Heterogeneous distribution of glycosyltransferases in the endoplasmic reticulum of castor bean endosperm

Endoplasmic reticulum membranes stripped of attached ribosomes were isolated from homogenates of germinating castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation. The isolated endoplasmic reticulum fraction was further separated into two major membrane subfractions by centrifugation on a flotation gradient. Both subfractions appeared to be derived from the endoplasmic reticulum inasmuch as they share several enzymic markers including cholinephosphotransferase, NADH-cytochrome c reductase, and glycoprotein fucosyl-transferase and phase separation of membrane polypeptides using Triton X-114 revealed a striking similarity in both their hydrophilic and hydrophobic protein components. The endoplasmic reticulum membrane subfractions contain glycoproteins which were readily labeled by incubating intact endosperm tissue with radioactive sugars prior to fractionation.

Castor bean endosperm endoplasmic reticulum apparently exhibits a degree of enzymic heterogeneity, however, since the enzymes responsible for the synthesis of dolicholpyrophosphate N-acetylglucosamine and dolicholmonophosphate mannose together with their incorporation into the oligosaccharide-lipid precursor of protein N-glycosylation were largely recovered in a single endoplasmic reticulum subfraction.

     

Isolation of a factor, from the capsule of a granulosis virus, synergistic for a nuclear-polyhedrosis virus of the armyworm

When the capsules of a granulosis virus are fed together with the polyhedra of a nuclear-polyhedrosis virus to larvae of the armyworm, Pseudaletia unipuncta, the former enhances the infectivity of the latter virus, a synergistic interaction. The enhancement of infectivity depends upon the concentration of the polyhedra and the capsules. The factor responsible for the synergistic activity in the capsule can be dissolved in alkaline solution, separated from the virus particles by centrifugation, and further purified by Sephadex G-200 gel filtration with 4 m urea. The fraction obtained from Sephadex filtration and containing the synergistic factor can be separated into two components by disc-electrophoresis with 8 m urea. Both components possess synergistic activity. The ID₅₀ of the synergistic factor corresponds to 0.0015 OD₂₈₀. Its optimum pH is 8.5. Synergism is most evident when the factor is fed to larvae together with the polyhedra or is fed 24 hr prior to the ingestion of the polyhedra. The factor appears to be a simple or a conjugated protein of the capsule.     

Interrogating the Dimerization Interface of the Prion Protein Via Site-Specific Mutations to p-Benzoyl-L-Phenylalanine

Transmissible spongiform encephalopathies are centered on the conformational transition of the prion protein from a mainly helical, monomeric structure to a β-sheet rich ordered aggregate. Experiments indicate that the main infectious and toxic species in this process are however shorter oligomers, formation of which from the monomers is yet enigmatic. Here, we created 25 variants of the mouse prion protein site-specifically containing one genetically-incorporated para-benzoyl-phenylalanine (pBpa), a cross-linkable non-natural amino acid, in order to interrogate the interface of a prion protein-dimer, which might lie on the pathway of oligomerization. Our results reveal that the N-terminal part of the prion protein, especially regions around position 127 and 107, is integral part of the dimer interface. These together with additional pBpa-containing variants of mPrP might also facilitate to gain more structural insights into oligomeric and fibrillar prion protein species including the pathological variants.     

Analysis of 953 Human Proteins from a Mitochondrial HEK293 Fraction by Complexome Profiling

Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification – mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.     

Chemical composition and rate of synthesis of the larval salivary secretion of the fly Rhynchosciara americana

The salivary secretion of Rhynchosciara americana was chemically analysed. The secretion shows a yellow colour, with a pH of 7·5 and protein as its major component (94·5 per cent of the secretion dry weight). Carbohydrates are minor components of the secretion which amount to 3·4 per cent of the secretion dry weight, of which 2·3 per cent are neutral carbohydrates and 1·1 per cent are galactosamine. The major amino acids present in the secretion proteins are aspartic acid, glycine, serine, and glutamic acid. The salivary secretion proteins can be separated into eleven protein fractions by urea-acrylamide gel electrophoresis from which nine fractions are PAS positive. The salivary pigment moves together with the protein fraction No. 8, which is quantitatively the most important one, and has spectral characteristics identical to a haemolymph pigment. The higher rate of gland protein labelling by ¹⁴C-phenylalanine determined in vivo and in vitro occurs around the middle of the spinning stage at the same time as the appearance of the large chromosomal puffs. The rôle of the salivary secretion in cocoon production is discussed.     

