Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M_w-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.     

Cloning and Characterization of Extracellular Metal Protease Gene of the Algicidal Marine Bacterium Pseudoalteromonas sp. Strain A28

The gene (empI) encoding an extracellular metal protease was isolated from a Pseudoalteromonas sp. strain A28 DNA library. The recombinant EmpI protein was expressed in E. coli and purified. Paper-disk assays showed that the purified protease had potent algicidal activity. A skim milk-polyacrylamide gel electrophoresis protease assay showed that the 38-kDa band of protease activity, which co-migrated with purified EmpI and was sensitive to 1,10-phenathroline, was detected in the extracellular supernatant of A28.     

Induction of Hydrolytic Enzymes in Brassica campestris in Response to Pathovars of Xanthomonas campestris

Inoculation of mature leaves of turnip (Brassica campestris) with the incompatible Xanthomonas campestris pv vitians resulted in the induction of β-1,3-glucanase and chitinase/lysozyme (CHL) activity. No increase in the basal activity of β-1,3-glucanase was observed after inoculation of leaves with heat- or rifampicin-killed X. c. vitians, Escherichia coli, or sterile water. Inoculation with the compatible X. campestris pv campestris resulted in a slower induction of glucanase than that seen with X. c. vitians. In contrast, all bacteria caused an induction of CHL activity. One major β-1,3-glucanase (molecular mass 36.5 kilodaltons, isoelectric point [pl] ~8.5) was purified from both inoculated and untreated leaves by ion-exchange chromatography. The enzyme degraded laminarin by an endo-glycolytic mechanism. Two major CHL isozymes (CHL 1 and CHL 2, molecular mass 30 kilodaltons and pl 9.4 and 10.2, respectively) were purified from X. c. vitians inoculated leaves by affinity chromatography on a chitin column followed by ion-exchange chromatography. Both enzymes degraded chitin by an endo-glycolytic mechanism although the ratio of lysozyme to chitinase specific activities for CHL 1 and CHL2 were different. The induction of CHL 1 was associated with the hypersensitive reaction caused by X. c. vitians whereas all other treatments induced largely CHL 2.     

水杨酸对水稻防卫反应酶系的系统诱导（简报）

经 10 .0 μg·ml-1水杨酸 (SA)喷雾处理后稻苗叶片中苯丙氨酸解氨酶 (PAL)、过氧化物酶 (PO)和多酚氧化酶 (PPO)活性都迅速增强 ,处理后 12～ 2 4h达到高峰。未经SA处理的叶片中PAL、PO和PPO酶活性则在处理后 4 8h达到高峰 ,且活性增加明显低于SA处理的叶片。处理和未处理叶片中木质素含量都迅速增加。这些生理指标的时序变化与SA诱导稻苗抗白叶枯病的表现基本吻合。     

Binding and Functional Properties of Five Extrinsic Proteins in Oxygen-evolving Photosystem II from a Marine Centric Diatom,Chaetoceros gracilis

Oxygen-evolving photosystem II (PSII) isolated from a marine centric diatom, Chaetoceros gracilis, contains a novel extrinsic protein (Psb31) in addition to four red algal type extrinsic proteins of PsbO, PsbQ′, PsbV, and PsbU. In this study, the five extrinsic proteins were purified from alkaline Tris extracts of the diatom PSII by anion and cation exchange chromatographic columns at different pH values. Reconstitution experiments in various combinations with the purified extrinsic proteins showed that PsbO, PsbQ′, and Psb31 rebound directly to PSII in the absence of other extrinsic proteins, indicating that these extrinsic proteins have their own binding sites in PSII intrinsic proteins. On the other hand, PsbV and PsbU scarcely rebound to PSII alone, and their effective bindings required the presence of all of the other extrinsic proteins. Interestingly, PSII reconstituted with Psb31 alone considerably restored the oxygen evolving activity in the absence of PsbO, indicating that Psb31 serves as a substitute in part for PsbO in supporting oxygen evolution. A significant difference found between PSIIs reconstituted with Psb31 and with PsbO is that the oxygen evolving activity of the former is scarcely stimulated by Cl⁻ and Ca²⁺ ions but that of the latter is largely stimulated by these ions, although rebinding of PsbV and PsbU activated oxygen evolution in the absence of Cl⁻ and Ca²⁺ ions in both the former and latter PSIIs. Based on these results, we proposed a model for the association of the five extrinsic proteins with intrinsic proteins in diatom PSII and compared it with those in PSIIs from the other organisms.     

