Analysis of globulin maturation in developing sunflower seeds

The synthesis of major storage globulin polypeptides has been examined in developing seeds of sunflower(Helianthus annuus L.). Analyses of total proteins and purified globulins, also called helianthinin, by gel electrophoresis and immunoelectrophoresis have shown that a burst of protein synthesis and accumulation occurs around 10 d after flowering. There is no mature globulin before that time and only small amounts of precursor forms can be detected. Thus, 10–12 d after flowering appears to be a transition period during which genetic information for the globulin becomes actively expressed. Immunoelectrophoresis has confirmed that globulin is the main storage protein, at seed maturation, accounting up to 70 % of total proteins per kernel. Pulse chase experiments have shown that synthesis initially involves the formation of high molecular mass precursors and that storage proteins are post-translationally processed. Intermediary products, with molecular mass higher than early translational products, can be detected, together with mature globulin polypeptides.     

The separation and comparison of albumin complexes of seed proteins in three cultivars ofPhaseolus vulgaris

Complexes of water soluble proteins (albumins) were investigated in three cultivarsof Phaseolus vulgaris, viz: Yeltruská Saxa, Vainica Saavegra B, and Krupnaya sakharnaya. The first two cultivars exhibit haemagglutinating activity against rabbit erythrocytes, but have different elution profiles on Sephadex G-100. Their individual peaks have a different subunit composition, as revealed by SDS gel electrophoresis, as well as a different immunoelectrophoretic pattern, although proteins I and II of the specificity Veltruská Saxa are present in both cultivars. The cultivar Krupnaya sakharnaya expressively differs from the preceding lectin cultivars; it has no erythroagglutinating activity, its albumin complex has a high-molecular component, absent in the preceding ones, and has no lectin peak in the region of molecular mass of 100 000 to 200 000. Immunoelectrophoresis gave no evidence of protein I and II of the specificity Veltruská Saxa.     

Sequence interrelationships of the subunits of vicilin from pea seeds

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than M_r～50 000 (i.e., M_r 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within M_r～50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than M_r～50 000 arose by endoproteolytic cleavage of parent molecules within the M_r～50 000 group. Cleavage in different M_r 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than M_r～50 000, with the possible exception of the M_r34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the M_r～50 000 group.     

Purification and properties of thiosulfate reductase from Desulfovibrio gigas

Thiosulfate reductase of the dissimilatory sulfate-reducing bacterium Desulfovibrio gigas has been purified 415-fold and its properties investigated. The enzyme was unstable during the different steps of purification as well as during storage at-15°C. The molecular weight of thiosulfate reductase estimated from the chromatographic behaviour of the enzyme on Sephadex G-200 was close to 220 000. The absorption spectrum of the purified enzyme exhibited a protein peak at 278 nm without characteristic features in the visible region. Thiosulfate reductase catalyzed the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate, and exhibited tetrathionate reductase activity. It did not show sulfite reductase activity. The optimum pH of thiosulfate reduction occurred between pH 7.4 and 8.0 and its K _m value for thiosulfate was calculated to be 5·10^-4 M. The sensitivity of thiosulfate reductase to sulfhydryl reagent and the reversal of the inhibition by cysteine indicated that one or more sulfhydryl groups were involved in the catalytic activity. The study of electron transport between hydrogenase and thiosulfate reductase showed that the most efficient coupling was obtained with a system containing cytochromes c ₃ (M _r =13000) and c ₃ (M _r =26000).     

Isolation and characterization of glutamine synthetase from the diazotroph Azospirillum brasilense

• 1.1. Glutamine synthetase was purified from the diazotroph Azospirillum brasilense.
• 2.2. The holoenzyme with a M_r of 630,000 is composed of 12 subunits of M_r 52,000.
• 3.3. A modified subunit of M_r 53,000 was also found by electrophoresis under denaturing conditions.
• 4.4. It is shown that the M_r 53,000 species is the adenylylated subunit.
• 5.5. The apparent K_m values for glutamate, ATP and ammonia were 2.5 ± 0.3 mM, 200 ± 20 μM and42 ± 2 μM, respectively.
• 6.6. Levels of glutamine synthetase activity in A. brasilense cells varied by a factor of 8 depending on the nitrogen source and its concentration in the growth medium.

     

Preclinical evaluation of caprylic acid-fractionated IgG antivenom for the treatment of Taipan (Oxyuranus scutellatus) envenoming in Papua New Guinea

Background

Snake bite is a common medical emergency in Papua New Guinea (PNG). The taipan, Oxyuranus scutellatus, inflicts a large number of bites that, in the absence of antivenom therapy, result in high mortality. Parenteral administration of antivenoms manufactured in Australia is the current treatment of choice for these envenomings. However, the price of these products is high and has increased over the last 25 years; consequently the country can no longer afford all the antivenom it needs. This situation prompted an international collaborative project aimed at generating a new, low-cost antivenom against O. scutellatus for PNG.

