Anti-Bcl-2 family members,zfBcl-x<Subscript>L</Subscript> and zfMcl-1a,prevent cytochrome <Emphasis Type="Italic">c</Emphasis> release from cells undergoing betanodavirus-induced secondary necrotic cell death

Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 μg/ml) was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 μg/ml) completely blocked viral protein B2 expression, and partly blocked protein A, protein α, and a pro-apoptotic death protein (Bad) expression. Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2) may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins, zfBcl-x_L and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members. J.-L. Wu and J.-R. Hong contributed equally to the research.     

Neuroprotective Effects of Chalcones from <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> on 6-Hydroxydopamine-Induced Cytotoxicity in Rat Mesencephalic Cells

In the present work, we showed that a chalcone-enriched fraction (CEF) isolated from the stem bark of a Brazilian medicinal plant, Myracrodruon urundeuva, presents neuroprotective actions on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death, in rat mesencephalic cells. In the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assay, which is an index of cell viability, CEF (1–100 μg/ml) reversed in a concentration-dependent manner the 6-OHDA-induced cell death. While cells exposed to 6-OHDA (40 μM) showed an increased concentration of thiobarbituric acid reactive substances (TBARS), the pretreatment with CEF (10–100 μg/ml) significantly decreased the 6-OHDA-induced TBARS formation, indicative of a neuroprotection against lipoperoxidation. Furthermore, the drastic increase of nitrite levels induced by 6-OHDA, indicative of nitric oxide formation and free radicals production, was prevented by CEF. Double staining with acridine orange/ethidium bromide showed that cultures exposed to 6-OHDA (40 and 200 μM) presented an increase of apoptotic and necrotic cell numbers in a concentration-dependent manner. CEF (100 μg/ml) protected cells from apoptosis and necrosis and increased number of cells presenting a normal morphology. The immunohistochemical analysis for tyrosine hydroxylase (TH) positive neurons indicated that 6-OHDA (40 and 200 μM) caused a concentration-dependent loss of TH+ and TH− neurons. CEF protected both cells types from 6-OHDA-induced cell death. All together, our results demonstrated neuroprotective effects of chalcones, which are able to reduce oxidative stress and apoptotic injury caused by 6-OHDA. Our findings suggest that chalcones could provide benefits, along with other therapies, in neurodegenerative injuries, such as Parkinson’s disease.     

Minimum inhibitory concentration (MIC) of crude preparations of Brevibacillus laterosporus SA14 bioactive material compared to vancomycin and oxacillin, against clinical isolates of methicillin-resistant Staphylococcus aureus

Secondary metabolites, particularly bioactive compounds, from probiotic bacteria, are good candidates for replacing antibiotics to which bacteria have become resistant. In order to compare bioactive crude material from strain SA14 of Brevibacillus laterosporus with two antibiotics, the MICs of this bioactive crude and those of antibiotics vancomycin and oxacillin, against methicillin-resistant Staphylococcus aureus (MRSA), were determined. The result indicated that the MIC (3.6–19.2 μg/ml) of bioactive crude was higher than vancomycin (MIC = 1.28–2.56 μg/ml) when tested against MRSA. Interestingly, all tested strains of MRSA were susceptible to bioactive crude and were approximately 5.2-fold more potent than oxacillin (MIC > 100 μg/ml). Its activity against MRSA gives support for further evaluation, and the development of this substance for therapeutic use.     

A novel bacterial pathogen (Enterobacter sp.) isolated from the leaf roller, Caloptilia theivora of tea of Darjeeling foothills

Enterobacter sp. was isolated from the diseased and dead caterpillars of the tea leaf roller (Caloptilia theivora) from the Darjeeling foothill region. When the vegetative form of the bacterium was applied via food, mortality of C. theivora showed an LC₅₀ value at 363.1 μg/ml (bacterial wt./vol. of water) with fiducial limits 363.25 and 362.94 μg/ml respectively. The LT₅₀ values for C. theivora were 6 days for 100 μg/ml, 5.96 days for 300 μg/ml, 5.81 days for 500 μg/ml, 4.96 days for 750 μg/ml and 4.61 days for 1,000 μg/ml concentrations. The finding would enable one to contemplate development of a microbial pesticide using this novel Enterobacter sp. DD01 for control of the leaf rolling pest.     