Lytic Enzymes of Trichoderma and Their Role in Plant Defense from Fungal Diseases: A Review

Lytic enzymes of mycoparasitic fungi of the genus Trichoderma, capable of suppressing a number of fungal phytopathogens that originate in air or soil, are reviewed. The topics analyzed include (1) regulation of production of chitinases, -1,3-glucanases, and proteases; (2) molecular and catalytic properties of purified enzymes; and (3) their in vitro ability to degrade cell walls and inhibit spore germination or germ-tube elongation in various phytopathogenic fungi. Among the results summarized are reports of cloning and expression of genes coding for certain lytic enzymes of Trichoderma spp. These genes are used for obtaining plant transgenes with increased resistance to fungal diseases and Trichoderma transformants that produce higher levels of one lytic enzyme (a chitinase or protease) and thereby exhibit a more pronounced ability to suppress phytopathogenic fungi.     

Response of the Ubiquitous Pelagic Diatom Thalassiosira weissflogii to Darkness and Anoxia

Thalassiosira weissflogii, an abundant, nitrate-storing, bloom-forming diatom in the world’s oceans, can use its intracellular nitrate pool for dissimilatory nitrate reduction to ammonium (DNRA) after sudden shifts to darkness and anoxia, most likely as a survival mechanism. T. weissflogii cells that stored 4 mM ¹⁵N-nitrate consumed 1.15 (±0.25) fmol NO₃ ^- cell^-1 h^-1 and simultaneously produced 1.57 (±0.21) fmol ¹⁵NH₄ ⁺ cell^-1 h^-1 during the first 2 hours of dark/anoxic conditions. Ammonium produced from intracellular nitrate was excreted by the cells, indicating a dissimilatory rather than assimilatory pathway. Nitrite and the greenhouse gas nitrous oxide were produced at rates 2-3 orders of magnitude lower than the ammonium production rate. While DNRA activity was restricted to the first few hours of darkness and anoxia, the subsequent degradation of photopigments took weeks to months, supporting the earlier finding that diatoms resume photosynthesis even after extended exposure to darkness and anoxia. Considering the high global abundance of T. weissflogii, its production of ammonium and nitrous oxide might be of ecological importance for oceanic oxygen minimum zones and the atmosphere, respectively.     

Induction of Enzymes and Phenolic Compounds Related to the Natural Defence Response of Netted Melon Fruit by a Bio-elicitor

Fungal disease in netted melon fruit is an important factor affecting their postharvest quality and therefore an important cause of large economic losses around the world. Among the alternatives to control fungal diseases, the induction of the natural defence response (NDR) in fruits is promising. The objective of this study was to induce the NDR in netted melon treated with a bio-elicitor formulated from Fusarium oxysporum growth in a potato dextrose agar enriched with netted melon skin. Netted melon fruits (cv 'Primo') were not treated (C), untreated and inoculated with F. oxysporum (IN), treated with a bio-elicitor (FES), or treated with the bio-elicitor and inoculated (FES + IN). After treatments, fruits were stored for 8 days at 20°C with 90–92% relative humidity. Melon was sampled every 2 days at 20°C to evaluate the development of Fusarium rot symptoms as disease index percentage (DI), changes in phenolic compounds, changes in phenylalanine ammonia-lyase (PAL) activity, chitinase activity (ChA) and β-1,3-glucanase activity (GA). It was found that DI in netted melon fruit was significantly reduced in the FES + IN as compared with the IN treatment. FES + IN and FES treatments showed the highest increase of phenolic acids. Higher levels of PAL activity were observed in the treatments IN, FES, and FES + IN with respect to C, after 4 days of storage. A large increase in ChA activity was observed in the treatments IN, FES and FES + IN after 6 days of storage. No differences in GA activity were found among FES, FES + IN and C treatments throughout storage. IN treatment showed the highest increase in GA activity after 4 days of storage. It is concluded that the bio-elicitor activates the NDR as measured by the increase in phenolic acids synthesis, PAL and ChA enzymes activity, in a similar way as the infection by the living pathogen.     