Methodology/Principal Findings

A new monospecific equine whole IgG antivenom, obtained by caprylic acid fractionation of plasma, was prepared by immunising horses with the venom of O. scutellatus from PNG. This antivenom was compared with the currently used F(ab'')₂ monospecific taipan antivenom manufactured by CSL Limited, Australia. The comparison included physicochemical properties and the preclinical assessment of the neutralisation of lethal neurotoxicity and the myotoxic, coagulant and phospholipase A₂ activities of the venom of O. scutellatus from PNG. The F(ab'')₂ antivenom had a higher protein concentration than whole IgG antivenom. Both antivenoms effectively neutralised, and had similar potency, against the lethal neurotoxic effect (both by intraperitoneal and intravenous routes of injection), myotoxicity, and phospholipase A₂ activity of O. scutellatus venom. However, the whole IgG antivenom showed a higher potency than the F(ab'')₂ antivenom in the neutralisation of the coagulant activity of O. scutellatus venom from PNG.

Conclusions/Significance

The new whole IgG taipan antivenom described in this study compares favourably with the currently used F(ab'')₂ antivenom, both in terms of physicochemical characteristics and neutralising potency. Therefore, it should be considered as a promising low-cost candidate for the treatment of envenomings by O. scutellatus in PNG, and is ready to be tested in clinical trials. Author Summary Snake bite envenoming represents an important public health hazard in Papua New Guinea (PNG). In the southern lowlands of the country the majority of envenomings are inflicted by the taipan, Oxyuranus scutellatus. The only currently effective treatment for these envenomings is the administration of antivenoms manufactured in Australia. However, the price of these products in PNG is very high and has steadily increased over the last 25 years, leading to chronic antivenom shortages in this country. As a response to this situation, an international partnership between PNG, Australia and Costa Rica was initiated, with the aim of generating a new, low-cost antivenom for the treatment of PNG taipan envenoming. Horses were immunised with the venom of O. scutellatus from PNG and whole IgG was purified from the plasma of these animals by caprylic acid precipitation of non-immunoglobulin proteins. The new antivenom, manufactured by Instituto Clodomiro Picado (Costa Rica), was compared with the currently available F(ab'')₂ antivenom manufactured by CSL Limited (Australia). Both were effective in the neutralisation of the most relevant toxic effects induced by this venom, although the whole IgG antivenom showed a higher efficacy than the F(ab'')₂ antivenom in the neutralisation of the coagulant activity.     

Phosphoenolpyruvate synthetase inMethanobacterium thermoautotrophicum

The thermophilic autotrophMethanobacterium thermoautotrophicum assimilates CO₂ via a novel pathway rather than via the Calvin cycle. The central intermediate of this pathway is acetyl CoA which is reductively carboxylated to pyruvate. Cell extracts of the organism contained phosphoenolpyruvate synthetase with a specific activity of 100 nmol min^-1 mg^-1 protein (65°C). Pyruvate kinase and pyruvate, phosphate dikinase were not detected. Phosphoenolpyruvate synthetase was partially purified (50-fold) and the following reaction stoichiometry was established: $${\text{Pyruvate + ATP + H}}_{\text{2}} {\text{O }} \to {\text{ Phosphoenolpyruvate + AMP + P}}_{\text{i}} $$ The enzyme activity was depedent on free Mg²⁺ ions, NH ₄ ⁺ or K⁺ ions, and SH-groups. Mn²⁺, but not Ca²⁺, could partially substitute for Mg²⁺; Na⁺ could not substitute for K⁺ or NH ₄ ⁺ . The pH-optima,V _max-values and the apparentK _M-values for the substrates of the enzyme in both directions were determined. Thermodynamic, kinetic and regulatory features indicate that, in vivo, the enzyme functions in the direction of phosphoenolpyruvate synthesis from pyruvate. Not only is the synthesis of phosphoenolpyruvate via the PEP synthetase reaction energetically favorable; the enzyme also catalyzed this synthesis 100 times faster than the reverse reaction, the apparentK _M value for pyruvate (40 μM) being low and the apparentK _M value for phosphate (100 mM) being high. Furthermore, AMP, ADP, PP and α-ketoglutarate were inhibitors of PEP synthesis, indicating that the enzyme activity may be controlled in vivo. The role of phosphoenolpyruvate synthetase in autotrophic CO₂ assimilation pathway ofMethanobacterium, as expected from previous labelling studies, is confirmed.     