Responses ofGanoderma lucidum to heavy metals

The lelvels of seven heavy metals and their toxicity towardGanoderma lucidum under various cultivation conditions were assessed. The contents of Mn, Cu, Zn, Cd, Hg, Pb and U in the fruitbodies of cultivatedG. lucidum, and sawdust substrates were determined to be at trace levels for U, 0.01–0.1 μg/g for Cd and Hg, and 1–5 μg/g for Pb, 10–120 μg/g for Mn, Cu and Zn. The effects of heavy metals, on the growth of mycelia ofG. lucidium in pure cultures were examined over a wide range of concentrations (10–3,000 μg/ml), and their toxicities were found to decrease in the order: Hg>Cd>Cu>U>Pb>Mn=Zn. The translocation and accumulation of Zn from contaminated substrates (at 10 μg/g) in fruitbodies were investigated by using⁶⁵Zn tracer, andG. lucidum was found to take up Zn with an efficiency of >60%, leading to accumulation of >100 μ/g, in fruitbodies and >80 μ/g Zn in basidiospores.     

Cry2A toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> expressed in insect cells are toxic to two lepidopteran insects

The cry2Aa and cry2Ab genes from a Brazilian Bacillus thuringiensis strain were introduced into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in order to evaluate the heterologous proteins expression in insect cells and their toxicity to different insects. The recombinant viruses (vAcCry2Aa and vSynCry2Ab) were amplified in Trichoplusia ni (BTI-Tn5B1-4) cells and used to infect Spodoptera frugiperda larvae. Total extracts from S. frugiperda infected with the recombinant viruses were analysed by SDS-PAGE, which detected the presence of polypeptides around 65 kDa. Cuboid-shaped protein crystals were observed in insect extracts by light and scanning electron microscopy. Bioassays, using the heterologous proteins showed toxicity against second instar A. gemmatalis larvae (Cry2Aa) with a LC₅₀ of 1.03 μg/ml and second instar S. frugiperda larvae (Cry2Ab) with a LC₅₀ of 3.45 μg/ml. No toxic activity was detected for Aedes aegypti and Culex quinquenfaciatus.     

Growth and fruitbody formation ofGanoderma lucidum on media supplemented with vanadium, selenium and germanium

Responses of mycelia ofGanoderma lucidum to vanadium, selenium and germanium were examined over a wide range of concentrations (10–1, 120 μg/ml) in pure culture. Se and V were found to be highly toxic, but Ge was not toxic at the levels tested.Ganododerma lucidum cultivated on substrates of sawdust with V (30–80 μg/g) developed mature fruitbodies, but the bioaccumulation of V was quite low (2.5–7 μg/g in pileus, 12.5–21.5 μg/g in stipe and <1 μg/g in basidiospores). Se as Na₂SeO₄ labeled with⁷⁵Se was effectively taken up from substrates and accumulated in fruitbodies (mainly in pileus), then depleted by discharge of basidiospores. Ge as GeCl₄ labeled with⁷⁷Ge was easily uptaken and translocated into fruitbodies.     

Antioxidant and antimicrobial actions of the clubmoss <Emphasis Type="Italic">Lycopodium clavatum</Emphasis> L.

Purpose of the present study was to evaluate antioxidant, antibacterial, antifungal, and antiviral activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts as well as the alkaloid fraction of Lycopodium clavatum L. (LC) from Lycopodiaceae growing in Turkey. Antioxidant activity of the LC extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method at 0.2 mg/ml using microplate-reader assay. Antiviral assessment of LC extracts was evaluated towards the DNA virus Herpes simplex (HSV) and the RNA virus Parainfluenza (PI-3) using Madin-Darby Bovine Kidney (MDBK) and Vero cell lines. Antibacterial and antifungal activities of the extracts were tested against standard and isolated strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis as well as the fungi; Candida albicans and C. parapsilosis. All of the extracts possessed noteworthy activity against ATCC strain of S. aureus (4 μg/ml), while the LC extracts showed reasonable antifungal effect. On the other hand, we found that only the chloroform extract was active against HSV (16–8 μg/ml), while petroleum ether and alkaloid extracts inhibited potently PI-3 (16–4 μg/ml and 32–4 μg/ml, respectively). However, all of the extracts had insignificant antiradical effect on DPPH. In addition, we also analyzed the content of the alkaloid fraction of the plant by capillary gas chromatography-mass spectrometry (GC-MS) and identified lycopodine as the major alkaloid.     

