Possible role for protein kinase C in the pathogenesis of inborn errors of metabolism

Protein kinase C (PKC) is a ubiquitous enzyme family implicated in the regulation of a large number of short- and long-term intracellular processes. It is hypothesized that modulation of PKC activity may represent, at least in part, a functional link between mutations (genotype) that lead to the pathological accumulation of naturally occurring compounds that affect PKC activity and perturbation of PKC-mediated substrate phosphorylation and cellular function in the corresponding diseases (phenotype). This model provides a unifying putative mechanism by which the phenotypic expression of some inborn errors of metabolism may be explained. Recent studies in a cell-free system of human skin fibroblasts support the hypothesis that alteration of PKC activity may represent the functional link between accumulation of sphingolipids and fatty acyl-CoA esters, and perturbation of cell function in sphingolipidoses and fatty acid oxidation defects, respectively. Further studies will elucidate the effects of these alterations on PKC-mediated short- and long-term cellular functions in these diseases, as well as the possible role of PKC in the pathogensis of other diseases. © 1995 Wiley-Liss, Inc.     

Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom

Summary Two-dimensional ¹H NMR techniques were used to determine the spatial structure of ectatomin, a toxin from the venom of the ant Ectatomma tuberculatum. Nearly complete proton resonance assignments for two chains of ectatomin (37 and 34 amino acid residues, respectively) were obtained using 2D TOCSY, DQF-COSY and NOESY experiments. The cross-peak volumes in NOESY spectra were used to define the local structure of the protein and generate accurate proton-proton distance constraints employing the MARDIGRAS program. Disulfide bonds were located by analyzing the global fold of ectatomin, calculated with the distance geometry program DIANA. These data, combined with data on the rate of exchange of amide protons with deuterium, were used to obtain a final set of 20 structures by DIANA. These structures were refined by unrestrained energy minimization using the CHARMm program. The resulting rms deviations over 20 structures (excluding the mobile N- and C-termini of each chain) are 0.75 ? for backbone heavy atoms, and 1.25 ? for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two α-helices and a hinge region of four residues; this forms a hairpin structure which is stabilized by disulfide bridges. The hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-α-helical bundle structure.     

A novel approach to the analysis of the initiation of embryo development in gramineae

An in vivo model system to study the initiation of embryo development is presented. From the so-called Salmon system of wheat (alloplasmic lines with a 1BL-1RS chromosome translocation), three completely isogenic and homozygous lines were produced by selection for uniformity in about 20 selfing/backcross generations as well as between sublines of doubled haploids. The line (aestivum)-Salmon is male fertile and sexual. The lines (caudata)-Salmon and (kotschyi)-Salmon are male sterile and have a parthenogenetic capacity of about 90%. The expression of nuclear-cytoplasmic male sterility is different for the two parthenogenetic lines. The initiation of autonomous embryo development at defined developmental stages of the ovaries and the maximum degree of parthenogenesis are identical in both parthenogenetic lines as proved by the auxin test and progeny analyses. The protein patterns from ovary extracts of the three isogenic lines were identical for more than 200 spots of 2-D polyacrylamide gels, confirming their homogeneity. However, one protein (P 115.1) was found 3 days before and during anthesis only in ovaries of the parthenogenetic lines. It seems to be involved in the initiation of parthenogenesis.     

Demonstration of high opioid-like activity in isolated peptides from wheat gluten hydrolysates

Because of a possible relationship between schizophrenia and celiac disease, a condition in some individuals who are sensitive to wheat gluten proteins in the diet, there has been interest in observations that peptides derived from wheat gluten proteins exhibit opioid-like activity in in vitro tests. To determine the origin of the peptides exhibiting opioid activity, wheat proteins were fractionated by size (gel filtration), by charge differences (ion exchange chromatography) and by differences in hydrophobicity (reversed-phase HPLC). These fractions were hydrolyzed by pepsin or pepsin and trypsin and the resulting peptides separated by gel filtration chromatography. The separated peptides were tested for opioid-like activity by competitive binding to opioid receptor sites in rat brain tissue in the presence of tritium-labeled dihydromorphine. The peptides showed considerable differences in activity; while some peptides exhibited no activity, 0.5 mg of the most active peptides were equivalent to 1 nM of morphine in the binding assay. The most active peptides were derived from the gliadin fraction of the gluten complex.     

Effect of low-molecular-weight proteins on protein (lysozyme) binding to isolated brush-border membranes of rat kidney

Filtered proteins including the low-molecular-weight protein lysozyme are reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with binding of the protein to the brush-border membrane. The binding of ¹²⁵I-labelled egg-white lysozyme (EC 3.2.1.17) to isolated brush-border membranes of rat kidney and the effect of several low-molecular weight proteins on that binding was determined. The Scatchard plot revealed a one-component binding type with a dissociation constant of 5.3 μM and 53.0 nmol/mg membrane protein for the number of binding sites. The binding of the cationic lysozyme was inhibited competitively by the addition of cationic cytochrome c to the incubation medium, while the neutral myoglobin had no effect. The anionic β-lactoglobulin A inhibited the lysozyme binding in a noncompetitive manner. These data suggest that the binding takes place between positively charged groups of the protein molecule and negative sites on the brush-border membrane, and, the competition between the cationic cytochrome c and the cationic lysozyme for the binding sites may be responsible for the inhibitory effect of cytochrome c on renal lysozyme reabsorption. The binding step at the brush-border membrane appears to be cation-selective.     

