Carbon-Mercaptooctadecane/Carboxylated Multi-walled Carbon Nanotubes Composite Based Genosensor for Detection of Bacterial Meningitis

Human brain bacterial meningitis is a life-threatening disease mainly caused by Neisseria meningitidis, lead to several complications including damage of brain or even death. The present available methods for diagnosis of meningitis have one or more limitations. A rmpM gene based genosensor was fabricated by immobilizing 5′-amino modified 19-mer single stranded DNA probe onto carbon-mercaptooctadecane/carboxylated multi-walled carbon nanotubes composite electrode and hybridized with 2.5–40 ng/6 μL of single stranded genomic DNA (ssG-DNA) of N. meningitidis from cerebrospinal fluid (CSF) of the suspected meningitis patients. The electrochemical response was measured by using cyclic voltammetry and differential pulse voltammetry (DPV) using 1 mM methylene blue as redox indicator in 30 min (including a response time of 1 min) at 25 °C. The sensitivity of the genosensor was 3.762 (μA/cm²)/ng and limit of detection was 2 ng of ssG-DNA of N. meningitidis with DPV. The genosensor has specificity only to N. meningitidis and does not hybridize with the genomic DNA of any other possible pathogen in human CSF. The immobilization of the probe and hybridization of the ssG-DNA were characterized by using electrochemical impedance in presence of 5 mM potassium ferricyanide and scanning electron microscopy. The genosensor loses only 12 % of its original DPV current on storage at 4 °C for 6 months. Carbon composite based electrochemical array can be constructed to detect multiple bacterial meningitis suspected patient CSF samples during an outbreak of the disease.     

Quick Diagnosis of Human Brain Meningitis Using Omp85 Gene Amplicon as a Genetic Marker

The usual diagnosis of life-threatening human brain bacterial meningitis are expensive, time consuming or non-confirmatory. A quick PCR based diagnosis of meningitis in cerebrospinal fluids (CSF) using specific primers of virulent Omp85 gene of Neisseria meningitidis can detect as low as 1.0 ng of genomic DNA (G-DNA) in 80 min for confirmation of bacterial meningitis caused by N. meningitidis infection. The 257 bp amplicon of Omp85 gene does not show homology with other suspected pathogens in CSF and can be used as a specific genetic marker for diagnosis of the disease.     

Nano-Au/cMWCNT Modified <Emphasis Type="Italic">speB</Emphasis> Gene Specific Amperometric Sensor for Rapidly Detecting <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> causing Rheumatic Heart Disease

A specific 5′ NH₂ labeled DNA probe of speB gene was immobilized onto the gold nanoparticles/carboxylated multi walled carbon nanotubes (Nano-Au/cMWCNT) screen printed electrode using EDC/NHS cross linking chemistry. This was followed by hybridization with 0.5–50 ng/6 µl of single stranded genomic DNA Streptococcus pyogenes infected patient throat swab samples. Electrochemical amperometric assay was deciphered by using cyclic voltammetry (CV) with methylene blue a redox indicator. The sensor had a sensitivity of 104.7 µA cm^?2 ng^?1 using CV with a R² of 0.907 and 0.01 ng/6 µl as the limit of detection (LOD). The modified electrode surface morphology was characterized using scanning electron microscopy. The stability of the electrode was seen at 4 °C for 180 days having 6% loss in the initial current. The sensor is speB gene specific and can detect the pathogen within 30 min.     

Characterization and constitutive expression of a novel endo-1,4-β-d-xylanohydrolase from Aspergillus niger in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl10 from Aspergillus niger, encoding a 308-residue mature xylanase belonging to glycosyl hydrolase family 10, was constitutively expressed in Pichia pastoris. The recombinant Xyl10 exhibited optimal activity at pH 5.0 and 60 °C with more than 50 % of the maximum activity from 40 to 70 °C. It retained more than 90 % of the original activity after incubation at 60 °C (pH 5.0) for 30 min and more than 74 % after incubation at pH 3.0–13.0 for 2 h (25 °C). The specific activity, K _m and V _max values for purified Xyl10 were, respectively, 3.2 × 10³ U mg^?1, 3.6 mg ml^?1 and 5.4 × 10³ μmol min^?1 mg^?1 towards beechwood xylan. The enzyme degraded xylan to a series of xylooligosaccharides and xylose. The recombinant enzyme with these properties has the potential for various industrial applications.     

