Determination of the amounts of the protein synthesis initiation and elongation factors in wheat germ

Previous work by Browning et al. (Browning, K. S., Lax, S. R., Humphreys, J., Ravel, J. M., Jobling, S. A., and Gehrke, L. (1988) J. Biol. Chem. 263, 9630-9634) indicated that wheat germ extracts do not contain sufficient amounts of some of the protein synthesis initiation factors to obtain optimal translation of all mRNAs. In this investigation, a quantitative enzyme-linked immunosorbent assay was used to determine the amounts of eukaryotic initiation factors (eIF) 2, 3, 4A, 4F, and (iso)4F as well as the amounts of 40 S ribosomal subunits and elongation factors (EF) 1 alpha and 2 present in wheat germ extracts. EF-1 alpha is present in the highest amount (approximately 5% of the total protein), and eIF-4F is present in the lowest amount (approximately 0.03% of the total protein). The micromolar amounts of the factors and ribosomes are as follows: EF-1 alpha, 34; EF-2, 5.2; eIF-2, 1.5; eIF-3, 0.7; eIF-4A, 3.0, eIF-4F, 0.09; eIF-(iso)4F, 0.8; and 40 S ribosomal subunits, 3.2. The molar ratios of the factors to 40 S ribosomal subunits are approximately 11:1 for EF-1 alpha, 1.6:1 for EF-2, 0.45:1 for eIF-2, 0.2:1 for eIF-3, 0.9:1 for eIF-4A, 0.03:1 for eIF-4F, and 0.25:1 for eIF-(iso)4F. These findings strongly suggest that the concentrations of the initiation factors, particularly those factors required for the binding of mRNA to ribosomes, may play a major role in regulating the translation of mRNAs within the cell.     

Sodium cholate-induced changes in the conformation and activity of rat pancreatic cholesterol esterase

Pancreatic cholesterol esterase (CEase) regulates dietary cholesterol absorption and is activated in the presence of trihydroxy bile salts while remaining inactive monohydroxy bile salts. CEase from rat pancreas has been purified by ammonium sulfate precipitation, hydroxylapatite chromatography, and gel filtration on Sephacryl S-200/S-300 columns connected in series, and its homogeneity and Mr (55,418 +/- 288) have been determined by sedimentation equilibrium centrifugation. The effects of tri-, di-, and monohydroxy bile salts on the conformation of the purified enzyme in buffer solution and in an in vitro assay system were studied by circular dichroism spectropolarimetry. The CD spectrum of the enzyme in solution shows a curve shape suggestive of an alpha-helicity, but low mean residue ellipticity (MRE) values may indicate an important beta-turn contribution. Sodium cholate, a trihydroxy bile salt, induces a decrease in the negative MRE values of the enzyme in solution at bile salt concentrations of 70-100 nM, with no further spectral changes at concentrations as high as 1 mM. Sodium cholate concentrations higher than 1 microM also induce an increase in the enzyme's negative MRE values under activity assay conditions, which reverts toward its original value once the reaction reaches equilibrium. These latter changes are interpreted as induced by substrate binding to the enzyme followed by partial substrate depletion after the reaction reaches equilibrium. Sodium deoxycholate, a dihydroxy bile salt, induces unstable transient increases and decreases in the MRE values of CEase in buffer solution and under activity assay conditions. These changes are bile salt concentration-dependent and may reflect self-association of the protein. Sodium taurolithocholate, a monohydroxy bile salt, does not affect the CD spectrum of CEase, and neither the di- or the monohydroxy bile salt activates the enzyme.     

Disulfiram administration affects substance P-like immunoreactive and monoaminergic neural systems in rodent brain.