Minimum inhibitory concentration of drugs againstMycobacterium leprae as determined by anin vitro assay

The observations that liveMycobacterium leprae after entry into cultured peritoneal macrophages from mice, reduced the EA rosetting macrophages, have been exploited to determine the minimum inhibitory concentration of diamino diphenyl sulphone and rifampicin. Diamino diphenyl sulphone showed a minimum inhibitory concentration of 0.028 μg/ml and rifampicin 0.11μg/ml when given externally. However, there was accumulation of diamino diphenyl sulphone inside the macrophages. At an external concentration of 0.028 μg/ml the concentration inside the macrophage was 0.5μg/ml. The minimum inhibitory concentration for diamino diphenyl sulphone in this assay system is higher by several folds and that for rifampicin is slightly lower, than what is reported earlier with mice foot pad experiments. The minimum inhibitory concentration reported in this assay system is quite close to what is observed forin vitro inhibition ofMycobacterium lufu with both the drugs     

Measurement of particle diameter of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> microcapsule by spray drying and analysis on its microstructure

Lactobacillus acidophilus, as a probiotic, is widely used in many functional food products. Microencapsulation not only increases the survival rate of L. acidophilus during storage and extends the shelf-life of its products, but also optimal size microcapsule makes L. acidophilus have an excellent dispersability in final products. In this paper, L. acidophilus was microencapsulated using spray drying (inlet air temperature of 170°C; outlet air temperature of 85–90°C). The wall materials used in this study were β-cyclodextrin and acacia gum in the proportion of 9:1 (w/w), and microcapsules were prepared at four levels of wall materials (15, 20, 25 and 30% [w/v]) with a core material concentration of 6% (v/v). The microcapsule diameters were measured by Malvern’s Mastersizer-2000 particle size analyzer. The results showed that the particle diameters of microcapsule were mostly within 6.607 μm and 60.256 μm and varied with 2.884–120.226 μm (the standard smaller microcapsule designated as <350 μm). Through comparison of microcapsule size and uniformity with different concentration of wall materials, we concluded that the optimal concentration of wall material was 20% (w/v), which gave microcapsule with a relatively uniform size (averaging 22.153 μm), and the number of surviving encapsulated L. acidophilus was 1.50 × 10⁹ c.f.u./ml. After 8 weeks storage at 4°C, the live bacterial number was above 10⁷ c.f.u./ml, compared with unencapsulated L. acidophilus, 10⁴–10⁵ c.f.u./ml. Through the observation of scanning electron microscopy, we found that the shapes of microcapsule were round and oval, and L. acidophilus cells located in the centre of microcapsule.     

Induction of apoptosis in cultured cells by extracts from shiitake (Lentinula edodes) mycelial culture broth

Extracts fromshiitake (Lentinula edodes) mycelial culture broth, by an organic solvent ethyl acetate, inhibited the proliferation of cultured cells. At lower concentrations (1.25–15 μg/ml), this inhibition, measured by the MTT assay, was dose- and cell line-dependent. Inhibition of tumor cells, such as Caski, SiHa, HeLa, HP-1 and A375, byL. edodes-436 extracts was stronger than inhibition of normal cells (3T3). At 20 μg/ml, the extracts induced changes in cell shape, DNA-fragmentation and the activation of caspase-3. The extracts also inhibited the binding of E2F protein to its promoter. The results suggest that extracts ofL. edodes culture broth contain substances that have the ability to induce apoptosis in the cultured cells.     