Gene Specific Impedimetric Bacterial DNA Sensor for Rheumatic Heart Disease

An impedimetric mga gene specific DNA sensor was developed by immobilization of single stranded DNA probe onto the screen printed modified gold-dendrimer nanohybrid composite electrode for early and rapid detection of S. pyogenes in human throat swab samples causing rheumatic heart disease. Electrochemical impedance response was measured after hybridization with bacterial single stranded genomic DNA (ssG-DNA) with probe. The sensor was found highly specific to S. pyogenes and can detect as low as 0.01 ng ssDNA in 6 µL sample only in 30 min. The nanohybrid sensor was also tested with non-specific pathogens and characterized by FTIR. An early detection of the pathogen S. pyogenes in human can save damage of mitral and aortic heart valves (rheumatic heart disease) by proper medical care.     

Genosensor for rapid,sensitive, specific point-of-care detection of H1N1 influenza (swine flu)

A 5′ amine group-linked haemagglutinin (HA) gene-specific probe was attached over the surface of a working electrode to develop a rapid, specific, and sensitive point of care detection assay for H1N1 (swine flu) in human respiratory nasal swabs. The probe was attached with a cysteine covered screen-printed gold electrode via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). The electrochemical assay was performed using differential pulse voltammetry with the use of the redox indicator methylene blue for the detection of different concentrations of the single-stranded viral genome. The developed genosensor showed high sensitivity for H1N1 influenza virus with a detection limit of 0.002 ng/6 μL of viral nucleic acid in the sample. Samples were analysed by quantitative real-time Polymerase Chain Reaction as well as by conventional PCR. The genosensor showed high specificity, as no cross-reaction was observed with the heterologous nucleic acid of different pathogens (Salmonella typhi, Neisseria meningitides, and Streptococcus pyogenes) and human DNA, and it was specific for H1N1 with a sensitivity of ∼49 μA cm⁻² ng^-1. Genosensor is based on a very simple methodology that can be followed based on its easy-to-access approach. It is quick and could be used as a point-of-care test for the detection of influenza virus within 30 min.     

2-Methylisoborneol production characteristics of <Emphasis Type="Italic">Pseudanabaena</Emphasis> sp. FACHB 1277 isolated from Xionghe Reservoir,China

The cyanobacterium Pseudanabaena sp. FACHB 1277, a 2-methylisoborneol (2-MIB) producer isolated from Xionghe Reservoir, was identified by molecular biological methods based on the 16S rDNA sequence. Pseudanabaena sp. FACHB 1277 is a planktonic freshwater species with relatively high 2-MIB per cell density value (7.76?×?10^?6 ng cell^?1) and specific growth rate (0.25?±?0.01 d^?1). The effects of temperature and light intensity on 2-MIB production of Pseudanabaena sp. FACHB 1277 were investigated. Of the six temperatures tested, 10, 15, 20, 25, 30, and 35 °C, the maximum total 2-MIB per cell density and minimum cell density were observed at 10 °C, while the total 2-MIB and dissolved 2-MIB (including extracellular and dissolved intracellular 2-MIB) increased with increasing temperature. Among the six tested light intensities (10, 25, 40, 55, 70, and 85 μmol photons m^?2 s^?1), the minimum total 2-MIB per cell density and maximum cell density were observed at 25 μmol photons m^?2 s^?1. The total 2-MIB and extracellular 2-MIB increased with light intensity increasing from 10 to 40 μmol photons m^?2 s^?1, while no significant increase was observed when the light intensity was higher than 40 μmol photons m^?2 s^?1. The maximum intracellular 2-MIB (including dissolved and bound) occurred at 25 μmol photons m^?2 s^?1. The present study indicates that increasing temperature could favor the conversion of bound intracellular to dissolved 2-MIB, while increasing light intensity stimulates the release of dissolved intracellular 2-MIB into the environment.     

