The Inhibition of Phloem Translocation by Ammonia

Bean plants (Phaseolus vulgaris L. cv. Fardenlosa Shiny) werelabelled with carbon-11 via their first trifoliate leaves when3-weeks-old and the transient inhibitions of translocation causedby the application of ammonium chloride solutions (10 mol m^–3)to a peeled region of stem were studied. At pH 6·5 theammonium was without effect. At pH 11·0 even a briefapplication inhibited translocation for many minutes, whilelonger applications inhibited translocation for considerablylonger. Solutions of 10 mol m^–3 sodium chloride were withouteffect at either pH. At pH 6·5 ammonium chloride solution contains predominantlyammonium ions (NH₄⁺) and at pH 11·0 predominantly dissolvedammonia gas (NH₃). Hence we conclude that phloem transport withinbean stems is inhibited by dissolved ammonia gas but not ammoniumions. Key words: Phloem translocation, transient inhibition, ammonia, ammonium ion     

The Electroshock-Induced Inhibition of Phloem Translocation

The transient inhibition of phloem translocation which is producedby an electric shock was studied by applying controlled-currentstimuli to short lengths of bean stem. Translocation was monitoredby observing the accumulation of carbon-11 label into the plantapex. The principal findings are: (i) For constant-current electricshocks whose (current) x (time) product was held constant at10 mC, those of higher current (and shorter duration) gave longerlasting inhibitions, (ii) Breaking a 5 s pulse into a trainof 100 ms pulses slightly shortened the duration of the inhibition,even though the same total charge was passed; however, the separationof the pulses within the train did not seem to matter. But,when the inhibition produced by a pair of 50 ms pulses was studied,the duration of inhibition increased with pulse spacing. (iii)Single pulses as short as 10 ms could produce a detectable inhibition.The duration of inhibition grew rapidly with pulse length forpulse lengths between 10 ms and 100 ms. (iv) When a pair of2.5 s pulses was applied, a longer inhibition was obtained ifthe two pulses were of opposite polarity. To explain these phenomena,a qualitative conceptual model is provided in terms of ion channelsin the phloem. In the experiments reported here and in all previous pulse-labellingexperiments from this laboratory, it has been noted that rapidlyincreasing stochastic variability in the count data made itextremely difficult to obtain useful results for a period ofmore than 2 h after loading, even if the number of counts perunit time remained fairly high. A quantitative theory for thislimitation is worked out and shown to agree with experimentaldata. Therefore, since biological variability from plant toplant makes it advisable to compare matched stimuli on the sameplant in order to detect trends reliably, the number of challengeswhich can usefully be applied following a single pulse-labellingwith a short-lived isotope is quite limited. Key words: Phloem translocation, transient inhibition, electric shock     

On the Mechanism of Proton Translocation by Respiratory Enzyme

The protonmotive function of the respiratory heme-copper oxidases is often described as the sum of two separate mechanisms: a proton pump plus an incomplete Mitchellian redox loop. However, these two functions may be mechanistically intertwined so that the uptake of protons to form water during the reduction of O₂ is a crucial part of the proton pump mechanism itself. This principle can be deduced from thermodynamic, kinetic, mechanistic, as well as from structural considerations, and was first proposed in conjunction with a histidine cycle model of proton translocation [Morgan, J. E., Verkhovsky, M. I., and Wikström, M. (1994). J. Bioenerg. Biomembr. 26, 599–608]. However, histidine cycle models go much further to suggest chemical details of how this principle might be applied.     

Mechanism of Chilling Injury in Sweet Potatoes

1. 1. Both amounts of lipid phosphorus and acid-insoluble nitrogen in the mitochondrial fraction from chilling-injured sweet potatoes (var. Okinawa 100) were larger than in the fraction from healthy sweet potatoes. The N-amount appeared to be increased more by chilling-injury than the P-amount.

2. 2. Sweet potato, a tropical plant, showed lower value of the degree of unsaturation of fatty acids in mitochondrial fraction than white potato, a temperate-zone plant.

3. 3. The amount of unsaturated fatty acids of C₁₆, C₁₈ and C₂₀ as percentage of the total fatty acids was higher in mitochondrial fractions from chilling-injured sweet potatoes (var. Okinawa 100 and var. Norin 1) than in mitochondrial fractions from healthy sweet potatoes. However, in the case of white potato mitochondrial fraction no detectable difference was observed between storage at 0~1°C and at 10~14°C.