Comparison of Vanadium-Rich Activity of Three Species Fungi of Basidiomycetes

A comparison of vanadium-rich activity of three species fungi of Basidiomycetes, Ganoderma lucidum, Coprinus comatus, and Grifola frondosa, was studied. By fermentation and atomic absorption spectroscopy analysis, the biomass of G. lucidum and G. frondosa declined rapidly when the concentration of vanadium exceeded 0.3% but the biomass of C. comatus did not decline rapidly until the concentration of vanadium exceeded 0.4% and the content of vanadium accumulated in the mycelia was 3529.3 μg/g. After the mice were administered (intragastrically) with vanadium-rich C. comatus, the blood glucose of alloxan-induced hyperglycemic mice was decreased (p < 0.05) and the body weight of the alloxan-induced hyperglycemic mice was increased gradually. Thus, we selected C. comatus to absorb vanadium and chose 0.4% as the optimal concentration of vanadium for the pharmacological works.     

A potential antimicrobial treatment against ESBL-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> using the tellurium compound AS101

Due to the extensive spread of antibiotic-resistant Klebsiella pneumoniae, the non-toxic immunomodulator, ammonium trichloro (dioxoethylene-o, o′) tellurate (AS101), was introduced for the first time in this study. Eleven strains of K. pneumoniae were tested: five were extended spectrum beta lactamase (ESBL)-producing strains and six were non-ESBL-producing strains. The MIC and MBC of ten strains were 9 μg/ml AS101 and 18 μg/ml for one strain. AS101 treatment inhibited bacterial growth in a dose-dependent manner on protein-rich media. No inhibition by AS101 was observed on poorer media. In combination with β-mercaptoethanol (2-ME) or cysteamine, AS101 inhibited bacterial growth in both types of media. Growth inhibition was also shown following AS101 treatment at both lag and log phases. Our data indicate that AS101 enters the bacterium through its porins, causing bacterial destruction. The mechanism of cell death was characterized using several techniques: (a) scanning electron microscopy showed that bacteria treated with AS101 or in combination with cysteamine exhibited evidence of cell-wall damage; (b) X-ray microanalysis demonstrated damage to Na/K pumps; and (c) transmission electron microscopy demonstrated cell lysis. These phenomena suggest that AS101 has antibacterial potential against K. pneumoniae infections. B. Sredni and Y. Nitzan were equal collaborators in this research.     

Antifungal potential of essential oil and various organic extracts of <Emphasis Type="Italic">Nandina domestica</Emphasis> Thunb. against skin infectious fungal pathogens

This study was undertaken to assess the in vitro antifungal potential of the essential oil and n-hexane, chloroform, ethyl acetate, and methanol extracts of Nandina domestica Thunb. against dermatophytes, the casual agents of superficial infections in animals and human beings. The oil (1,000 μg/disc) and extracts (1,500 μg/disc) revealed 31.1–68.6% and 19.2–55.1% antidermatophytic effect against Trichophyton rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, Trichophyton mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, Microsporum canis KCTC 6591, M. canis KCTC 6348, and M. canis KCTC 6349, respectively, along with their respective minimum inhibitory concentration values ranging from 62.5 to 500 and 125 to 2,000 μg/ml. Also, the oil had strong detrimental effect on spore germination of all the tested dermatophytic fungi as well as concentration and time-dependent kinetic inhibition of T. rubrum KCTC 6375. The present results demonstrated that N. domestica mediated oil and extracts could be potential sources of natural fungicides to control certain important dermatophytic fungi.     

Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

Bioactive metabolites from Alternaria brassicicola ML-P08, an endophytic fungus residing in Malus halliana

