Early effects of excess cadmium uptake in Phaseolus vulgaris

Abstract. Seedlings of Phaseolus vulgaris were exposed to solutions containing Cd²⁺ in the range 0 to 1 molm⁻³. Ethylene formation started following 3 h of exposure to 10⁻², 10⁻¹ and 1 mol m⁻³ Cd²⁺, peaked at 18 h and returned to a relatively low rate after 24 h. Cadmium-induced ethylene formation depended on the formation of 1-aminocyclopropane-1-carboxylic acid (ACC). Aminoethoxyvinylglycine (AVG, 0.1 mol m⁻³) inhibited ACC accumulation and ethylene production during exposure to 0.2 mol m⁻³ Cd²⁺.
Activity of soluble and ionically-bound peroxidase increased after 18 h of exposure to Cd²⁺ concentrations above 10⁻³ mol m⁻³ due to an increase in activity of cathodic isoperoxidases. Stimulation of soluble and ionically-bound peroxidase by 0.2 mol m⁻³ Cd²⁺ was reduced in the presence of 0.1 mol m⁻³ AVG.
Accumulation of soluble and insoluble ('ligninlike') phenolics was found in plants exposed to Cd²⁺ (10⁻² mol m⁻³ or above) in the presence or absence of AVG. Deposition of insoluble (autofluorescing) material occurred in cell walls around vessels and was associated with reduced expansion and water content of leaves.     

Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.     

Selective cleavage of glycyl bonds by papaya proteinase IV

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc-Ala-Ala-Gly-NHPhNO₂ is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in β-trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly-65 and Gly-23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV.     

Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations.

The rates of calcium transport and Ca2+-dependent ATP hydrolysis by rabbit skeletal muscle sarcoplasmic reticulum were stimulated by monovalent cations. The rate of decomposition of phosphoprotein intermediate of the Ca2+-dependent ATPase of sarcoplasmic reticulum was also increased by these ions to an extent that is sufficient to account for the stimulation of calcium transport and Ca2+-dependent ATPase activity. The order of effectiveness of monovalent cations tested at saturating concentrations in increasing rate of phosphoprotein decomposition is: K+, Na+ greater than Rb+, NH4+ greater than Cs+ greater than Li+, choline+, Tris+.     

STUDIES ON SEEDS : IV. Lipid Composition of Bean Cotyledon Vesicles

Lipid content has been determined for two types of lipid-rich vesicles isolated from bush bean cotyledon at 24 hr of germination. The larger, nonassociating vesicles are four to six times richer in triglyceride than the smaller vesicles which associate strongly among themselves, as well as with smooth membranes in the cell. The larger vesicles contain about 640 µmoles of phospholipid per gram of protein, while the smaller vesicles have only one-half to two-thirds as much phospholipid per gram of protein. The ratio of individual phospholipids in both kinds of vesicles is close to 20% phosphatidylethanolamine, 60% phosphatidylcholine, and 20% phosphatidylinositol. The fatty acid composition of all phospholipids is similar, and quite different from that of triglyceride, which contains twice as much linolenic acid and less than one-fourth as much palmitic acid. Pea cotyledon has quantitatively the same lipid content as bean cotyledon.     

Kinetochore microtubules and chromosome movement during prometaphase in Drosophila melanogaster spermatocytes studied in life and with the electron microscope

Prometaphase I chromosome behavior was examined in wild-type Drosophila melanogaster primary spermatocytes. Cine analysis of live cells reveals that bivalents exhibit complex motions that include (1) transient bipolar orientations, (2) simultaneous reorientation of homologous kinetochores, (3) movements not parallel to the spindle axis, and (4) movement along the nuclear membrane. — Kinetochores and kinetochore microtubule have been analyzed for bivalents previously studied in life. The results suggest that most chromosome motions (complex though they may be) can be explained by poleward forces acting on or through kinetochore microtubules that span the distance between the kinetochore and the vicinity of a pole. The results also suggest that the majority of short kinetochore microtubules may be remnants of previous microtubule-mediated associations between a kinetochore and a pole.     