     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Phylogenetic Distribution of Neuron-Specific Enolase

: Neurons and neuroendocrine cells contain a unique isoenzyme of the glycolytic enzyme enolase which is not found in other cells. This acidic enolase isoenzyme has been designated neuron specific enolase or NSE and is easily identified by its elution on DEAE sephadex. The present study shows that brain tissue from species such as yeast, fish and frog do not contain appreciable amounts of acidic “NSE-like” enolase suggesting that lower species do not have this neuronal isoenzyme.     

穿膜肽的结构特点和穿膜机制

穿膜肽（penetratin）是果蝇的触角足同源异形域的DNA结合结构域第三个片段的商品名,由16个氨基酸残基组成,它可以介导多种疏水大分子进入活体细胞质内而不破坏细胞膜的完整性;其最大的特点是可以介导多种大分子进入细胞内,并且无需外源能量,分子作为整体插入细胞内,穿膜过程不需解折叠。该发现开辟了药物进入细胞的新的介导途径。该文介绍穿膜肽的结构特点、穿膜机制、应用及局限性。     

茄子幼苗耐低温性生理机制研究

选择耐低温性不同的6份茄子高代自交系幼苗为材料,进行白天10℃/夜间7℃低温处理10d,调查其耐低温性、生长指标(株高、茎粗、鲜重、干重)、生理指标(POD、SOD、APX活性及脯氨酸含量),以探讨低温对茄子幼苗生长过程中的生长和生理指标的影响.结果显示:(1)各材料的上述性状在常温下均差异不显著,而在低温处理下差异极显著;与对照相比较,幼苗株高、茎粗、鲜重和干重下降,而POD、SOD、APX活性及脯氨酸含量上升.(2)各材料指标的升降幅度与其低温耐受性密切相关,耐低温性强的材料生长指标下降幅度较小,而生理指标提高幅度较大,耐低温性弱的材料则表现相反.(3)各指标间相比较,茄子耐低温性与株高、茎粗、干重、POD活性、SOD活性呈显著正相关,与其余性状的相关未达显著水平.研究表明,低温使茄子幼苗的生长受到一定程度的抑制,但耐低温材料能够通过调节自身的保护酶系统活性来减轻低温的伤害,维持植物体的正常生长及生理代谢功能.     

Phylogenetic Distribution and Identification of Fin-winged Fruits

Fin-winged fruits have two or more wings aligned with the longitudinal axis like the feathers of an arrow, as exemplified by Combretum, Halesia, and Ptelea. Such fruits vary in dispersal mode from those in which the fruit itself is the ultimate disseminule, to schizocarps dispersing two or more mericarps, to capsules releasing multiple seeds. At least 45 families and more than 140 genera are known to possess fin-winged fruits. We present an inventory of these taxa and describe their morphological characters as an aid for the identification and phylogenetic assessment of fossil and extant genera. Such fruits are most prevalent among Eudicots, but occur occasionally in Magnoliids (Hernandiaceae: Illigera) and Monocots (Burmannia, Dioscorea, Herreria). Although convergent in general form, fin-winged fruits of different genera can be distinguished by details of the wing number, texture, shape and venation, along with characters of persistent floral parts and dehiscence mode. Families having genera with fin-winged fruits and epigynous perianth include Aizoaceae, Apiaceae, Araliaceae, Asteraceae, Begoniaceae, Burmanniaceae, Combretaceae, Cucurbitaceae, Dioscoreaceae, Haloragaceae, Lecythidiaceae, Lophopyxidaceae, Loranthaceae, and Styracaceae. Families with genera having fin-winged fruits and hypogynous perianth include Achariaceae, Brassicaceae, Burseraceae, Celastraceae, Cunoniaceae, Cyrillaceae, Fabaceae, Malvaceae, Melianthaceae, Nyctaginaceae, Pedaliaceae, Polygalaceae, Phyllanthaceae, Polygonaceae, Rhamnaceae, Salicaceae sl, Sapindaceae, Simaroubaceae, Trigoniaceae, and Zygophyllaceae. This survey has facilitated the identification of fossil winged fruits such as Combretaceae and Araliaceae in the late Cretaceous of western North America and provides additional evidence toward the identification of various Cenozoic fossils including Brassicaceae, Fabaceae, Polygonaceae, Rutaceae, and Sapindaceae.     