High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus

NZ2114, a new variant of plectasin, was overexpressed in Pichia pastoris X-33 via pPICZαA for the first time. The total secreted protein of fermentation supernatant reached 2,390 mg/l (29 °C) and 2,310 mg/l (25 °C), and the recombinant NZ2114 (rNZ2114) reached 860 mg/l (29 °C) and 1,309 mg/l (25 °C) at 96 h induction in a 5-l fermentor, respectively.The rNZ2114 was purified by cation exchange chromatography, and its yield was 583 mg/l with 94.8 % purity. The minimal inhibitory concentration (MIC) of rNZ2114 to four ATCC strains of Staphyloccocus aureus was evaluated from 0.028 to 0.90 μM. Meanwhile, it showed potent activity (0.11–0.90 μM) to 20 clinical isolates of MRSA. The rNZ2114 killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in Mueller-Hinton medium within 6 h when treated with 4?×?MIC. The postantibiotic effect of rNZ2114 to S. aureus ATCC 25923 and ATCC 43300 was 18.6–45.6 and 1.7–3.5 h under 1×, 2×, and 4× MIC, respectively. The fractional inhibitory concentration index (FICI) indicated a synergistic effect between rNZ2114 and kanamycin, streptomycin, and vancomycin against S. aureus ATCC 25923 (FICI?=?0.125), and additivity between rNZ2114 and ampicillin, spectinomycin (FICI?=?0.625), respectively. To S. aureus ATCC 43300 [methicillin-resistant S. aureus (MRSA)], rNZ2114 showed a synergistic effect (FICI?=?0.125–0.3125) with kanamycin, ampicillin, streptomycin, and vancomycin, and antagonism with spectinomycin (FICI?=?8.0625). The rNZ2114 caused only less than 0.1 % hemolytic activity in the concentration of 128 μg/ml, and showed a good thermostability from 20 to 80 °C. In addition, it exhibited the highest activity at pH 8.0. These results suggested that large-scale production of NZ2114 is feasible using the P. pastoris expression system, and it could be a new potential antimicrobial agent for the prevention and treatment of S. aureus especially for MRSA infections.     

Plant regeneration from cell suspension-derived protoplasts of Populus × beijingensis

A protocol for plant regeneration from cell suspension-derived protoplasts of Populus × beijingensis is described. Protoplasts were isolated from cell suspension cultures 6 d after subculture and further cultured in liquid NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 0.6 M glucose, 9.05 μM 2,4-dichlorophenoxyacetic acid, and 0.89 μM 6-benzyladenine at a density of 2?×?10⁵ protoplasts per milliliter. The initial plating efficiency and final plating efficiency recorded after 10 and 30 d reached 33.7 and 1.07%, respectively. The proliferated calli transferred to regeneration medium supplemented with 2.22 μM 6-benzyladenine and 0.54 μM α-naphthaleneacetic acid gave the highest rate of shoot formation (44.4%). All protoplast-derived shoots were able to form roots on half-strength MS medium supplemented with 2.46 μM indole-3-butyric acid.     

Winter bloom of picoeukaryotes in Hungarian shallow turbid soda pans and the role of light and temperature

The seasonal dynamics of picophytoplankton communities in shallow turbid alkaline pans in Hungary was studied between July 2006 and May 2007. Similarly to other aquatic environments in the temperate zone, dominance of picocyanobacteria was observed in summer and that of picoeukaryotes in winter. The mild winter in 2006–2007, with midday water temperatures of 5–10°C, resulted in large winter phytoplankton blooms (maximum chlorophyll a concentration 800 μg l^?1) in the shallow pans. The phytoplankton was composed of single-celled picoeukaryotes and had a maximum of 108 × 10⁶ cells ml^?1 in Büdös-szék pan, 50 × 10⁶ cells ml^?1 in Kelemen-szék pan in April 2007, and 47 × 10⁶ cells ml^?1 in Zab-szék pan in March 2007. In order to explain the winter dominance of picoeukaryotes, we isolated picoeukaryotic and picocyanobacterial strains and determined the temperature and light dependence of their photosynthesis. Under temperatures <15°C, the photosynthetic activity of the picoeukaryotic strain was higher and their light utilization was better than those of the picocyanobacterial strain. The results indicate that low temperature and light intensity in winter provide a competitive advantage to picoeukaryotes, while higher temperatures and light intensity are more favorable for picocyanobacteria.     

