endothelin or endothelin-like peptides. This structure plays an important role towards the design of endothelin receptor selective agonists and antagonists.

The preparation of D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,4,5-trisphosphate in milligram quantities from a readily available starting material.

The optimisation of a reaction for the conversion of glycerophosphoinositols to phosphoinositols is described. This reaction has been used in a scheme, described in detail, for the formation of D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,4,5-trisphosphate in mg quantities from a readily available preparation of mixed phosphoinositides. An optimised procedure is also detailed for the recovery of these products to high yield and purity. The identity of the products has been confirmed both by high resolution anion-exchange column chromatography and by 1H nuclear magnetic resonance studies. We report for the first time the 1H nuclear magnetic resonance spectrum for D-myo-inositol 1,4-bisphosphate.

Stereochemistry of poly(methyl methacrylate) acrylic resin denture base material.

Nuclear magnetic resonance (NMR) 13C has been used to determine the tacticity of poly(methyl methacrylate) (PMMA) denture base materials. Curing cycle has no effect on tacticity. A tendency towards a mainly syndiotactic arrangement is shown. Industrially produced PMMA showed the same tacticity as the dental products.

A linear endothelin-1 analogue: solution structure of ET-1[Aib1,3,11,15, Nle7] by nuclear magnetic resonance spectroscopy and molecular modelling.

Two-dimensional nuclear magnetic resonance techniques and a combination of distance geometry and molecular dynamics calculations were utilised to determine the three dimensional solution structure of an ET-1 analogue, ET-1[Aib1,3,11,15, Nle7], in a methanol-d3/water co-solvent. The modelled structure shows that the peptide folds into a consistent alpha-helical conformation between residues Ser4-His16 while the C-terminus prefers no fixed conformation. Our studies confirm that the disulphide links which are normally associated with the endothelin family of neuropeptides are not important for the formation of a helical conformation in solution. This full length, modified, synthetic linear ET-1 analogue plays a vital role towards designing endothelin receptor agonists. Structure activity relationships are discussed in terms of the conformational features of the calculated structure.

Sulphated compounds attenuate beta-amyloid toxicity by inhibiting its association with cells.

Agents that interfere with the toxic effects of beta-amyloid protein may be therapeutically useful against Alzheimer's disease. We reported recently that several sulphated glycosaminoglycans and sulphonated dyes attenuate the toxic effects of beta-amyloid fragments beta 25-35 and beta 1-40 in two clonal cell lines. We now demonstrate that this protective effect is due to interference with beta-amyloid cell association rather than effects on beta-amyloid structure. Using an enzyme-linked immunoabsorbance assay to detect cell-associated beta 1-40, we found in a range of compounds a strong correlation between inhibition of HeLa cell association of beta 1-40 and attenuation of cellular toxicity as measured by inhibition of 3-[4,5-dimethylthia-zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. In contrast, effects on peptide structure, as measured by Congo red binding, were generally inconsistent with the attenuating effects of the compounds on cellular toxicity. These results suggest that by binding beta-amyloid these agents prevent its interaction with cells.

Sulfonated dyes attenuate the toxic effects of beta-amyloid in a structure-specific fashion.

We recently reported that several sulfate-containing glycosaminoglycans, a class of compounds associated with the beta-amyloid plaques of Alzheimer's disease, attenuate the toxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. The amyloid-binding sulfonated dye Congo Red was shown to have a similar effect. Using two clonal cell lines, we now demonstrate that several sulfonated dyes attenuate beta-amyloid toxicity and that the protective effect appears specific for compounds whose sulfonate groups can interact with the beta-pleated structure of aggregated amyloid. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.

Sulfated glycosaminoglycans and dyes attenuate the neurotoxic effects of beta-amyloid in rat PC12 cells.

Glycosaminoglycan (GAG)-containing proteoglycans are associated with the neuritic plaques and cerebrovascular beta-amyloid deposits of Alzheimer's disease as well as with the amyloid deposits of prion and other disorders. GAGs and other sulfate-containing compounds have previously been shown to bind beta-amyloid peptide in vitro, suggesting possible effects of beta-amyloid deposition and/or toxicity in vivo. Using reduction of the redox dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure beta-amyloid neurotoxicity in rat pheochromocytoma PC12 cells, several polysulfated GAGs and synthetic sulfate-containing compounds were found to attenuate the neurotoxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.