A Relay Mechanism for Phloem Translocation

Munch's pressure-flow mechanism is the most popular hypothesisfor phloem translocation; however, difficulties with it arenot resolved. The metabolic pumping hypotheses on the otherhand often run into complications with the high flux-densityrequirement of translocation. A ‘relay’ mechanismis proposed for solute translocation in plants, which is a hybridbetween pressure flow and one or another of the, at present,alternative candidates for the task of driving phloem translocation.Such a compromise may make sense of the confusing experimentaland ultrastructural evidence. phloem transport, Münch pressure flow mechanism     

Mechanism of tRNA Translocation on the Ribosome

During the translocation step of the elongation cycle of peptide synthesis two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. Translocation is catalyzed by the elongation factor G (EF-G) and requires GTP hydrolysis. The fundamental biochemical features of the process were worked out in the 1970–80s, to a large part by A.S. Spirin and his colleagues. Recent results from pre-steady-state kinetic analysis and cryoelectron microscopy suggest that translocation is a multistep dynamic process that entails large-scale structural rearrangements of both ribosome and EF-G. Kinetic and thermodynamic data, together with the structural information on the conformational changes in the ribosome and EF-G, provide a detailed mechanistic model of translocation and suggest a mechanism of translocation catalysis by EF-G.     

Translocation and Intracellular Distribution of Tritiated Gibberellin A3

The localization of tritium-radioactivity in dwarf kidney bean plants (Phaseolus vulgaris) of ³H-gibberellm A₃(³H-GA₃) applied in a large quantity was investigated in advance of the study on GA₃ metabolism in this plant. Immediately after the application of ³H-GA₃, the radioactivity was distributed uniformly in the top of this plant; no further transportation of the radioactivity into the growing apical region from mature leaves and stems was the observed as the growth stage proceeded. An investigation on the intracellular localization of the radioactivity demonstrated that most part of the radioactivity was found in the cellular soluble fraction, while no radioactivity was detected in such subcellular particles as nuclei, mitochondria and microsomes. Examinations of the occurrence of GA₃ bound with such macromolecules as RNA and protein gave negative results.     

Mechanism for Inhibition of Deoxyribonuclease Activity by Antisera

The activity of bovine DNase, but not that of porcine DNase, is inhibited by antisera against bovine DNase, and vice versa. Inhibition of DNase is found in the immunoglobulin G-containing fractions, as shown by ion exchange chromatography. Inactive DNase, carboxymethylated specifically at the active site His¹³⁴, competes with active DNase and reverses the antisera inhibition of DNase, suggesting that the epitode responsible for inhibition does not contain the active site His¹³⁴. Alignment of the sequences of DNase of these two species shows that the greatest variation occurs between residues 153 and 163, within which are three consecutive peptide bonds, Lys-Trp-His-Leu, that are readily cleaved by trypsin, chymotrypsin, or thermolysin. The 8-hr digest of DNase by each of these three proteases has lost the ability to reverse antisera inhibition. The degree of antisera inhibition varies with the metal ion used as the activator for DNase-catalyzed reactions. When Mn²⁺, Co²⁺, or Mg²⁺ plus Ca²⁺ are used as activators, inhibition is approximately 50%. When pBR322 plasmid is used as substrate, gel electrophoresis shows that the DNase-catalyzed DNA hydrolysis produces a significant amount of double-strand cuts with Mn²⁺, Co²⁺, or Mg²⁺ plus Ca²⁺ as activators and antisera inhibit DNase action only on double-strand cuts. With only Mg²⁺ as the activator no double-strand cuts are observed, either in the presence or absence of antisera, and the DNase activity is not significantly inhibited. We conclude that antisera inhibition is due to antibody binding of the DNase polypeptide chain within residues 153 and 163. These residues are not crucial for catalysis, but are required for DNA binding, which results in double-strand cuts.     

Microscopic Mechanism of Antibiotics Translocation through a Porin

OmpF from the outer membrane of Escherichia coli is a general porin considered to be the main pathway for beta-lactam antibiotics. The availability of a high-resolution crystal structure of OmpF and new experimental techniques at the single-molecule level have opened the way to the investigation of the microscopic mechanisms that allow the passage of antibiotics through bacterial pores. We applied molecular dynamics simulations to investigate the translocation process of ampicillin (Amp) through OmpF. Using a recent algorithm capable of accelerating molecular dynamics simulations we have been able to obtain a reaction path for the translocation of Amp through OmpF. The mechanism of passage depends both on the internal degrees of freedom of Amp and on interactions of Amp with OmpF. Understanding this mechanism would help us design more efficient antibiotics and shed light on nature's way of devising channels able to enhance the transport of molecules through membranes.     