Spatiotemporal Variation of Bacterial Assemblages in a Shallow Subtropical Coastal Lagoon in Southern Brazil

A study on the bacterioplankton of Conceição Lagoon (27°34′ S–48°27′ W), Southern Brazil, was carried out in July 2005 (austral winter) and January 2006 (austral summer) to characterize the bacterial spatiotemporal distribution and to determine the heterotrophic and photoautotrophic bacterial dominance in hypoxic/oxic stratified waters. Bacterial abundance increased significantly (p?5 (winter) to 3.21?×?10⁶ cells mL^?1 (summer), heterotrophic coccus/rod-shaped (HCR) cells from 7.00?×?10⁴ to 3.60?×?10⁶ cells mL^?1, and heterotrophic filamentous (HF) bacteria from 2.90?×?10³ to 2.74?×?10⁵ cells mL^?1. Bacterial biovolumes also increased in summer with mean biovolumes of CCY ranging from 0.38 to 1.37 μm³, HCR cells from 0.31 to 1.12 μm³, and HF from 3.32 to 11.34 μm³. Principal component analysis showed that salinity, temperature, and light were the abiotic factors that better explained the temporal variability of bacterial assemblages. Bacterial heterotrophy dominated in the lagoon, excepted by the southern and part of central sector in January 2006, when autotrophic-dominated microbial community occurred. Spatially, bacterial assemblages were influenced by nutrient gradient, oxygen, and salinity with a positive relationship between biovolumes and nutrients and a negative relationship between abundance of coccus cyanobacteria and nutrients. area revealed a singular temporal pattern with hypoxic bottom waters in winter and oxygen-rich waters appearing in summer related with the availability of light and predominant microbes. Thus, oxygen consumption/production is likely to be regulated by the amount of light reaching the bottom, stimulating the production of oxygen by oxygenic phototrophs.     

A selective temperature-sensitive defect in viral RNA expression in cells infected with a ts transformation mutant of murine sarcoma virus

We investigated the nature of the defect in the temperature-sensitive mutant of Moloney murine sarcoma virus (Mo-MuSV), termed ts110. This mutant has a temperature-sensitive defect in a function required for maintenance of the transformed state. A nonproducer cell clone, 6m2, infected with ts110 expresses P85 and P58 at 33°C, the transformed temperature, but only P58 is detected at the restrictive temperature of 39°C. Shift-up (33°C → 39°C) and in vitro experiments have established that P85 is not thermolabile for immunoprecipitation. Previous temperature-shift experiments (39°C → 33°C) have shown that P85 synthesis resumes after a 2–3 hr lag period. Temperature shifts (39°C → 33°C) performed in the presence of actinomycin D prevented the synthesis of P85, whereas P58 synthesis did not decline for 5 hr, suggesting that P58 and P85 are translated from different mRNAs. The shift-up experiments also indicated that, once made, the RNA coding for P85 can function at the restrictive temperature for several hours. MuSV-ts110-infected cells superinfected with Mo-MuLV produced a ts110 MuSV-MuLV mixture. Sucrose gradient analysis of virus subunit RNAs revealed a ～28S and a ～35S peak. Electrophoresis of the ～28S poly(A)-containing RNA from ts110 virus in methyl mercuric hydroxide gels resolved two RNAs with estimated sizes of 1.9 × 10⁶ and 1.6 × 10⁶ daltons, both smaller than the wild type MuSV-349 genomic RNA (2.2 × 10⁶ daltons). RNA in the ～28S size class from virus preparations harvested at 33°C was found to translate from P85 and P58, whereas, the ～35S RNA yielded helper virus Pr63^gag. In contrast, virus harvested at 39°C was deficient in P85 coding RNA only. Peptide mapping experiments indicate that P85 contains P23 sequences, a candidate Moloney mouse sarcoma virus src gene product. Taken together, these results suggest that two virus-specific RNAs are present in ts 110-infected 6m2 cells and rescued ts110 pseudotype virions at 33°C, one coding for P85, whose expression can be interfered with by shifting the culture to 39°C; the other coding for P58, whose expression is unaffected by temperature shifts. P85 is a candidate gag-src fusion protein, while P58 contains gag sequences only.     