Coagulation factor V exists uncomplexed in bovine plasma

Bovine coagulation factor V has been examined immunochemically to ascertain whether the coagulant polypeptide (h) with M_r = 290 000–330 000 is complexed in plasma with a second immunochemically distinct polypeptide (I₂) of M_r = 400 000. Antiserum containing antibodies to h and l₂ detects the l₂ polypeptide eluting earlier than the h chain on gel filtration of plasma with either added calcium or EDTA, consistent with the behavior of a higher molecular weight noninteracting species. An immobilized monospecific antibody to l₂ removes only the l₂ polypeptide from a purified factor V preparation containing both h and l₂. Moreover, while a monospecific antibody to the h chain was able to precipitate purified radioactively labelled h chain alone or mixed with plasma, the l₂ antibody was unable to precipitate radioactively labelled h chain even after attempted recombination of the h chain with l₂ present in plasma. These studies indicate that the l₂ polypeptide is not complexed to the h chain in a purified system or in plasma and reinforce the conclusion that factor V is a single polypeptide chain uncomplexed in plasma.     

Relation of some M1 characters to the frequency of M2 mutants inArabidopsis thaliana

The relation of the M₁ root length and the frequency of M₁ chlorophyll chimeras to the sterility grade and to the frequency of M₂ mutants ofArabidopsis thaliana is demonstrated.     

Functional characterization of single-chain factor X from rat liver

¹⁴C-Labeled single-chain factor X prepared by vitamin K-dependent carboxylation in vitro was partially purified by adsorption to BaSO₄ and chromatography on DEAE-Sephacel. Known activators of factor X were analyzed for their effect on the single-chain molecule. ¹⁴C-Labeled factor X antigens were recovered immunochemically from incubation mixtures and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation with trypsin resulted in the generation of factor Xa clotting activity, and the ¹⁴C-labeled product migrated after reduction with an apparent molecular weight of 22,500 ± 1500 (mean ± 1 SD). The light chain produced by factor Xa was similar to that produced by trypsin (M_r 24,500 ± 1500; mean ± 1 SD). Incubation of single-chain factor X with factor VII and thromboplastin, factor IXa, or the factor X activating enzyme from Russell's viper venom gave a reducible product with a light chain of higher apparent molecular weight (M_r 37,000–38,000). Incubation with factor VII and thromboplastin also resulted in the generation of factor Xa clotting activity. Incubation of single-chain factor X with platelets resulted in the binding of about 20% of the ¹⁴C. The bound ¹⁴C-labeled factor X antigen released by freezing and thawing in the presence of EDTA was reduced to give a ¹⁴C-labeled polypeptide with M_r 31,000. Walker 256 tumor cells bound about 30% of the ¹⁴C. The bound material, after reduction, gave a ¹⁴C-labeled polypeptide with M_r 23,000.     

Mitochondrial proline dehydrogenase deficiency in hyperprolinemic PRO/Re mice: Genetic and enzymatic analyses

Genetic analyses, involving backcross and F₂ matings, demonstrate that the type I hyperprolinemia of PRO/Re mice is caused by an abnormal allele at a single locus designated pro-1. Mice homozygous for this allele (pro-1 ^b /pro-1^b) possess a deficiency in the activity of component 1 of mitochondrial proline dehydrogenase. In liver mitochondria of normal C57BL/6J mice, two proline dehydrogenase activity components are demonstrable by electrophoretic resolution of Triton X-100 solubilized extracts. In mitochondria of PRO/Re mice, the activity of component 1 is not readily detectable. Residual proline dehydrogenase activity in PRO/Re mitochondria appears, therefore, to be due in large measure to activity component 2 which is more stable to incubation at 40 C, exhibits slower electrophoretic mobility, and is less reactive to menadione. Kinetic analyses demonstrate a K _m (proline) for the Triton X-100 solubilized enzyme activities of PRO/Re and C57BL/6J liver mitochondria of 0.4 M and 2.9×10^?3 M, respectively. C57BL/6J enzyme activity is inhibited by high substrate concentration. The activity of component 1 was not detected in other subcellular fractions of PRO/Re liver obtained by differential centrifugation. Abnormal control of respiratory chain function in PRO/Re mitochondria appears to involve primarily proline oxidation, as indicated by the level of activity of several inner membrane enzymes.     