A total of 48 strains were isolated from the normal tissues of Malus halliana and the EtOAc extracts of their cultures were subjected to primary antimicrobial screening against four test bacteria and three fungi. As a result, 22 strains exhibited antimicrobial activity against at least one test microbe. Among them, Alternaria brassicicola ML-P08 showing strong activity (MICs: 0.31–2.50 mg/ml) was selected for further investigation on its secondary metabolites. Bioassay-guided fractionation of the EtOAc extract of its liquid culture afforded seven compounds, which were identified as alternariol (1), alternariol 9-methyl ether (2), altechromone A (3), herbarin A (4), cerevisterol (5), 3β,5α-dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6) and 3β-hydroxy-(22E,24R)-ergosta-5,8,22-trien-7-one (7), respectively, by spectral means (MS, IR, ¹H- and ¹³C-NMR). In vitro antimicrobial assay showed that compound 3 was substantially active against Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens and Candida albicans with the MICs of 3.9, 3.9, 1.8, and 3.9 μg/ml, respectively. Compound 4 also showed pronounced antifungal activity against Trichophyton rubrum and C. albicans with MICs of both 15.6 μg/ml. In addition, compound 1 exhibited strong xanthine oxidase inhibitory activity with the IC₅₀ of 15.5 μM, comparable to that of positive control, allopurinol (IC₅₀: 10.7 μM).     

Repair of UVB-Damaged Skin by the Antioxidant Sulphated Flavone Glycoside Thalassiolin B Isolated from the Marine Plant Thalassia testudinum Banks ex König

Daily topical application of the aqueous ethanolic extract of the marine sea grass, Thalassia testudinum, on mice skin exposed to UVB radiation resulted in a dose-dependent recovery of the skin macroscopic alterations over a 6-day period. Maximal effect (90%) occurred at a dose of 240 μg/cm², with no additional effects at higher doses. Bioassay-guided fractionation of the plant extract resulted in the isolation of thalassiolin B (1). Topical application of 1 (240 μg/cm²) markedly reduces skin UVB-induced damage. In addition, thalassiolin B scavenged 2,2-diphenyl-2-picrylhydrazyl radical with an EC₅₀ = 100 μg/ml. These results suggest that thalassiolin B is responsible for the skin-regenerating effects of the crude extract of T. testudinum. Erik L. Regalado and María Rodríguez have contributed equally to this work and should be considered as first authors.     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antimicrobial, antitumor and in vivo cytotoxicity of actinomycetes inhabiting marine shellfish

Sixty-three actinomycete strains isolated from the marine shellfish Donax trunculus anatinus were phenotypically identified as ten genera, in addition to two unidentified strains. Their metabolic extracts exhibited wide antimicrobial activities towards 11 reference and clinical cultures; and 17.5% showed antitumor activities with solid tumor selectivity of four Nocardioides, Kitasatosporia and Streptomyces strains. Streptomyces 23-2B was particularly noted for its high antitumor activity against Ehrlich’s ascites carcinoma with plateau inhibitory effect at 500, 250 and 50 μg/ml concentrations, promising solid tumor selectivity and high cytotoxicity to human carcinoma of liver (HEPG2), cervix (HELA) and breast (MCF7) (IC₅₀: 3.89, 9.4 and 10 μg/ml, respectively). In vivo cytotoxicity of S.23-2B metabolites showed common sign of unimpaired kidney and liver functions, as indicated from non-significant elevation in serum enzymatic activities, urea, creatinine, total protein and albumin levels in response to 0.5 and 5 μg/g doses after alternate-day injection for 2 weeks. Microorganisms associated with the marine shellfish are suggested to be potential source of bioactive metabolites.     

Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli

The antibacterial activity and acting mechanism of silver nanoparticles (SNPs) on Escherichia coli ATCC 8739 were investigated in this study by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with SNPs. The experimental results indicated 10 μg/ml SNPs could completely inhibit the growth of 10⁷ cfu/ml E. coli cells in liquid Mueller–Hinton medium. Meanwhile, SNPs resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that SNPs were able to destroy the permeability of the bacterial membranes. When the cells of E. coli were exposed to 50 μg/ml SNPs, many pits and gaps were observed in bacterial cells by transmission electron microscopy and scanning electron microscopy, and the cell membrane was fragmentary, indicating the bacterial cells were damaged severely. After being exposed to 10 μg/ml SNPs, the membrane vesicles were dissolved and dispersed, and their membrane components became disorganized and scattered from their original ordered and close arrangement based on TEM observation. In conclusion, the combined results suggested that SNPs may damage the structure of bacterial cell membrane and depress the activity of some membranous enzymes, which cause E. coli bacteria to die eventually.