Continuous biodegradation of parathion by immobilized Sphingomonas sp. in magnetically fixed-bed bioreactors and evaluation of the enzyme stability of immobilized bacteria

Magnetically-modified Sphingomonas sp. was prepared using covalent binding of magnetic nanoparticles on to the cell surface. The magnetic modified bacteria were immobilized in the fixed-bed bioreactors (FBR) by internal and external magnetic fields for the biodetoxification of a model organophosphate, parathion: 93 % of substrate (50 mg parathion/l) was hydrolyzed at 0.5 ml/min in internal magnetic field fixed-bed bioreactor. The deactivation rate constants (at 1 ml/min) were 0.97 × 10^?3, 1.24 × 10^?3 and 4.17 × 10^?3 h^?1 for immobilized bacteria in external and internal magnetic field fixed-bed bioreactor and FBR, respectively. The deactivation rate constant for immobilized magnetically modified bacteria in external magnetic field fixed-bed bioreactor (EMFFBR) was 77 % lower than that of immobilized cells by entrapping method on porous basalt beads in FBR at 1 ml/min. Immobilized magnetic modified bacteria exhibited maximum enzyme stability in EMFFBR.     

Development and Padronization of Three Multiplex PCRs for the Diagnosis of Chlamydia trachomatis, Toxoplasma gondii, Herpes Simplex Viruses 1 and 2, and Cytomegalovirus

To develop multiplex PCRs (mPCRs) that allows simultaneous diagnosis of the infectious agents Chlamydia trachomatis, Toxoplasma gondii, HSV 1/2, and Cytomegalovirus (CMV). The study included patients with clinical suspicion of these agents, and clinical samples were blood, cerebrospinal fluid, urine, vaginal swabs, and amniotic fluid. After the extraction of DNA, this was used as a template in amplification by PCR of selected genes. The following conditions were tested: primer concentration, MgCl₂ concentration, and annealing temperature. Three mPCRs were developed: multiplex I (CMV, HSV 1/2), multiplex II (CMV, HSV 1/2, T. gondii), and multiplex III (C. trachomatis, T. gondii, HSV 1/2, and CMV). The primer pairs used were shown to be specific for each infectious agent, and the specificity of mPCR assays was 100 %. Both the reactions of the monoplex PCR and mPCR produced a detection limit of 2 × 10^?5 to 6 × 10^?7 ng/μl of different DNAs. Upon conclusion, amplified products of expected size were obtained in 3 different reactions, and all the infectious agents were detected simultaneously in each mPCR. The concordant results of the study suggest that mPCR can be a powerful tool to improve the diagnostics of infectious diseases.     

A rapid,simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples

A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62 °C for 60 min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5 × 10⁰ copies/μL, 100 times more than that of conventional PCR (5 × 10² copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis.     

Electrochemical selective detection of dopamine on microbial carbohydrate-doped multiwall carbon nanotube-modified electrodes

Microbial carbohydrate-doped multiwall carbon nanotube (MWNT)-modified electrodes were prepared for the purpose of determining if 4-(2-aminoethyl)benzene-1,2-diol (3,4-dihydroxyphenylalanine; dopamine) exists in the presence of 0.5 mM ascorbic acid, a representative interfering agent in neurotransmitter detection. The microbial carbohydrate dopants were α-cyclosophorohexadecaose (α-C16) from Xanthomonas oryzae and cyclic-(1 → 2)-β-d-glucan (Cys) from Rhizobium meliloti. The cyclic voltammetric responses showed that the highest sensitivity (5.8 × 10^?3 mA cm^?2 μM^?1) is attained with the Cys-doped MWNT-modified ultra-trace carbon electrode, and that the α-C16-doped MWNT-modified glassy carbon electrode displays the best selectivity to dopamine (the approximate peak potential separation is 310 mV).     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.     

Efficient protoplast isolation and transient gene expression system for <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrid cultivar ‘Ruili Beauty’

With the release of the Phalaenopsis equestris (Schauer) Rchb.f. genome database, more in-depth studies of Phalaenopsis spp. will be carried out in the future. Transient gene expression in protoplasts is a useful system for gene function analysis, which is especially true for Phalaenopsis, whose stable genetic transformation is difficult and extremely time-consuming. In this study, juvenile leaves from aseptic Phalaenopsis seedlings were used as the starting material for protoplast isolation. After protocol refinement, the highest yield of viable protoplasts [5.94 × 10⁶ protoplasts g^?1 fresh weight (FW)] was achieved with 1.0% (w/v) Cellulase Onozuka R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M D-mannitol, with an enzymolysis duration of 6 h. As indicated by transient expression of green fluorescent protein (GFP), a transformation efficiency of 41.7% was achieved with 20% (w/v) polyethylene glycol (PEG-4000), 20 μg plasmid DNA, 2 × 10⁵ mL^?1 protoplasts, and a transfection duration of 30 min. The protocol established here will be valuable for functional studies of Phalaenopsis genes.     