3-O-methyl-D-galactose residues in lycophyte primary cell walls.

Acid hydrolysis of cell wall-rich material from young leaves of the lycophyte Selaginella apoda (L.) Spring yielded substantial amounts of 3-O-methyl-D-galactose (1) in addition to the usual major monosaccharides (glucose, galactose, arabinose, xylose and galacturonic acid). The yield of 1 approximately equalled that of galacturonic acid. Compound 1 was identified as 3-O-methylgalactose by its 1H and 13C NMR spectra, and shown to be the D-enantiomer by its susceptibility to D-galactose oxidase. Compound 1 was detected in acid hydrolysates of the alcohol-insoluble residues from young leaves of all lycophytes tested, both homosporous (Lycopodium, Huperzia and Diphasiastrum) and heterosporous (Selaginella). It was not detectable in the charophyte green algae Coleochaete scutata, Chara coralina or Klebsormidium flaccidum, any bryophytes [a hornwort (Anthoceros), four liverworts and three mosses], or any euphyllophytes [a psilopsid (Psilotum), a horsetail (Equisetum), eusporangiate and leptosporangiate ferns, the gymnosperm Gnetum, and diverse angiosperms]. A high content of 1 is thus an autapomorphy of the lycophytes.

Pulcherosine, an oxidatively coupled trimer of tyrosine in plant cell walls: its role in cross-link formation.

An oxidatively coupled trimer of tyrosine has been isolated from hydrolysates of primary cell walls of a tomato cell culture. UV-absorption, fluorescence and 1H NMR spectra showed that the trimer was pulcherosine, composed of isodityrosine and tyrosine oxidatively coupled via a biphenyl linkage such that the aromatic core is 2,2'-dihydroxy-3-phenoxybiphenyl. Pulcherosine could act as an intermediate in the conversion of isodityrosine to the tetramer, di-isodityrosine. Steric considerations show that the three tyrosine units of pulcherosine could not be near-neighbour residues within a single polypeptide chain. Pulcherosine therefore forms inter-polypeptide cross-links and/or wide intra-polypeptide loops.

N alpha- and N epsilon-D-galacturonoyl-L-lysine amides: properties and possible occurrence in plant cell walls.

Three representatives of a novel class of amide (isopeptide) glycoconjugates have been synthesised: N alpha-D-galacturonoyl-L-lysine and N epsilon-D-galacturonoyl-L-lysine and N epsilon-D-polygalacturonoyl-L-lysine. Galacturonoyl-lysine amide bonds were labile in 2 M trifluoroacetic acid at 120 degrees and in alkali, but relatively stable in cold acid. The amide bonds were resistant to digestion by Driselase, Pronase and trypsin. The polysaccharide backbone of N epsilon-D-polygalacturonoyl-L-lysine was hydrolysed by Driselase to yield two major ninhydrin-positive compounds which were shown by 1H and 13C NMR spectroscopy to be tri- and tetra-alpha-(1-->4)-D-galacturonoyl-L-lysines. To investigate the possible natural occurrence of N-galacturonoyl isopeptide bonds, we fed cell-suspension cultures of spinach and tomato with D-[6-14C]glucuronic acid, which radio-labels pectic polysaccharides. The radioactive cell walls were digested with, sequentially, Driselase, mild acid, and proteinases. On electrophoresis at pH 2.0, several of the radioactive digestion-products were cathodic. Some of the cathodic products yielded [14C]galacturonic acid upon complete acid hydrolysis. The existence of these products is compatible with the presence of novel N-galacturonoyl isopeptide bonds, which could serve as cross-links in plant cell walls.

The Coping Strategies Questionnaire: a large sample, item level factor analysis.