Phylogenetic Distribution of Potential Cellulases in Bacteria

Many microorganisms contain cellulases that are important for plant cell wall degradation and overall soil ecosystem functioning. At present, we have extensive biochemical knowledge of cellulases but little is known about the phylogenetic distribution of these enzymes. To address this, we analyzed the distribution of 21,985 genes encoding proteins related to cellulose utilization in 5,123 sequenced bacterial genomes. First, we identified the distribution of glycoside hydrolases involved in cellulose utilization and synthesis at different taxonomic levels, from the phylum to the strain. Cellulose degradation/utilization capabilities appeared in nearly all major groups and resulted in strains displaying various enzyme gene combinations. Potential cellulose degraders, having both cellulases and β-glucosidases, constituted 24% of all genomes whereas potential opportunistic strains, having β-glucosidases only, accounted for 56%. Finally, 20% of the bacteria have no relevant enzymes and do not rely on cellulose utilization. The latter group was primarily connected to specific bacterial lifestyles like autotrophy and parasitism. Cellulose degraders, as well as opportunists, have multiple enzymes with similar functions. However, the potential degraders systematically harbor about twice more β-glucosidases than their potential opportunistic relatives. Although scattered, the distribution of functional types, in bacterial lineages, is not random but mostly follows a Brownian motion evolution model. Degraders form clusters of relatives at the species level, whereas opportunists are clustered at the genus level. This information can form a mechanistic basis for the linking of changes in microbial community composition to soil ecosystem processes.     

Phylogenetic Distribution and Evolutionary History of Bacterial DEAD-Box Proteins

DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation.     

Mechanism of Inhibition of Translation Termination by Blasticidin S

Understanding the mechanisms of inhibitors of translation termination may inform development of new antibacterials and therapeutics for premature termination diseases. We report the crystal structure of the potent termination inhibitor blasticidin S bound to the ribosomal 70S?release factor 1 (RF1) termination complex. Blasticidin S shifts the catalytic domain 3 of RF1 and restructures the peptidyl transferase center. Universally conserved uridine 2585 in the peptidyl transferase center occludes the catalytic backbone of the GGQ motif of RF1, explaining the structural mechanism of inhibition. Rearrangement of domain 3 relative to the codon-recognition domain 2 provides insight into the dynamics of RF1 implicated in termination accuracy.     

Phylogenetic Distribution and Significance of the Hypothalamic Releasing Hormones

SYNOPSIS. The mammalian hypothalamic releasing factors regulatingthyroid, gonadal and adrenal function as well as growth hormonesecretion have been isolated, characterized and their nucleotidesequences determined. In general, their hypophysiotropic effectsare replicated in lower vertebrates though thyrotropin releasinghormone (TRH) does not appear to stimulate thyroid functionin amphibia and fish. The releasing factors, or peptides structurallyrelated to these substances, are found throughout the CNS ofall vertebrates where they likely function as neurotransmittersor neuromodulators. High concentrations of TRH and other neuralpeptides includingsauvagine, which is related to corticotropinreleasing factor (CRF) and has CRF-like activity, are foundin amphibian skin, a neural crest derived tissue. mRNA extractedfrom the skin of Xenopus laevis was cloned and led to the identityof the DNA sequence of pre-pro TRH. Molecular variants of somatostatinhave been recognized from studies on the pancreatic islets ofthe anglerfish and catfish. Within mammalian species there isheterogeneity of growth hormone releasing factor (GRF), the44 (and 40) amino acid peptides isolated from a human(h) pancreatictumor. In the teleost brain-pituitary, 2 distinct hGRF-likeneuronal systems are present. Additionally, various molecularforms of hGRF exist in the fish brain showing structural changesfrom the human variety.     

Mechanism of Inhibition of Jack Bean alpha-Mannosidase by Swainsonine

The indolizidine alkaloid, swainsonine, was previously shown to be a potent inhibitor of lysosomal and jack bean α-mannosidase (Dorling, Huxtable, Colegate 1980 Biochem J 191: 649-651). We examined the effects of various concentrations of this alkaloid on a number of commercially available glycosidases and found swainsonine to be quite specific for α-mannosidase (50% inhibition at 1-5 × 10⁻⁷ molar). Optimum inhibition was observed after a 2-minute preincubation of enzyme and inhibitor. Lineweaver-Burk plots of substrate concentration versus velocity in the presence of various amounts of swainsonine showed considerable curvature at high substrate concentrations, suggesting that swainsonine may be a competitive inhibitor that binds tightly to the enzyme and is only slowly removed. Periodate oxidation of swainsonine completely destroyed its inhibitory activity.