Zif,the Zoocin A Immunity Factor,Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a d-alanyl-l-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three l-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.During streptococcal peptidoglycan synthesis, monomer subunits are generated inside the cell, with nonribosomal peptidyl transferases responsible for the addition of amino acids onto the epsilon amino group of lysine in the subunits. These nonribosomal peptidyl transferases are part of the FemABX family of proteins, some of which have been implicated in penicillin resistance (5, 26). In Streptococcus pneumoniae peptidoglycan synthesis, MurM attaches either an l-alanine or an l-serine to the epsilon amino group of lysine, and MurN then adds an l-alanine (11, 26).Zoocin A is a d-alanyl-l-alanine endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18). The N-terminal catalytic domain (CAT) has high degrees of similarity to several other bacteriolytic endopeptidases, including the staphylolytic enzyme lysostaphin. The C-terminal target recognition domain (TRD), which facilitates binding of the enzyme to peptidoglycan (1), has very little similarity to any characterized conserved domain.Producer cell immunity to zoocin A is due to zif (zoocin A immunity factor), which is adjacent to zooA on the chromosome and is transcribed divergently (4). Zif has high degrees of similarity to MurM and MurN and also to the lysostaphin resistance protein and other FemABX-like immunity proteins (23). Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6, 9, 15, 25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A. In the present study, Zif was shown to insert an additional l-alanine into the peptidoglycan cross bridges, which inhibited both binding of the zoocin A TRD and the ability of the zoocin A CAT to hydrolyze the cross bridge.     

The Mode of Action of Toxic Protein in Wheat and Barley on Brewing Yeast

The mode of action of the toxic protein isolated from wheat on brewing yeast was investigated, and the following results were obtained: (1) The toxin inhibits respiration and fermentation of the yeast, and causes death of the cell in a few min (6 min) at a concentration of 4 ppm. (2) At a lower concentration (0.4 ppm), the toxin inhibits incorporation of sugars without causing death of the cells. (3) Potassium ion, phosphate ion, protein and nucleotides leak from the cell upon treatment with toxin at a lower concentration (0.4 ppm). (4) A directly proportional relationship exists between the lowest lethal concentration of the toxin and the yeast cell population. (5) The toxin is adsorbed onto the cell wall and cell membrane.

According to these results, the toxin seems to react with functional site(s) of the cell membrane causing changes in the permeability of the membrane and resulting in cell death.     

嵊山岛海洋药用生物资源状况调查

对嵊山岛礁生物资源以及邻近海域的微藻资源调查,共发现121种潮间带生物,其中有28种海洋药用生物,包括红条毛肤石鳖(Acanthochiton rubrolineatus)、马粪海胆(Hem icentiotus pulcherrimus)、海萝(Gloiopeltis furcata)等,其中多数是海洋中药或可制成中成药,嵊山岛丰富的海绵资源具有筛选天然活性物质的潜力。嵊山岛邻近海域的产毒藻类包括产麻痹性贝毒(PSP)的链状亚历山大藻(Alexandrium catenatum)、塔玛亚历山大藻(Alexandrium tamarens),产腹泻性贝毒(DSP)的倒卵形鳍藻(D inophysis forti)、具尾鳍藻(D inophysis caudata);产记忆缺失性贝毒(ASP)的尖刺拟菱形藻(Pseudo-Nitzschia pungens)、多列拟菱形藻(Pseudo-Nitzschia multiseries)和多纹拟菱形藻(Pseudo-Nitzschia multistriata);产神经性贝毒(NSP)的短凯伦藻(Kerina breve)以及红色裸甲藻(Gymnodinium sangium)、米氏凯伦藻(Kerina m ikim oto)、环状异甲藻(Heterocapsa circularisqua-ma)等能产生生理活性物质的藻类,其中嵊山岛邻近海域产石房蛤毒素和神经毒素的有毒藻类可能具有药用开发价值。     

The Mode of Action of a Carboxypeptidase from Malt

Certain methionine auxotrophs of Arthrobacter paraffineus and Bacillus species produce large amounts of O-acetylhomoserine (OAH). The methionine requirement of these auxotrophs could be satisfied by either cystathionine or homocysteine but not by homoserine. The cell-free extacts from the auxotrophs were found to be deficient in cystathionine ?-synthase activity. OAH and O-succinylhomoserine (OSH) could replace methionine in the auxotrophs which are deficient in homoserine-O-transacetylase. A methionine auxotroph of Corynebacterium glutamicum also produced OAH, and the blocked step in the auxotroph appeared to be between cystathionine and homocysteine.

