Homology of plasmids in strains of unicellular Cyanobacteria.

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 10⁶ and 32.7 × 10⁶. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 10⁶ and 24 × 10⁶. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 10⁶ and 2.0 × 10⁶, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.     

Precursors of chloroplast ribosomal RNA in Euglena gracilis

Incorporation of ³²P into mature chloroplast rRNA species of MW 1.1 × 101 and 0.56 × 10⁶ has been followed in Euglena gracilis by pulse and pulse chase experiments. Mature rRNA species have precursors of MW 1.16 × 10⁶ ± 0.01 × 10⁶ and 0.64 × 106 ± 0.01 × 10⁶ resp. These precursors have base composition and hydridization properties similar to those of the mature, rRNA species. No evidence of a single common precursor to these molecules was found. Rifampicin did not affect the synthesis of chloroplast rRNA.     

δ-Aminolevulinic acid synthase of Euglena gracilis: Physical and kinetic properties

Physical and kinetic properties of δ-aminolevulinic acid synthase from wild-type and aplastidic strains of Euglena gracilis have been determined. Michaelis constants for glycine, succinyl-CoA and pyridoxal phosphate are 8.5 × 10^?3m, 2.5 × 10^?5m, and 2.9 × 10^?6m, respectively. Optimum reaction pH is 7.8, and maximal product yield during a 30-min incubation occurs at 40 °C. Activity in frozen cell extracts remains constant for 5 days, then falls slowly to one-third of the initial value after 3 months. Enzyme activity rapidly declines irreversibly in the absence of pyridoxal phosphate. Agarose gel chromatography of the native enzyme yields a single band of activity at an elution volume corresponding to a molecular weight of 138,000. δ-Aminolevulinic acid synthase obtained from green wild-type strain Z cells is identical in its physical properties to that obtained from white aplastidic mutant strain W₁₄ ZNalL cells.     

The relationship between frameshift mutagenicity and DNA-binding affinity in a series of acridine-substituted derivatives of the experimental antitumour drug 4′-(9-acridinylamino)methanesulphonanilide (AMSA)

The mutagenicity and DNA-binding affinity of members of a series of acridine-substituted derivatives of 4′-(9-acridinylamino)methanesulphonanilide (AMSA) have been compared. The series includes compounds ranging from highly active to inactive in the L1210 murine leukaemia. Binding to DNA was measured by an ethidium displacement technique, with a correction being made for acridine-induced quenching of ethidium. Mutagenicity was assessed by measuring the reversion frequencies of the frameshift tester strain Salmonella typhimurium TA1537 in liquid culture. The results indicate that maximum mutagenicity is found in a “window” of DNA-binding affinities between 10⁶ and 5 × 10⁶ M^?1 (determined at 0.01 ionic strength). Compounds with binding affinities below 10⁶ M^?1 generally lacked both antitumour and mutagenic activity, whereas those with affinities above 5 × 10⁶ M^?1 were active against L1210 leukaemia but virtually inactive in inducing frameshift mutations.     

Purification and characterization of selenium-glutathione peroxidase from hamster liver

Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 μmol cumene hydroperoxide reduced/min/mg of protein at 37 °C, pH 7.6, and 0.25 mm GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 °C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k₁ (t-butyl hydroperoxide), 7.06 × 10⁵ mm min^?1; k₁ (cumene hydroperoxide), 1.04 × 10⁶ mm^?1 min^?1; k₁ (p-menthane hydroperoxide), 1.2 × 10⁶ mm^?1 min^?1; k₁ (diisopropylbenzene hydroperoxide), 1.7 × 10⁶ mm^?1 min^?1; k₁ (linoleic acid hydroperoxide), 2.36 × 10⁶ mm^?1 min^?1; k₁ (ethyl hydroperoxide), 2.5 × 10⁶ mm^?1 min^?1; and k₁ (hydrogen peroxide), 2.98 × 10⁶ mm^?1 min^?1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.     

Production of phenolic compounds in callus cultures of tea plant under the effect of 2,4-D and NAA

The effect of hormone-like compounds at different concentrations: 2,4-D (2 × 10^?6; 2 × 10^?5; and 2 × 10^?4M) and 1-NAA (2 × 10^?7; 2 × 10^?6; 2 × 10^?5; 4 × 10^?5, and 6 × 10^?5 M) on the growth and production of phenolic compounds, including flavans and lignin, was investigated in callus culture of tea plant (Camellia sinensis L., a highly productive strain IFR ChS-2). The growth of the culture was vigorous, and production of phenolic compounds therein was efficient in the medium containing 2 × 10^?5 M 2,4-D. Substitution of 1-NAA for 2,4-D in all the cases decelerated the growth of the culture. These changes were more pronounced when 2 × 10^?7 and 2 × 10^?6 M 1-NAA was used; in this case, biomass accumulation decreased by 1.5–2.0 times as compared with control material growing on the medium with 2 × 10^?5 M 2,4-D. In the presence of 1-NAA, the content of total soluble phenolic compounds and flavans in the calli rose by 30% on the average as compared with control material. Accumulation of lignin remained essentially the same. Therefore, the replacement of 2,4-D with 1-NAA in the nutrient medium used for the growing of highly productive strain of tea plant callus did not induce considerable changes in its ability to produce phenolic compounds.     