Identification and virulence characterization of entomopathogenic fungus <Emphasis Type="BoldItalic">Lecanicillium attenuatum</Emphasis> against the pea aphid <Emphasis Type="BoldItalic">Acyrthosiphon pisum</Emphasis> (Hemiptera: Aphididae)

An entomopathogenic fungal strain was originally isolated on artificial medium from the corpse of a pea aphid (Acyrthosiphon pisum Harris) collected at Jingzhou, China (N30°21′18.15″, E112°08′41.63″). Based on tests of the morphological, physiological and biochemical characteristics and analysis of internal transcribed spacer (ITS) sequences, it was considered to be a strain of Lecanicillium attenuatum Zare & W. Gams. Therefore, the strain was designated L. attenuatum YZU 151121. The activity of the biological agents under study was determined at 26 °C and 90% relative humidity. The number of A. pisum killed was increased by increasing the concentration of L. attenuatum. The results demonstrated that L. attenuatum YZU 151121 showed a high efficacy against 3rd-instar nymphs (LC₅₀ = 2.91 ± 0.365 × 10⁵ conidia/ml) and adults (LC₅₀ = 3.12 ± 0.398 × 10⁶ conidia/ml) after 6 days of exposure. Crude extract from this strain was tested for contact toxicity and showed high activity in 3rd-instar nymphs and adults, with LC₅₀ values of 251.34 ± 49.54 and 315.46 ± 87.66 mg/l, respectively. In addition, crude extract at a concentration of 200 mg/l could significantly reduce fecundity in adults. These results revealed that the strain YZU 151121 may be useful in biopesticides for controlling pea aphid.     

Transition state stabilization by deaminases: Rates of nonenzymatic hydrolysis of adenosine and cytidine

Nonenzymatic rates of hydrolytic deamination of adenosine and cytidine by acids and bases analogous to side chains of naturally occurring amino acids are compared with the rates of uncatalyzed deamination in water and with the rates of the hydroxide- and hydrogen ion-catalyzed reactions. For adenosine, hydroxide ion is an effective catalyst, with a second-order rate constant of 7.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C and an energy of activation of 19.9 kcal/mol. Acid-catalyzed deamination of adenine proceeds with a second-order rate constant of 1.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C. At concentrations of 1 m and at pH values corresponding to their respective pK_a values, dimethylamine, acetate, selenide, imidazole, phosphate, and zinc(II) do not enhance the rate of deamination of adenosine beyond that observed in water, and 2-mercaptoethanol produces only a modest rate enhancement. The uncatalyzed rate of adenosine deamination in water is 8.6 × 10⁻⁹ s⁻¹ at 85°C: extrapolation to 37°C and comparison with k_cat for rat hepatoma adenosine deaminase yield a rate enhancement by the enzyme of approximately 2 × 10¹²-fold. 1,6-Dimethyladenosine, the conjugate acid of which has a pK_a value much higher than that of adenosine, is not readily deaminated, suggesting that the uncatalyzed deamination of adenosine does not proceed by hydroxide ion attack on the rare protonated form of adenosine, but rather by attack on the neutral species. Deamination of cytidine is catalyzed most effectively by hydroxide ion, with a second-order rate constant of 4.5 × 10⁻⁴ m⁻¹ s⁻¹ at 85°C and an energy of activation of 28.5 kcal/mol. The uncatalyzed rate of deamination of cytidine in water, which also exhibits an energy of activation of 28.5 kcal/mol, is 8.8 × 10⁻⁸ s⁻¹ at 85°C. Comparison of the rate extrapolated to 25°C with k_cat for bacterial cytidine deaminase gives a rate enhancement for the enzyme of 4 × 10¹¹-fold. The C-5 proton of the pyrimidine ring of cytidine does not exchange with solvent during alkaline hydrolysis, suggesting that deamination under these conditions does not involve prior addition of water across the 5,6 double bond.