Cell-free extracts of A. paraffineus, C. glutamicum and Bacillus species catalyzed the formation of OAH from acetyl-CoA and homoserine, while a corresponding reaction with succinyl-CoA was not detected. Cystathionine γ-synthases in extracts of C. glutamicum and Bacillus species were specific for OAH, while the enzyme in extract of A. paraffineus was rather specific for OSH though it reacted with OAH to a certain extent.

These results indicate that the biosynthesis of l-methionine in these bacteria involves OAH.     

The Use of Cellulase in Inhibiting Biofilm Formation from Organisms Commonly Found on Medical Implants

A study was made of the use of cellulase to inhibit biofilm formation by a pathogenic bacterium commonly found in medical implants. A Pseudomonas aeruginosa biofilm was grown on glass slides in a parallel flow chamber for 4 d with glucose as the nutrient source. Biofilm development was assessed by measuring the colony forming units (CFU) and biomass areal density. Biofilm was grown at pH 5 and 7 in the presence of three different cellulase concentrations, 9.4, 37.6 and 75.2 units ml^{m 1}. In addition, a control study using deactivated cellulase was performed. The results show that cellulase is effective in partially inhibiting biomass and CFU formation by P. aeruginosa on glass surfaces. The effect of cellulase depended on concentration and was more effective at pH 5 than pH 7. The experiment was further extended by investigating the effect of cellulase on the apparent molecular weight of purified P. aeruginosa exopolysaccharides (EPS). The observation of EPS using size exclusion chromatography showed a decrease in apparent molecular weight when incubated with enzyme. An increase in the amount of reducing sugar with time when the purified EPS were incubated with enzyme also supports the hypothesis that cellulase degrades the EPS of P. aeruginosa. While cellulase does not provide total inhibition of biofilm formation, it is possible that the enzyme could be used in combination with other treatments or in combinations with other enzymes to increase effectiveness.     

离子束对生物体的作用原理及应用

离子束是指一束具有一定能量的质量数小于或等于4的带电离子束,离子束注入技术是生物物理技术,具有生理损伤小、突变谱广和突变频率高等特点。离子束与生物体的相互作用是我国具有独立知识产权的生物物理技术,我国科学家在上世纪80年代已经发现了离子束注入的生物效应,并将这一原理应用于植物诱变育种。本文主要概述了低能离子束注入对生物体的作用原理,以及该技术在植物育种、微生物品种改良和遗传改良上的应用,最后还小结了离子束注入技术在研究领域存在的问题并对其未来发展方向提出展望。     

ArhGAP15, a Rac-specific GTPase-activating Protein,Plays a Dual Role in Inhibiting Small GTPase Signaling

Signaling from small GTPases is a tightly regulated process. In this work we used a protein microarray screen to identify the Rac-specific GAP, ArhGAP15, as a substrate of the Rac effectors Pak1 and Pak2. In addition to serving as a substrate of Pak1/2, we found that ArhGAP15, via its PH domain, bound to these kinases. The association of ArhGAP15 to Pak1/2 resulted in mutual inhibition of GAP and kinase catalytic activity, respectively. Knock-down of ArhGAP15 resulted in activation of Pak1/2, both indirectly, as a result of Rac activation, and directly, as a result of disruption of the ArhGAP15/Pak complex. Our data suggest that ArhGAP15 plays a dual negative role in regulating small GTPase signaling, by acting at the level of the GTPase itself, as well interacting with its effector, Pak kinase.     