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.     

Screening of high toxic Metarhizium strain against Plutella xylostella and its marking with green fluorescent protein

Entomopathogenic fungus is proposed to be one of the best biocontrol agents against the destructive insect pest Plutella xylostella. In this study, we tested the virulence of 11 Metarhizium strain isolates against P. xylostella using a leaf dipping method, and found one strain, named 609, which had displayed the highest pathogenicity. Bioassay results showed that the accumulated corrected mortality rate was 86.7 % on the eighth day after inoculation with a spore concentration 1 × 10⁸ conidia/mL, and that the time to 50 % lethality was 5.7-day. The strain was identified as Metarhizium anisopliae var. acridum by internal transcribed spacer (ITS) region sequencing. A green fluorescent protein (GFP) marker containing vector, camben-gfp, was constructed and delivered into strain 609 by Agrobacterium tumefaciens-mediated transformation. Six positive clones expressing GFP were selected and tested for toxicity against P. xylostella, all of which displayed the same toxicity as the parental wild type strain. The survival rate of transformant T1 was investigated by monitoring GFP levels at 4-day intervals after inoculation into soil. We found that the concentration of Metarhizium spores decreased sharply from 1 × 10⁷ conidia/g to 1 × 10⁶ conidia/g in the first 5 days after inoculation. The decreasing trend then stabilized and the spore count declined to approximately 1 × 10⁴–10⁵ conidia/g after 1 month. The results of this study indicate that the expression of gfp gene in strain 609 does not alter the virulence capability of Metarhizium. This strain will therefore be useful for the control of P. xylostella and as a tool to study molecular biology properties and monitor colonization of M. anisopliae in the field.     

A characterization of alpha-bungarotoxin binding in the brain of the horseshoe crab, Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin to membrane fragments prepared from Limulus brain tissue has been investigated. Toxin binding approaches saturation in the range of 30 to 40 nm, with maximum binding of 2 to 6 pmol/mg of protein. The saturation kinetics and the rate of displacement of bound toxin are consistent with multiple toxin binding sites. Pharmacological studies show that binding is inhibited by both cholinergic agonists and antagonists, I₅₀′s for inhibition by d-tubocurarine, nicotine, decamethonium, carbachol, and atropine are 2 × 10^?6, 7 × 10^?6, 2 × 10^?5, 6 × 10^?4, and 3 × 10^?4m, respectively. Nicotinic ligands inhibited binding much more effectively than muscarinic ligands. Toxin binding activity was solubilized with Triton X-100. Velocity sedimentation analysis of the solubilized activity revealed three separate components. Seventy to eighty percent of the binding activity had a sedimentation coefficient of 8.6 S. The remaining activity was composed of two components with sedimentation coefficients of 15.1 and 17 S.     

The kinetics of oxidation of hemerythrin species—linear free energy relationships

The second-order rate constants (M^?1sec^?1, 25°C, pH 8.2, I = 0.15 M) for the oxidation to (semi-met)₀of deoxyhemerythrin from Phascolopsis gouldii (P.g.) and Themiste zostericola (T.z.) have been determined for Fe(CN)₅(4-NH₂py)^2? (3.6 × 10⁴ T.z.,2.8 × 10²P.g.),Fe(CN)₅NH₃^2?(2.4 × 10⁴ T.z.), Fe(CN)₆^3? (1.0 × 10⁵ T.z.,1.4 × 10²P.g.), Fe(CN)₅PPh₃^2? (7.3 × 10⁵T.z.), and Fe(CN)₄dipy- (~6 × 10⁶ T.z.,7.5 × 10⁴ P.g.). Corresponding rate constants for the oxidation of (semi-met)_R to met are: Fe(CN)₅(4-NH₂py)^2? (1.2 × 10³ P.g.), Fe(CN)₆^3? (3.4 × 10⁵ T.z., 4.5 × 10 Fe(CN)₅PPh₃^2? (4.4 × 10⁴P.g.), Fe(CN)₄dipy^? (1.7 × 10⁵P.g.), and Coterpy₂³⁺ (5.1 P.g.) The rates of oxidation of deoxy- and (semi-met)_R myohemetythrin by Fe(CN)₆^3? were too rapid for stopped-flow measurement. The Marcus relationship for cross-reactions was successfully applied to these data.     