OBJECTIVE: The Coping Strategies Questionnaire (CSQ), a measure of coping in chronic pain patients, was subjected to item-level exploratory factor analysis. SUBJECTS: A sample of 965 chronic pain patients were used in the analysis. RESULTS: Principal components analysis using a varimax rotation procedure identified nine factors that accounted for 54.5% of the variance. Of these nine factors, the first five represent subscales of the original CSQ subscales. The catastrophizing subscale replicated with significant loadings for all six original items, and ignoring sensations replicated with five of six items. Factors representing reinterpreting pain sensations, coping self-statements, and diverting attention subscales also appeared. The items from the praying and hoping subscale split into separate praying and hoping factors (factors 6 and 8). When reliability coefficients were calculated, factors 7 through 9 had unacceptably low internal consistency and thus were not considered stable factors. Correlations between factors 1 through 6 and other measures of psychological and physical functioning were calculated in the construct validation portion of this study. Previously found relationships were replicated in that the correlations between CSQ factor scores and measures of pain, depression, and disability were in the same direction in this data set as those previously reported.

Bias effects in three common self-report pain assessment measures.

OBJECTIVE: Past research has shown response biases to influence the accuracy of results from self-report measures. In pain assessment, where a percentage of patients have financial and other reasons to minimize or exaggerate psychological disturbance, it becomes especially important to identify the influence of response bias in self-report of adjustment. This study investigated the susceptibility of three commonly used self-report pain assessment measures to response bias. DESIGN: This study used a within-subjects (asymptomatic subjects) design with two experimental conditions and nonequivalent control group (chronic pain patients). SUBJECTS: Experimental group: 40 students enrolled in an occupational therapy program at a major southeastern United States university. CONTROL GROUP: 200 subjects referred to a multidisciplinary pain clinic at a major teaching hospital. MEASURES: Coping Strategies Questionnaire, Multidimensional Pain Inventory, and Pain Beliefs and Perceptions Inventory. RESULTS: With few exceptions, asymptomatic subjects scored significantly differently on these measures while portraying themselves as either coping well or coping poorly. In addition, when using the "coping poorly" response set, asymptomatic subjects reproduced scores similar to those of symptomatic chronic pain patients. CONCLUSION: The susceptibility to manipulation appeared constant across the three measures, a finding that highlighted the difficulties clinicians and researchers encounter in accurate interpretation of results from these measures in the absence of validity indicators. This study also emphasizes the ease with which subjects with sufficient motivation can present themselves in an untruthful and manipulative manner and can generate scores that are, on their own, difficult to distinguish from those of a group of typical chronic pain patients.

Development of ET(B) selective agonists: solution structure of a linear endothelin-1 analogue, ET-1 [Cys(Acm)(1,15), Ala3, Leu7, dAsp8, Aib11].

The solution structure of a synthetic ET(B) selective agonist, ET-1[Cys(Acm)(1,15), Ala3, Leu7, dAsp8, Aib11] has been solved by 1H NMR and molecular modelling studies. Such solution structures of linear modified peptides in aqueous methanol are being used in an ongoing program of research designed to assist in an understanding of the basic structural requirements for the biological activity of vasoconstrictors. The resulting structure of this peptide is characterised by an alpha-helical conformation between residues Leu6-His16 and by N- and C-termini which assume no defined conformation. A knowledge of the solution structures of this and related peptides, which are ET(B) selective agonists, are proving to be important in the understanding of how they interact with the ET(B) receptor.

13C-NMR determination of the molecular dynamics of cholesterol in dimyristoylphosphatidylcholine vesicles.