Avian Riboflavinuria IX. Qualitative Action of a Mutant Gene in Chicken on Riboflavin-Binding Protein Synthesis

Laying chickens normally produce a riboflavin-binding protein (RBP) essential for the transfer of vitamin B₂ to the egg. A mutant line of chickens (rdrd) is incapable of synthesizing functional RBP. The rdrd laying hens contain a component in the liver and the magnum of the oviduct which crossreacts with rabbit antiserum specific for RBP. No crossreacting material (CRM) was observed in eggs or blood of rdrd hens. CRM is absent in extracts of livers of rdrd males, but it can be induced in roosters of this genotype by estradiol. CRM appears to be less immunogenic on a weight basis than RBP, and it carries less net negative charge. No measurable riboflavin-binding capacity was found for newly isolated or degraded CRM.     

Action of Inhibitors of RNA and Protein Synthesis on Cell Enlargement

Further studies with inhibitors of protein synthesis are presented to support the conclusion, drawn from work with chloramphenicol, that protein synthesis is a critical limiting factor in auxin-induced cell expansion. The indoleacetic acid-induced elongation of oat coleoptile sections was strongly inhibited by dl-p-fluorophenylalanine, and the inhibition is antagonized by phenylalanine. Puromycin at 10(-4)m very strongly inhibited the indoleacetic acid-induced growth of oat coleoptile and artichoke tuber sections and exerted a less powerful effect on pea stem sections. As found earlier with chloramphenicol, concentrations of puromycin effective in inhibiting the growth of coleoptile sections had quantitatively similar effects on protein synthesis, as measured by the incorporation of C(14)-leucine into protein of the coleoptile tissue. Several analogues of RNA bases were also tested, but while 8-azaguanine very strongly inhibited growth of artichoke tuber disks, 6-azauracil was the only one of this group clearly inhibitory to growth in coleoptile or pea stem sections. Actinomycin D actively inhibited both elongation and the incorporation of C(14)-leucine into protein in oat coleoptile sections. Inhibition of the 2 processes went closely parallel. Actinomycin D also powerfully inhibited growth of artichoke tuber disks. All the compounds effective in inhibiting growth generally inhibited the uptake of leucine as well.The possibility that auxin causes cell enlargement in plants by inducing the synthesis of a messenger RNA and of one or more new but unstable enzymes, is discussed. Possible but less favored alternative explanations are: A) that auxin induces synthesis of a wall protein, or B) that the continued synthesis of some other unstable protein (by a process independent of auxin) may be a prerequisite for cell enlargement.     

The Mode of the Herbicidal Action of Benzyl N,N-Dialkyldithiocarbamates

The mode of the herbicidal action of benzyl N,N-dialkyldithiocarbamates was examined. These compounds did not inhibit the Hill reaction nor increase ATP-ase activity. However, they enhanced the respiration of plants and strongly inhibited oxidative phosphorylation. There was a linear relationship between Taft’s σ* constants of N-alkyl substituents of benzyl N,N-dialkyldithiocarbamates and median P/O inhibition concentrations of these dithiocarbamates.     

Protein Stability and Folding Kinetics in the Nucleus and Endoplasmic Reticulum of Eucaryotic Cells

We measure the stability and folding relaxation rate of phosphoglycerate kinase (PGK) Förster resonance energy transfer (FRET) constructs localized in the nucleus or in the endoplasmic reticulum (ER) of eukaryotic cells. PGK has a more compact native state in the cellular compartments than in aqueous solution. Its native FRET signature is similar to that previously observed in a carbohydrate-crowding matrix, consistent with crowding being responsible for the compact native state of PGK in the cell. PGK folds through multiple states in vitro, but its folding kinetics is more two-state-like in the ER, so the folding mechanism can be modified by intracellular compartments. The nucleus increases PGK stability and folding rate over the cytoplasm and ER, even though the density of crowders in the nucleus is no greater than in the ER or cytoplasm. Nuclear folding kinetics (and to a lesser extent, thermodynamics) vary less from cell to cell than in the cytoplasm or ER, indicating a more homogeneous crowding and chemical environment in the nucleus.     