Proteomic Analysis of Protease Resistant Proteins in the Diabetic Rat Kidney

Glycation induced protein aggregation has been implicated in the development of diabetic complications and neurodegenerative diseases. These aggregates are known to be resistant to proteolytic digestion. Here we report the identification of protease resistant proteins from the streptozotocin induced diabetic rat kidney, which included enzymes in glucose metabolism and stress response proteins. These protease resistant proteins were characterized to be advanced glycation end products modified and ubiquitinated by immunological and mass spectrometry analysis. Further, diabetic rat kidney exhibited significantly impaired proteasomal activity. The functional analysis of identified physiologically important enzymes showed that their activity was reduced in diabetic condition. Loss of functional activity of these proteins was compensated by enhanced gene expression. Aggregation prone regions were predicted by in silico analysis and compared with advanced glycation end products modification sites. These findings suggested that the accumulation of protein aggregates is an inevitable consequence of impaired proteasomal activity and protease resistance due to advanced glycation end products modification.One of the foremost causes of diabetic complications is formation of sugar-derived substances called advanced glycation end products (AGEs),¹ which affect target cell through altered protein structure- function, matrix-matrix/matrix-cell interaction, and by activation of receptor for AGE (RAGE) signaling pathway (1). Although the accumulation of AGEs is a slow process in healthy individuals, their formation is markedly accelerated in diabetes because of hyperglycemia (2). AGE-modified proteins are thermostable and resistant to denaturation. The stability of proteins is believed to be because of additional negative charge (highly oxidized state) brought by AGE modification of proteins, which may contribute to protease resistance (3). Glycation induced protease resistance has been studied in collagen (4–6) and amyloid (7). In addition to glycation, impairment in the proteasomal function may facilitate accumulation of protease resistant protein aggregates in diabetes. Proteasome mediated protein degradation is a central quality control mechanism in the cell. Activity of proteasome is affected during aging (8) and physiological disorders like diabetes (9) resulting in accumulation of ubiquitinated protein aggregates. In muscle extract of diabetic rats, accumulation of toxic glycated proteins was observed because of decreased proteasomal activity (6–9). This proteolytic system is of particular importance in protecting cells against adverse conditions, such as heat shock, glycation, or oxidative stress. However, when the generation of damaged proteins exceeds the capacity of the cell to degrade them, they are progressively accumulated leading to cytotoxicity (10). Severely aggregated, cross-linked, and oxidized proteins are poor substrates for degradation and inhibit the proteasomal activity (11).The kidney is one of the main organs affected in diabetes caused by accumulation of AGEs. Proteins of extracellular matrix, kidney, as well as proteins from circulation, get AGE modified and trapped in the kidney (12). Both intracellular and extracellular AGEs have been observed in the diabetic kidney. Extracellular AGEs interact with the RAGE leading to apoptosis and inflammation (13), whereas intracellular AGEs are formed because of various dicarbonyls. Eventually, both types of the AGEs contribute to kidney damage (14). Furthermore, methyl glyoxal, a highly reactive dicarbonyl covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney (15). Together AGE modification and decreased proteasomal function may be responsible for the accumulation of protease resistant proteins (PRPs) in the diabetic kidney. In our previous study, we have reported the presence of AGE modified proteins in the kidney of the streptozotocin (STZ) induced diabetic rat (12). The current work is inspired by a DARTS (drug affinity responsive target stability) approach, wherein the drug targets are relatively less susceptible to protease action on drug binding (16). A similar approach was adopted here to identify protease resistant proteins from the diabetic kidney. These proteins were characterized to be AGE modified and ubiquitinated by Western blot analysis and mass spectrometry. Functional characterization and expression analysis of some of the identified proteins was performed to gain insight into the consequences of these modifications in diabetes. Further, aggregation prone regions in these proteins were predicted by the in silico approach. These findings shed light on the role of identified PRPs in diabetic complications.     

The Cellular Ataxia Telangiectasia-Mutated Kinase Promotes Epstein-Barr Virus Lytic Reactivation in Response to Multiple Different Types of Lytic Reactivation-Inducing Stimuli

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor β (TGF-β) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53- and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect.     