The biosynthetic enzyme peptidylglycine alpha-amidating monooxygenase catalyzes the formation of a variety of biologically active alpha-amidated peptides from respective COOH-terminal glycine-extended peptide precursors. Peptidylglycine alpha-amidating monooxygenase activity is dependent on copper, ascorbate, and molecular oxygen and is inhibited by the relatively selective copper chelator N,N-diethyldithiocarbamate or its disulfide dimer disulfiram (Antabuse). In the present study, chronic disulfiram treatment (100 mg/kg/day, for 12-25 days) resulted in significant changes in several neurochemical parameters in the mouse central nervous system, including levels of substance P-like, unamidated substance P-Gly-like, and protease-generated substance P-Gly-Lys-like immunoreactivities (SP-LI, SP-G-LI, and SP-G-K-LI, respectively). Combined high performance liquid chromatography/radioimmunoassay analyses of the extracted SP-LI, SP-G-LI, and SP-G-K-LI species indicated very similar chromatographic and immunochemical behavior as demonstrated for chemically authentic peptide standards. Additionally, changes in levels of monoamines and their metabolites were observed after drug administration. Complementary immunohistochemical analyses using affinity-purified anti-SP-G sera localized these drug-induced changes in levels of immunoreactive unamidated precursor to neural elements that normally express SP. As a functional corollary to alterations in neurochemical parameters, we observed significant disulfiram-induced increases in pain thresholds, potentiated by capsaicin treatment. Overall, our results indicate that the observed changes in steady state levels of immunoreactive SP and of the immature COOH-terminal extended forms of SP may reflect compensatory biosynthetic and posttranslational processing events in SP-containing neural systems after pharmacological challenge.     

Salt tolerant sugarbeet progeny from tissue cultures challenged with multiple salts

Lines of sugarbeet (Beta vulgaris L.) tolerant of multiple salts was accomplished by an in vitro multiple salt challenge. Petioles were placed on RV medium amended with 5 different salts along with Murashige and Skoog base salts for one month. Surviving shoots were cultured on RV medium to obtain petioles for subsequent challenges. During the first, second and third challenges, organogenically regenerated shoots developed from 5%, 46%, and 80% of the petioles, respectively. After the third multiple salt challenge, tolerant shoots were rooted and transplanted in soil. Salt was added to this soil at 1.0% by weight and plants were observed for 2 months. The ten most salt tolerant plants were vernalized to obtain seed. The R₁ seed and controls were planted in soil containing 0%, 0.61% or 0.77% multiple salts per dry soil weight. Emergence of R₁ seedlings was significantly greater than the controls under salt stress. Multiple salt tolerant R₁ plants were maintained in salt amended soil to the 8–10 leaf stage and appeared as healthy and vigorous as the control growing in salt free soil.Contribution from Missouri Agricultural Experiment Station Journal Series No. 10948. University of Missouri, Columbia, MO, USA Mention of trade names does not constitute a guarantee or warranty of the product by University of Missouri of Holly Sugar Corporation and does not imply their approval to the exclusion of other products.     

Linkage disequilibrium studies in multiple endocrine neoplasia type 1 (MEN1)

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary. The MEN1 gene has been localised to a 2-Mb region of chromosome 11q13 by meiotic mapping studies in MEN1 families. Such studies may have a limited resolution of approximately 1 cM (i.e. 1 Mb) and we have therefore investigated 96 MEN1 families (40 British, 17 French, 12 Finnish, 7 Swedish, 7 Dutch, 7 North American, 2 Australian, 1 New Zealand, 1 German, 1 Spanish and 1 Danish) for linkage disequilibrium, in order to facilitate a finer mapping resolution. We have utilised five microsatellite DNA sequence polymorphisms from the candidate region and have accurately determined their allele sizes, which ranged from 161 bp to 272 bp. The heterozygosity and number of alleles (given in brackets), respectively, at the loci were: D11S1883 (76%, 11), D11S457 (55%, 5), PYGM (94%, 18), D11S1783 (10%, 4) and D11S449 (87%, 16). Allelic association was assessed by Chi-square 2 ×n contingency tables, by Fisher exact 2 ×n contingency tables and by a likelihood-based approach. The results of haplotype analysis revealed 91 different affected haplotypes in the 96 families, an identical affected haplotype being observed in no more than two families. These results indicate the absence of an ancestral affected haplotype. Significant linkage disequilibrium (P < 0.005) could be established amongst the microsatellite loci but not between the loci and MEN1 in either the total population or in any of the geographical sub-populations. The absence of linkage disequilibrium between MEN1 and the polymorphic loci is probably the result of the occurrence of multiple different disease-causing mutations in MEN1. Received: 1 April 1997 / Accepted: 25 June 1997