Macrocyclic diterpene esters of the cytotoxic fraction from Euphorbia kamerunica

The ether-soluble resin of the latex of Euphorbia kamerunica was separated into a non-polar and a polar fraction by gradient elution column chromatography. From the non-polar fraction three macrocyclic diterpene esters were isolated by adsorption and partition preparative TLC methods. These compounds were identified as 3,7,8-triacetyl-ingol-12-tigliate,3,7-diacetyl-ingol-12-tigliate and ingol-tetra-acetate by means of spectroscopic data, together with hydrolysis and partial synthesis.     

In vitro oxidation of indoleacetic Acid by soluble auxin-oxidases and peroxidases from maize roots

Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl₂ and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [³H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.     

Functional Characterization of Pseudomonas Contact Dependent Growth Inhibition (CDI) Systems

Contact-dependent inhibition (CDI) toxins, delivered into the cytoplasm of target bacterial cells, confer to host strain a significant competitive advantage. Upon cell contact, the toxic C-terminal region of surface-exposed CdiA protein (CdiA-CT) inhibits the growth of CDI^- bacteria. CDI⁺ cells express a specific immunity protein, CdiI, which protects from autoinhibition by blocking the activity of cognate CdiA-CT. CdiA-CT are separated from the rest of the protein by conserved peptide motifs falling into two distinct classes, the “E. coli”- and “Burkholderia-type”. CDI systems have been described in numerous species except in Pseudomonadaceae. In this study, we identified functional toxin/immunity genes linked to CDI systems in the Pseudomonas genus, which extend beyond the conventional CDI classes by the variability of the peptide motif that delimits the polymorphic CdiA-CT domain. Using P. aeruginosa PAO1 as a model, we identified the translational repressor RsmA as a negative regulator of CDI systems. Our data further suggest that under conditions of expression, P. aeruginosa CDI systems are implicated in adhesion and biofilm formation and provide an advantage in competition assays. All together our data imply that CDI systems could play an important role in niche adaptation of Pseudomonadaceae.     

A functional genomic analysis of Arabidopsis thaliana PP2C clade D

In the reference dicot plant Arabidopsis thaliana, the PP2C family of P-protein phosphatases includes the products of 80 genes that have been separated into ten multi-protein clades plus six singletons. Clade D includes the products of nine genes distributed among three chromosomes (APD1, At3g12620; APD2, At3g17090; APD3, At3g51370; APD4, At3g55050; APD5, At4g33920; APD6, At4g38520; APD7, At5g02760; APD8, At5g06750; and APD9, At5g66080). As part of a functional genomics analysis of protein phosphorylation, we retrieved expression data from public databases and determined the subcellular protein localization of the members of clade D. While the nine proteins have been grouped together based upon primary sequence alignments, we observed no obvious common patterns in expression or localization. We found chimera with the GFP associated with the nucleus, plasma membrane, the endomembrane system, and mitochondria in transgenic plants.     

Wagyu–Angus cross improves meat tenderness compared to Angus cattle but unaffected by mild protein restriction during late gestation