Computational Modeling of Tumor Response to Drug Release from Vasculature-Bound Nanoparticles

Systemically injected nanoparticle (NPs) targeting tumor vasculature offer a venue for anti-angiogenic therapies as well as cancer detection and imaging. Clinical application has been limited, however, due to the challenge of elucidating the complex interplay of nanotechnology, drug, and tumor parameters. A critical factor representing the likelihood of endothelial adhesion is the NP vascular affinity, a function of vascular receptor expression and NP size and surface-bound ligand density. We propose a theoretical framework to simulate the tumor response to vasculature-bound drug-loaded NPs and examine the interplay between NP distribution and accumulation as a function of NP vascular affinity, size, and drug loading and release characteristics. The results show that uniform spatial distribution coupled with high vascular affinity is achievable for smaller NPs but not for larger sizes. Consequently, small (100 nm) NPs with high vascular affinity are predicted to be more effective than larger (1000 nm) NPs with similar affinity, even though small NPs have lower drug loading and local drug release compared to the larger NPs. Medium vascular affinity coupled with medium or larger sized NPs is also effective due to a more uniform distribution with higher drug loading and release. Low vascular affinity hampered treatment efficacy regardless of NP size, with larger NPs additionally impeded by heterogeneous distribution and drug release. The results further show that increased drug diffusivity mainly benefits heterogeneously distributed NPs, and would negatively affect efficacy otherwise due to increased wash-out. This model system enables evaluation of efficacy for vascular-targeted drug-loaded NPs as a function of critical NP, drug, and tumor parameters.     

Microwaves in the Induction of Immune Response to VI-Antigen

In this work the possibility of using microwaves (MW) for immuno-modulation in the immunization of animals with thymus-independent antigen was studied. The projection zones of the thyroid and adrenal glands of the test animals were subjected to the action of decimeter MW, while the corresponding zones of control animals were subjected to imitation MW. The endocrine activity of rabbits was estimated by radioim-mune methods. Vi-antigen was shown to be a thymus-independent antigen for rabbits, according to the results of fluorescent probes to study the structural rearrangements in surfaces of thymocyte membranes and their nuclei, which reflect early changes during the physiological activation of cells. The irradiation by MW on the projection zone of the thyroid was accompanied by a decrease in the glucocorticoid activity of the adrenal cortex and a simultaneous pronounced immunostimulating effect. MW irradiation of the zone of the adrenal glands was accompanied by immunosuppression in combination with enhanced glucocorticoid activity of the adrenal cortex.     

The Role of Membrane-Bound Enzymes in an Early Response of Aleurone Tissue to Gibberellic Acid

Treatment of aleurone layers of barley seed with gibberellicacid increases the observable phosphorylcholine glyceride transferaseactivity in a membrane fraction prepared from extracts of thealeurone cells. This gibberellic acid-dependent increase inglyceride transferase activity requires neither RNA synthesisnor protein synthesis. Membrane fractions prepared from mixturesof extracts of gibberellic acid-treated layers and control layershave a specific activity of glyceride transferase higher thanexpected on the basis of simple addition of the activities fromthe two sources. Therefore, some kind of activation is occurring.     

Response of the Photosynthetic Apparatus of the Diatom Thallassiosira weisflogii to High Irradiance Light

The response of the photosynthetic apparatus to high irradiance illumination (440–2200 W/m²) was studied in the diatom Thallassiosira weisflogii by fluorescence methods. Changes in the photosynthetic apparatus were monitored by measuring characteristics of chlorophyll fluorescence F ₀, F _m, F _v/F _m, and qN for several hours after illumination of the alga with high-intensity light. Incubation of the alga with 2 mM DTT, an inhibitor of de-epoxidase of carotenoids in the diadinoxanthin cycle, led to a decrease in the nonphotochemical quenching of chlorophyll fluorescence and a drop in the F _v/F _m ratio, a characteristic that reflects the quantum efficiency of the functioning of the photosynthetic apparatus. Light-induced absorption changes associated with transformations of carotenoids of diadinoxanthin cycle were recorded in vivo in algal suspensions in the absence and in the presence of DTT. Using the microfluorometric method, we measured cell distribution over the efficiency of the primary processes of photosynthesis (F _v/F _m) after illumination. We found cells with a high tolerance of their photosynthetic apparatus to photooxidative damage. The relatively high tolerance of a portion of the cell population to high-light illumination can be related to light-induced transformation of carotenoids and to the functioning of other protective systems of the photosynthetic apparatus in diatoms.     