Occlusive Impedance Plethysmography: A Noninvasive Method of Diagnosis of Proximal Deep Vein Thrombosis

The purpose of this study was to assess and confirm the accuracy of impedance plethysmography (IPG) by the occlusive cuff method, in detecting proximal (popliteal, femoral and iliac) deep vein thrombosis in patients with symptomatic limbs. In 27 patients 30 consecutive limbs were studied with concurrent venography and IPG. The IPG result was normal in 9 of 9 limbs which were normal on venography, and abnormal in 15 of 16 limbs which showed venographic evidence of proximal deep vein thrombosis (DVT). An abnormal IPG strongly suggests proximal DVT (predictive value 0.88). A normal IPG virtually rules out proximal DVT (predictive value 1.0).     

Taxonomic utility of a phylogenetic analysis of phosphoglycerate kinase proteins of Archaea, Bacteria, and Eukaryota: insights by Bayesian analyses

We studied 131 protein sequences of the essentially ubiquitous glycolytic enzyme 3-phosphoglycerate kinase (3-PGK) by Bayesian analyses in three Domains: 15 Archaea, 83 Bacteria, and 33 Eukaryota. The posterior distribution of phylogenetic trees developed were based on a uniform prior, the WAG model of protein evolution, Metropolis-Hastings sampling in a Markov chain Monte Carlo analysis, and a package of diagnostics to critically evaluate the validity of the analyses. The 15 Archaea separated with high posterior probability. The archaean Phyla Euryarchaeota and the apparently Euryarchaeota derived Crenarchaeota were monophyletic. The 33 Eukaryota separated into two main groups: the non-chlorophyllous forms with coherent sub-groupings of Euglenozoa, Alveolata, Fungi, and Metazoa and all the chlorophyllous species studied: the Plantae (Viridaeplantae), chlorophyllous Stramenopiles, and the chlorophyllous Bacteria. This association supports other opinions concerning the related lineage of cyanobacteria and the Plantae. The 3-PGK sequences from 83 Bacteria in almost every instance associated by their recognized taxal group: alpha-, beta-, gamma-, epsilon-proteobacteria, Chlamydia, Actinobacteridae, and Firmicutes. Firmicutes sequences were subdivided into three apparently monophyletic groups: the anaerobic Clostridia, the spore-forming Bacillales and a group containing the Mollicutes, Lactobacillales and non-spore-forming Bacillales. The 3-PGK-gene tree assemblage was notable both for its pervasive clustering in three Domains according to recognized taxonomic groupings of Class, Order, Family, and Genus. The 3-PGK enzyme or 3-PGK-like activity may have played a central role in the metabolism of the Universal Ancestor.     

Qri2/Nse4, a component of the essential Smc5/6 DNA repair complex

We demonstrate a role for Qri2 in the essential DNA repair function of the Smc5/6 complex in Saccharomyces cerevisiae. We generated temperature-sensitive (ts) mutants in QRI2 and characterized their properties. The mutants arrest after S phase and prior to mitosis. Furthermore, the arrest is dependant on the Rad24 checkpoint, and is also accompanied by phosphorylation of the Rad53 checkpoint effector kinase. The mutants also display genome instability and are sensitive to agents that damage DNA. Two-hybrid screens reveal a physical interaction between Qri2 and proteins that are non-Smc elements of the Smc5/6 DNA repair complex, which is why we propose the name NSE4 for the open reading frame previously known as QRI2. A key role for Nse4 in Smc5/6 function is likely, as overexpressing known subunits of the Smc5/6 complex suppresses nse4(ts) cell cycle arrest. The nse4(ts) growth arrest is non-lethal and unlike the catastrophic nuclear fragmentation phenotype of smc6(ts) mutants, the nucleus remains intact; replicative intermediates and sheared DNA are not detected. This could imply a role for Nse4 in maintenance of higher order chromosome structure.