Water Stress and Protein Synthesis: V. Protein Synthesis, Protein Stability, and Membrane Permeability in a Drought-sensitive and a Drought-tolerant Moss

The effects have been studied of water stress and desiccation on protein synthesis in the drought-tolerant moss Tortula ruralis and the drought-sensitive moss Hygrohypnum luridum. At any particular level of steady state water stress, the inhibition of protein synthesis was greater in H. luridum than in T. ruralis. Water stress-induced changes in the pattern of protein synthesis, as determined by the double label ratio technique, were minor in T. ruralis, but major in H. luridum. Proteins of both mosses were found to be stable during desiccation and subsequent rehydration. Changes in membrane permeability, as indicated by the leakage of amino acid, were observed during rehydration of desiccated moss and were dependent on the rate of desiccation. The leakage was small and reversible in T. ruralis but large and irreversible in H. luridum. Although H. luridum failed to recover from complete desiccation (80% loss in fresh weight), it was able to recover fully from steady state stress under conditions where a maximum loss of 55% in fresh weight was recorded.     

The Stress of Protein Misfolding: From Single Cells to Multicellular Organisms

Organisms survive changes in the environment by altering their rates of metabolism, growth, and reproduction. At the same time, the system must ensure the stability and functionality of its macromolecules. Fluctuations in the environment are sensed by highly conserved stress responses and homeostatic mechanisms, and of these, the heat shock response (HSR) represents an essential response to acute and chronic proteotoxic damage. However, unlike the strategies employed to maintain the integrity of the genome, protection of the proteome must be tailored to accommodate the normal flux of nonnative proteins and the differences in protein composition between cells, and among individuals. Moreover, adult cells are likely to have significant differences in the rates of synthesis and clearance that are influenced by intrinsic errors in protein expression, genetic polymorphisms, and fluctuations in physiological and environmental conditions. Here, we will address how protein homeostasis (proteostasis) is achieved at the level of the cell and organism, and how the threshold of the stress response is set to detect and combat protein misfolding. For metazoans, the requirement for coordinated function and growth imposes additional constraints on the detection, signaling, and response to misfolding, and requires that the HSR is integrated into various aspects of organismal physiology, such as lifespan. This is achieved by hierarchical regulation of heat shock factor 1 (HSF1) by the metabolic state of the cell and centralized neuronal control that could allow optimal resource allocation between cells and tissues. We will examine how protein folding quality control mechanisms in individual cells may be integrated into a multicellular level of control, and further, even custom-designed to support individual variability and impose additional constraints on evolutionary adaptation.     

Purification and Mode of Action of Synexin: A Protein Enhancing Calcium-Induced Membrane Aggregation

Synexin, a protein from the cytosol of the adrenal medulla, selectively increases the ability of Ca2+ to aggregate chromaffin granules and other membrane-bound particles. The ability of synexin to self-aggregate in the presence of Ca2+ can be employed in the purification of the protein by monitoring purification with parallel assays that utilize the aggregation of both chromaffin granule membranes and phosphatidylserine liposomes. It is shown that the enhancement of the Ca2+-induced aggregation of both liposomes and chromaffin granule membranes is a property associated with a 47,000 Mr protein. Trypsin inactivated synexin. We found that if granule membranes were well washed after trypsin treatment, they were still excellent substrates for synexin aggregation. This finding cannot be explained by extinction changes owing to synexin self-aggregation. The 47,000 Mr protein enhancement Ca2+ aggregation of phosphatidylserine liposomes containing up to 40% phosphatidylcholine, liposomes made from lipids extracted from chromaffin granule membranes, and trypsin-treated chromaffin granule membranes, thus suggesting that synexin activity in vivo may be independent of specific membrane proteins but dependent on the presence of acidic phospholipids in the membrane.