pGIAK1, a Heavy Metal Resistant Plasmid from an Obligate Alkaliphilic and Halotolerant Bacterium Isolated from the Antarctic Concordia Station Confined Environment

pGIAK1 is a 38-kb plasmid originating from the obligate alkaliphilic and halotolerant Bacillaceae strain JMAK1. The strain was originally isolated from the confined environments of the Antarctic Concordia station. Analysis of the pGIAK1 38,362-bp sequence revealed that, in addition to its replication region, this plasmid contains the genetic determinants for cadmium and arsenic resistances, putative methyltransferase, tyrosine recombinase, spore coat protein and potassium transport protein, as well as several hypothetical proteins. Cloning the pGIAK1 cad operon in Bacillus cereus H3081.97 and its ars operon in Bacillus subtilis 1A280 conferred to these hosts cadmium and arsenic resistances, respectively, therefore confirming their bona fide activities. The pGIAK1 replicon region was also shown to be functional in Bacillus thuringiensis, Bacillus subtilis and Staphylococcus aureus, but was only stably maintained in B. subtilis. Finally, using an Escherichia coli - B. thuringiensis shuttle BAC vector, pGIAK1 was shown to display conjugative properties since it was able to transfer the BAC plasmid among B. thuringiensis strains.     

盐生植物碱地肤对盐碱胁迫的生理响应特点

以天然盐生植物碱地肤(Kochia sieversiana)为材料,采用人工生态模拟方法对其施加0~480mmol/L的盐胁迫(摩尔比1∶1的NaCl和Na2SO4)和碱胁迫(摩尔比1∶1的NaHCO3和Na2CO3),测定了盐碱胁迫下的相对生长率(RGR)、叶绿素含量及丙二醛含量.结果表明:低浓度的盐胁迫(80mmol/L)对碱地肤的生长具有刺激作用,其在高达480mmol/L的盐胁迫或400mmol/L的碱胁迫下仍能存活并维持一定的生长,具有强抗盐性和强抗碱性;在相同盐浓度下,碱地肤受碱胁迫的伤害强度和对碱胁迫所做出的胁变反应均大于对盐胁迫.可见,碱地肤的抗盐性强于抗碱性,受碱胁迫的伤害大于盐胁迫,维持高含水量可能是碱地肤适应高强度盐碱胁迫的重要特性.     

RecA Expression in Response to Solar UVR in the Marine Bacterium Vibrio natriegens

Solar ultraviolet radiation may produce daily stress on marine and estuarine communities as cells are damaged and repair that damage. Reduction in the earth's stratospheric ozone layer has increased awareness of the potential effects that ultraviolet radiation may have in the environment, including how marine bacteria respond to changes in solar radiation. We examined the use of the bacterial RecA protein as an indicator of the potential of bacteria to repair DNA damage caused by solar UV irradiation using the marine bacterium Vibrio natriegens as a model. RecA is universally present in bacteria and is a regulator protein for the so-called Dark Repair Systems, which include excision repair, postreplication recombinational repair, and mutagenic or SOS repair. Solar UVB and UVA both reduced V. natriegens viability in seawater microcosms. After exposure to unfiltered solar radiation or radiation in which UVB was blocked, survival dropped below 1%, whereas visible light from which UVA and UVB had been filtered had no effect on survival. Using a RecA-specific antibody for detection, RecA protein was induced by solar radiation in a diel pattern in marine microcosms conducted in the Gulf of Mexico. Peak induction was observed at dusk each day. Although RecA expression was correlated with the formation of UVB-induced cyclobutyl pyrimidine dimers, longer wavelength UVA radiation also induced recA gene expression. Our results demonstrate that RecA-regulated, light-independent repair is an important component in the ability of marine bacteria to survive exposure to solar ultraviolet radiation and that RecA expression is a useful monitor of bacterial repair after exposure to solar UVR.