For the spring-calving beef herds, late gestation coincides with winter and early spring, when cows are dependent on feed supplements with low quality hay, which is hard to meet their nutrient requirements. However, the effects of deficiencies of metabolizable protein intake during late gestation on offspring beef quality are unclear. Wagyu cattle have excellent marbling, and insemination with Wagyu vs Angus semen is a practical option for beef producers to improve beef quality. To test, Angus cows (621 ± 73 kg) were selected and randomly separated into two groups. Each group was inseminated with either Angus or Wagyu semen. During the last 90 days of gestation, cows in each group were further separated and received either a low protein diet (85% of the NRC metabolizable protein requirement), which was a low quality hay-based diet common in northwestern region of the U.S., or an adequate protein diet (108% NRC requirement). All progeny was managed together and harvested at a final BW of 576.5 ± 16.6 kg. Wagyu-sired offspring had higher marbling scores and quality grades than Angus (P < 0.01). Protein supplementation did not affect Slice Shear Force (SSF) in either breeds (P = 0.60). However, Wagyu-sired cattle had lower SSF than Angus-sired (P < 0.01). In addition, Wagyu-sired cattle had higher intramuscular fat (P < 0.05) and total collagen content (P < 0.05), but Angus-sired had greater mature collagen cross-links, as shown by higher contents of Pyridinoline (P < 0.01) and Ehrlich Chromogen (P < 0.01). Consistently, the mRNA expression of enzymes catalyzing collagen cross-linking was higher in Angus-sired offspring, including Plod 1 (P < 0.05), Plod 2 (P = 0.08), and P4Hα 2 (P < 0.01). In conclusion, Wagyu-sired cattle had greater tenderness and marbling score compared to Angus-sired, which was associated with lower collagen cross-links. Feeding mature grass hay-based diet with relatively low protein content during late gestation had no major effect on beef quality of subsequent cattle.     

酿酒酵母FFC2146 胞内蛋白及胞外蛋白双向电泳条件优化及图谱建立

通过对酿酒酵母(Saccharomyces cerevisiae)的培养基、培养条件及蛋白质提取方案的优化,建立了酿酒酵母胞外和胞内蛋白双向电泳图谱制作方法。在YNB培养基中培养20 h,经过离心取上清-超滤-冻干可得到酿酒酵母胞外蛋白质样品;用SDS缓冲液悬浮酵母细胞-煮沸-超声-增溶,得到了酿酒酵母胞内蛋白质样品。经过双向电泳分离、硝酸银染色和PDQuest图像分析可以检测到了200多种酿酒酵母胞外蛋白和500多种酿酒酵母胞内蛋白。     

STUDIES ON INSULIN BIOSYNTHESIS : Subcellular Distribution of Leucine-H3 Radioactivity During Incubation of Goosefish Islet Tissue

Islet tissue from the goosefish (Lophius piscatorius) was incubated in the presence of leucine-H³. The tissue was then separated into subcellular fractions and the radioactivity determined in total acid alcohol-soluble proteins, insoluble proteins, and insulin. At any time, microsomal protein had a higher activity than secretion granule protein. Pulse-chase experiments further suggest the microsomes as primary sites of protein synthesis. The data are evidence for microsomal synthesis of insulin and for its subsequent transfer into the secretion granules.     

Evolution of Tertiary Structure of Viral RNA Dependent Polymerases

Viral RNA dependent polymerases (vRdPs) are present in all RNA viruses; unfortunately, their sequence similarity is too low for phylogenetic studies. Nevertheless, vRdP protein structures are remarkably conserved. In this study, we used the structural similarity of vRdPs to reconstruct their evolutionary history. The major strength of this work is in unifying sequence and structural data into a single quantitative phylogenetic analysis, using powerful a Bayesian approach.The resulting phylogram of vRdPs demonstrates that RNA-dependent DNA polymerases (RdDPs) of viruses within Retroviridae family cluster in a clearly separated group of vRdPs, while RNA-dependent RNA polymerases (RdRPs) of dsRNA and +ssRNA viruses are mixed together. This evidence supports the hypothesis that RdRPs replicating +ssRNA viruses evolved multiple times from RdRPs replicating +dsRNA viruses, and vice versa. Moreover, our phylogram may be presented as a scheme for RNA virus evolution. The results are in concordance with the actual concept of RNA virus evolution. Finally, the methods used in our work provide a new direction for studying ancient virus evolution.     

Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins

The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel β-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa     

Structural investigations of the extracellular polysaccharides elaborated by Beijerinckia mobilis

The extracellular mucilage from Beijerinckia mobilis, a member of the Azotobacteriaceae, after removal of contaminating protein, was separated into a neutral polysaccharide (N-2, 10%); a neutral, dialysable fraction (N-1, 5%), consisting of glucose and oligosaccharides containing glucose, arabinose, and rhamnose; and an acidic polysaccharide (85%). N-2 (mol. wt, 1900) was highly branched and comprised glucopyranose, mannopyranose, and arabinofuranose residues (1:1:1). The various linkages were determined. The acid fraction was a polymer of high molecular weight composed of L-guluronic acid (65%), D-glucose (15%), and D-glycero-D-mannoheptose (20%), together with acetic and pyruvic acids. From the results of methylation, periodate oxidation, and partial hydrolysis, a branched molecule with a backbone of guluronic acid and heptose, and side chains of glucose and guluronic acid is proposed. Pyruvic acid was found to be acetal-linked to 2?5% of the heptose residues. The similarities between this polysaccharide and that from the related species Azotobacter indicum are discussed.     

Resolution and reconstitution of succinate-cytochrome c reductase. Preparations and properties of high purity succinate dehydrogenase and ubiquinol—cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c₁ III complex).An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b-c₁ complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c₁ complex was separated into cytochrome b-c₁ III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate.The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low K_m ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C.Chemical composition analysis of cytochrome b-c₁ III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation.When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.     

Replication initiated at the origin (oriC) of the E. coli chromosome reconstituted with purified enzymes

A crude soluble enzyme system capable of authentic replication of a variety of oriC plasmids has been replaced by purified proteins constituting three functional classes: initiation proteins (RNA polymerase, dnaA protein, gyrase) that recognize the oriC sequence and presumably prime the leading strand of the replication fork; replication proteins (DNA polymerase III holoenzyme, single-strand binding protein, primosomal proteins) that sustain progress of the replication fork; and specificity proteins (topoisomerase I, RNAase H₁ protein HU) that suppress initiation of replication at sequences other than oriC, coated with dnaA protein. Protein HU and unidentified factors in crude enzyme fractions stimulate replication at one or more stages. Replication has been separated temporally and physically into successive stages of RNA synthesis and DNA synthesis.     

Partial In Vitro Digestion of Active Gliadin-Related Peptides in Celiac Disease

Peptides corresponding to residues 75–86 (RPQQPYPQPQPQ) and 75–85 of the A-gliadin structure, which were shown to be active in an animal model of celiac disease, were digested in vitro with small intestinal mucosa from children with celiac disease in remission and with mucosa from normal children. The products of digestion were separated into two fractions by gel permeation chromatography. Undigested residues (M _r > 400 fraction) from both peptides contained mainly glutamine, proline, and tyrosine, while the digested materials (M _r < 400 fraction) contained mainly proline, glutamine and arginine. Much larger amounts of undigested peptides were obtained from digestion with celiac mucosa than from normal mucosa. The results with peptide 75–86 indicated that the octapeptide 77–84 (QQPYPQPQ) was the main residual component and this peptide was shown to be active in the assay. Peptide 77–84 was also obtained as a residue from digestion of peptide 75–85, together with heptapeptide 77–83. The results lend further support for a primary mucosal defect in celiac disease and indicate that residual peptides in the small intestine of patients with the disease still retain appreciable toxicity.     

The structure and composition of the cyst wall of the metacercaria of Cloacitrema narrabeenensis (Howell & Bearup, 1967) (Digenea: Philophthalmidae).

The mstacercarial cyst of Cloacitrema narrabeenensis which is formed in the open is composed of four layers: an outermost layer of acid mucopolysaccharide, a layer of protein which is presumed to be tanned, a layer of neutral mucopolysaccharide and an innermost layer of keratinized protein. The two layers which together form the outer cyst wall can be split off by slight pressure from the two remaining layers which together form the inner cyst wall. In the centre of the ventral side of the inner cyst wall, the keratinized layer is incomplete and this ventral plug region is composed of neutral mucopolysaccharide. The cyst wall is therefore very similar to that of Fasciola hepatica, the main difference being that the order of the two layers of the outer cyst is reversed. General evolutionary and functional relationships of metacercarial cysts are discussed.