Studies on the structure, protein composition and aseembly of the neck of bacteriophage T4

We have developed an osmotic shock procedure which disconnects the tail from the head of intact bacteriophage T4, leaving the neck region attached to the tail. Purification of these necked tails permitted detailed structural observations of the neck and the collar/whisker complex attached to it, as well as comparison by gel electrophoresis with tails lacking the neck. Five or six neck proteins were found: N1 (M_r = 52,000; 39 copies/phage) is the product of the wac³ gene (Pwac), forms both the collar and six whiskers as a multimeric fibrous protein, and probably assembles onto phage after head to tail joining; N2 (M_r= 35,000; 5 to 6 copies/phage), N3 (M_r= 33,000; 17 copies/phage) identified here as P13, and N6 (M_r= 28,000; 10 to 11 copies/phage) are all assembled in heads prior to tail joining; N4 (M_r= 32,000; 6 to 9 copies/phage) is unusual in that it is present in wac or wac⁺ phage and necked tails but is absent from purified heads; N5 (M_r =29,000) is probably P14 and like N4 is not found in heads. However, while we find one to two copies of N5 per necked tail, we have not observed it in phage.An aberrant neck structure called the extension assembles on the distal end of the tail connector late (after 33 min, 30 °C) in head-defective, mutant-infected cells. The extension contains five of the six neck proteins (N2 is absent), and blocks head to tail joining in vitro. Mutations in genes 13 and 14, and the double mutant 49:Wac block extension assembly.Other results show that the wac mutant E727J is an amber lesion, and that Pwac can assemble on collarless, wac phage in vitro.     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

The cytochromes of a marine Beggiatoa

Naturally grown Beggiatoa filaments, occurring in massive near-mono-cultures at a “black smoker” wall of the Guaymas Basin hydrothermal vent site, were harvested and used for the analysis of their cytochromes. The cytochromes have been characterized by gel permeation chromatography, optical spectroscopy and redox potentiometry. Only c-type cytochromes were detected; a small, high potential cytochrome c that seems typical of its class, and a large complex (M_r 210,000) containing at least four thermodynamically distinct c-type hemes, which was partially dissociated by chromatography on DEAE-Sepharose. The hemes of the large complex have appropriate oxidation-reduction midpoint potentials (E_m7 +240 mV, +15 mV,-160 mV,-340 mV) to be involved in the metabolism of sulfide, which is presumed to be the source of reductant for this organism.     

Host-parasite relationship 2- cholinesterase activity of the larvae of the rice mothCorcyra cephalonica [Lep.: Pyralidae] parasitized byBracon hebetor [Hym.: Braconidae]

The parasitic waspBracon hebetor Say paralyses its host, the larvae of the rice mothCorcyra cephalonica Staint, before oviposition. The effect of injected venom on the activity of cholinesterase was studied. The ChE activity of the paralysed larvae showed a partial inhibition of about 40% than that of the normal larvae. Such inhibition in ChE activity interferes with the synaptic transmission and/or may result in neuromuscular block causing the observed paralysis.     

Vibrio madracius sp. nov. Isolated from Madracis decactis (Scleractinia) in St Peter & St Paul Archipelago,Mid-Atlantic Ridge,Brazil

Three novel isolates (A-354^T, A-328, and A-384) were retrieved from apparently healthy scleractinian Madracis decactis in the remote St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil. The novel isolates formed a distinct lineage based on the phylogenetic reconstruction using the 16S rRNA and pyrH gene sequences. They fell into the Mediterranei clade and their closest phylogenetic neighbour was V. mediterranei species, sharing upto 98.1 % 16S rRNA gene sequence similarity. Genomic analysis including in silico DDH, MLSA, AAI and genomic signature distinguished A-354^T from V. mediterranei LMG 19703 (=AK1) with values of 33.3, 94.2, 92 %, and 11.3, respectively. Phenotypically, the novel isolates can be differentiated from V. mediterranei based on the four following features. They do not grow at 8 % NaCl; use d-gluconic acid but not l-galactonic acid lactone as carbon source; and do not have the fatty acid C_18:0. Differentiation from both the other Mediterranei clade species (V. maritimus and V. variabilis) is supported by fifteen features. The novel species show lysine decarboxylase and tryptophan deaminase, but not gelatinase and arginine dihydrolase activity; produce acetoin; use α-d-lactose, N-acetyl-d-galactosamine, myo-Inositol, d-gluconic acid, and β-hydroxy-d,l-butyric acid; and present the fatty acids C_14:0 iso, C_15:0 anteiso, C_16:0 iso, C_17:0 anteiso, and C_17:1x8c . Whole-cell protein profiles, based on MALDI-TOF, showed that the isolates are not clonal and also distinguished them from the closes phylogenetic neighbors. The name Vibrio madracius sp. nov. is proposed to encompass these novel isolates. The G+C content of the type strain A-354^T (=LMG 28124^T=CBAS 482^T) is 44.5 mol%.