Mutant hemoglobin stability depends upon location and nature of single point mutation

The temperature dependence of the rates of heme release from the beta subunits of methemoglobin A and 5 beta mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the alpha 1/beta 2 interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.     

The role of triiodothyronine in the regulation of synthesis rate and translatable mRNA level of fatty acid synthetase in a preadipocyte cell line

Triiodothyronine (T₃) effects on the activity, rate of synthesis and mRNA content of the key lipogenic enzyme, fatty acid synthetase, were studied in differentiating ob₁₇ preadipocytes cloned from ob/ob mouse epididymal adipose tissue. During differentiation in the presence of insulin, a 6–10-fold increase in both fatty acid synthetase specific activity and synthesis rate were reproducibly observed and occurred concomitantly. The relative synthesis rate exhibited a progressive elevation from 0.5% at confluence to a maximum level of 2% in the presence of insulin. The rate of the enzyme degradation determined by pulse-chase experiments was similar in differentiating cells and insulin-untreated cells of the same age ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 40–42 h). Furthermore, the increase in the enzyme synthesis rate was preceded by a progressively elevating amount of mRNA encoding for this protein as detected by translation in a reticulocyte lysate cell-free system. It is thus suggested that the increment in total and neosynthesized fatty acid synthetase in essentially due to an increased enzyme synthesis, reflecting an increased relative content of its specific mRNA. T₃ included at a physiological concentration (1.5 nM) in the culture medium enhanced significantly both enzyme synthesis and its specific mRNA. The most important T₃ effect was an acceleration of both processes, a stimulation of the mRNA level being detected as early as day 3 post-confluence and maximum at day 5 when the effect on the synthetase synthesis rate and activity began to be enhanced. This suggests that T₃ would mainly affect fatty acid synthetase as a pretranslational level.     

Preparation and discharge of secretion in the subcommissural organ of the rat

Summary The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30° C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981).Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The periodically striated bodies, which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.Supported by grants from the Ministry for Science and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. B. Wolff, Mrs. S. Mehnert, Mrs. E. Siebert, Mrs. Ch. Schneider, and Mrs. I. Seifert is gratefully acknowledged     

Myxobacterial slime and proteolytic activity

An extracellular protein-polysaccharide-lipide (PPL) complex from exponentially growing cultures of Myxococcus virescens was purified by phosphate precipitation and gel chromatography. The high molecular weight slime polymer appeared homogenous upon isoelectric focusing. The PPL complex exhibited proteolytic activity against gelatin and the activity was only partly reduced by heat treatment. The function of the slime polymer as protein denatured was studied. The complex formed micelles similar to anionic detergents and it inhibited the precipitation and coagulation of proteins by trichloroacetic acid. Lysozyme was totally inactivated when treated with the PPL complex. By gel chromatography binding studies, the PPl complex was found to bind lysozyme in the ratio of 1 to 5.8 (w/w). After separation of added protein from the complex the anticoagulation effect on the protein remained. The biological function of the PPL complex was demonstrated with hemoglobin. When all susceptible peptide bonds in PPL-treated hemoglobin were hydrolyzed by trypsin only 20% in the urea-denatured protein were attacked. The combined role of slime and proteolytic activity is discussed.Abbreviations Used PPL protein-polysaccharide-lipide - TCA trichloroacetic acid - BSA bovine serum albumin - Tris tris-(hydroxymethyl)aminomethane - CMC critical micelle concentration - DNFB 2,4-dinitrofluorobenzene - DNP N-dinitrophenyl - SDS sodium dodecylsulphate - H.U. Hultin units     

The effect of copper and nitrogen on the amino acid composition of oat straw

Summary The effect of fertilization with nitrogen and copper on the amino acid composition of oat straw has been studied.The plants (Avena sativa cv Yielder) were grown in peat with a very low copper content and supplied with two levels of nitrogen (NH₄ or NO₃) and three levels of copper sulphate.The higher level of nitrogen stimulated growth only when copper was added, whereas, without copper, it had an adverse effect on growth and prevented grain formation altogether. The higher level of nitrogen increased the nitrogen content of the straw at all levels of copper, but particularly in plants receiving no copper.Total amino acids in the straw hydrolysate of copper sufficient oats accounted for about 50% of the total N and was about 20% higher in copper-deficient tissues. The addition of copper caused a decrease in the amounts of all amino acids. The relative proportions of most of the amino acids to glycine remained fairly constant. Threonine, serine, alanine, iso-leucine, histidine and arginine showed small significant differences with copper treatment, whereas valine, tyrosine, phenylalanine, proline, lysine and cysteic acid (derived from cysteine and cystine) showed no differences. The proportion of aspartic acid relative to glycine in the straw hydrolysate was greatly increased in copper deficient plants supplied with the higher level of nitrogen, particularly as ammonium. The proportion of glutamic acid was also increased by the higher level of nitrogen, but showed no effect of added copper. Most of the difference in aspartic acid could be accounted for as free asparagine. The possible reasons for higher proportions of asparagine are discussed in relation to the metabolism of the oat plant.