Using 13C-NMR measurements of T1, T2 and the nuclear Overhauser enhancement factor at 50.32, 90.56 and 150.87 MHz, we have measured the dynamics of cholesterol in dimyristoylphosphatidylcholine (DMPC) vesicles from 28 to 50 degrees C. Using the model-free approach of Lipari and Szabo, we have found that at 37 degrees C the motion of the rigid steroid ring can be described by an equal contribution from two effective motions with correlation times of 63 and 0.85 ns. The C26 and C27 carbon atoms of cholesterol were found to have an effective correlation time of 8 +/- 2 ps and a value for the square of the generalised order parameter of 0.03 +/- 0.01. The corresponding values for the C25 carbon atom were 17 +/- 4 ps and 0.09 +/- 0.02, showing slower motion and greater order for this carbon atom, which is nearer to the rigid steroid ring. Apart from the effect of vesicle size on T2, no concentration dependence of the dynamics of cholesterol was detected over the cholesterol concentration range 2-30 mol%. The order parameters and correlation times from the present 13C-NMR experiments are shown to be compatible with those from 2H-NMR experiments. This establishes the validity of the present approach, which we are currently extending to low concentrations of cholesteryl oleate in DMPC vesicles.

Cultured fish cells metabolize octadecapentaenoic acid (all-cis delta3,6,9,12,15-18:5) to octadecatetraenoic acid (all-cis delta6,9,12,15-18:4) via its 2-trans intermediate (trans delta2, all-cis delta6,9,12,15-18:5).

Octadecapentaenoic acid (all-cis delta3,6,9,12,15-18:5; 18:5n-3) is an unusual fatty acid found in marine dinophytes, haptophytes, and prasinophytes. It is not present at higher trophic levels in the marine food web, but its metabolism by animals ingesting algae is unknown. Here we studied the metabolism of 18:5n-3 in cell lines derived from turbot (Scophthalmus maximus), gilthead sea bream (Sparus aurata), and Atlantic salmon (Salmo salar). Cells were incubated in the presence of approximately 1 microM [U-14C]18:5n-3 methyl ester or [U-14C]18:4n-3 (octadecatetraenoic acid; all-cis delta6,9,12,15-18:4) methyl ester, both derived from the alga Isochrysis galbana grown in H14CO3-, and also with 25 microM unlabeled 18:5n-3 or 18:4n-3. Cells were also incubated with 25 microM trans delta2, all-cis delta6,9,12,15-18:5 (2-trans 18:5n-3) produced by alkaline isomerization of 18:5n-3 chemically synthesized from docosahexaenoic acid (all-cis delta4,7,10,13,16,19-22:6). Radioisotope and mass analyses of total fatty acids extracted from cells incubated with 18:5n-3 were consistent with this fatty acid being rapidly metabolized to 18:4n-3 which was then elongated and further desaturated to eicosatetraenoic acid (all-cis delta8,11,14,17,19-20:4) and eicosapentaenoic acid (all-cis delta5,8,11,14,17-20:5). Similar mass increases of 18:4n-3 and its elongation and further desaturation products occurred in cells incubated with 18:5n-3 or 2-trans 18:5n-3. We conclude that 18:5n-3 is readily converted biochemically to 18:4n-3 via a 2-trans 18:5n-3 intermediate generated by a delta3,delta2-enoyl-CoA-isomerase acting on 18:5n-3. Thus, 2-trans 18:5n-3 is implicated as a common intermediate in the beta-oxidation of both 18:5n-3 and 18:4n-3.

Analysis of biological fluids using 600 MHz proton NMR spectroscopy: application of homonuclear two-dimensional J-resolved spectroscopy to urine and blood plasma for spectral simplification and assignment.

The application of 600 MHz two-dimensional J-resolved 1H NMR spectroscopy (JRES) to the analysis of human urine and blood plasma is demonstrated. This method when applied at very high field gives a rapid means of simplifying and aiding the assignment of highly overlapped resonances of minor metabolites in biofluids. Using this approach, mixtures of drug and endogenous metabolites were identified in untreated urine samples, the signals of which were extensively overlapped in single pulse 600 MHz spectra. For untreated blood plasma samples the JRES experiment was also effective for the selective attenuation of signals from the plasma proteins thus revealing strong well-resolved signals from the low molecular weight components. For the first time it was shown to be possible to assign in detail the spectra region from 3 to 4 ppm in blood plasma, including the complete assignment of the signals from alpha- and beta-glucose. JRES spectra of plasma were much easier to interpret and had a much higher information content than equivalent one-dimensional Hahn spin-echo spectra, thus aiding the identification of non protein-bound low molecular weight metabolites in plasma.