The interaction of substrate-related ketals with bacterial and viral neuraminidases

Arthrobacter sialophilus neuraminidase catalyzes the hydration of 5-acetamido-2,6-anhydro-3, 5-dideoxy-d-glycero-d-galacto-non-2-enonic acid (2,3-dehydro-AcNeu) with K_m and k_cat values of 8.9 × 10^?4m and 6.40 × 10^?4 s^?1, respectively. The methyl ester of 2,3-dehydro-AcNeu as well as 2,3-dehydro-4-epi-AcNeu are also hydrated by the enzyme. The product resulting from the enzymatic hydration of 2,3-dehydro-AcNeu is N-acetylneuraminic acid. A series of derivatives of 2,3-dehydro-AcNeu (K_I 1.60 × 10^?6m) including 2,3-dehydro-4-epi-AcNeu (2.10 × 10^?4m) and 2,3-dehydro-4-keto-AcNeu (K_I = 6.10 × 10^?5 m) were each competitive inhibitors of the enzyme. The methyl esters of these ketal derivatives were also competitive enzyme inhibitors. Dissociation constants for these ketals were determined independently by fluorescence enzyme titrations which gave values similar to those found kinetically. These six relatives of 2,3-dehydro-AcNeu were also competitive inhibitors for the influenza viral neuraminidases. For the viral neuraminidases, the dissociation constant for 2,3-dehydro-AcNeu and its methyl ester were 2.40 × 10^?6 and 1.17 × 10^?3m, respectively. The interpretation placed upon the K_I values determined for these ketals against the Arthrobacter versus influenza neuraminidases is that the bacterial enzyme has a more flexible glycone binding site.     

一株溶藻细菌对海洋原甲藻的溶藻效应

从深圳大鹏湾南澳赤潮爆发海域的表层海水中分离得到1株对海洋原甲藻(Prorocentrum micans)具有溶藻活性的海洋细菌,菌株编号为N10。利用液相感染法研究了该溶藻细菌的溶藻效果和溶藻作用方式。结果表明,菌株N10能使藻细胞失去运动活性,并膨胀变形,细胞膜内物质聚集于一端,藻细胞最终破裂死亡。菌悬液接种到藻液中的量越大,初始细菌密度越高,其溶藻效果越强。菌悬液以1∶10的体积比接种到藻液中时,藻细胞在24 h的死亡率为83%,至72 h全部溶解死亡;体积比为1∶20的藻细胞在24 h的死亡率为71%,之后藻细胞密度略有波动,120 h时死亡率达77%;而体积比为1∶100的藻细胞密度在前24 h有所下降,死亡率达39%,之后藻细胞密度又开始明显上升;对照组的藻细胞密度均呈明显上升趋势。菌悬液过滤液和高温加热处理后的菌悬液过滤液对海洋原甲藻均无溶藻活性,表明菌株N10的溶藻方式为直接溶藻。通过16S rRNA序列分析并与GenBank数据进行同源性检索,并结合细菌形态及生理生化特征,菌株N10隶属于黄杆菌科(Flavobacteriaceae)中的Muricauda sp.。     

Changes in the mechanical properties of the extensible cuticle of Rhodnius through the fifth larval instar

The ultimate tensile strength (σ_UT) and the modulus of elasticity (E) of Rhodnius extensible cuticle are σ_UT = 2.20 × 10⁷ Nm^?2, E = 2.43 × 10⁸ Nm^?2 (unplasticised); σ_UT = 1.43 × 10⁷ Nm^?2, E = 9.45 × 10⁶ Nm^?2 (plasticised with 5HT) and σ_UT = 9.05 × 10⁶ Nm, E = 2.46 × 10⁶ Nm^?2 (plasticised in pH 5 buffer).The mechanical properties of cuticle from insects which have deposited additional layers of cuticle after they have been fed differ from those of cuticle from unfed insects. This is possibly due to the different composition of the additional cuticle: it is suggested that the post-feeding cuticle is providing protection and a template for the next instars cuticle.The maximum strain of extensible cuticle from starved insects is related to the amount of matrix protein present.     

Occurrence in vivo of "hidden breaks" at specific sites of 26 S ribosomal RNA of Musca carnaria.

A fragmentation process occurs in 26 S ribosomal RNA of mature cytoplasmic ribosomes of Musca carnaria. It consists of the sequential appearance of three “hidden breaks” that fragment 26 S rRNA (M_r = 1.42 × 10⁶) into four pieces with approximate molecular weights of 0.68 × 10⁶, 0.35 × 10⁶, 0.29 × 10⁶ and 0.096 × 10⁶, respectively. This fragmentation was not observed in 17 S rRNA (M_r = 0.74 × 10⁶).Extremely mild treatment of newly assembled ribosomes with pancreatic RNAase reproduces the 26 S rRNA fragmentation phenomenon in vitro in the same way as it occurs in vivo.This evidence is discussed in relation to the secondary structure of 26 S rRNA and its binding with specific ribosomal proteins.     

Studies on the culicine mosquito host range of Bacillus sphaericus and Bacillus thuringiensis var. israelensis with notes on the effects of temperature and instar on bacterial efficacy

Toxicity tests of three strains of Bacillus sphaericus against late instars of 12 culicine mosquito species indicated a wide range of susceptibility. Culex pipiens and C. salinarius were highly susceptible (LC₅₀s < 10⁴ spores/ml) to strain 1593, and C. pipiens and C. restuans were highly susceptible to strain 2013-4. The potency of strain SSII-1 was approximately one-tenth that of strains 1593 and 2013-4 against C. pipiens. Susceptibility of Aedes species to strain 1593 was highly variable. At temperatures ≥ 20°C, A. fitchii, A. intrudens, A. stimulans, and A. vexans were moderately to highly susceptible (LC₅₀s 6 × 10³−4 × 10⁴ spores/ml), A. triseriatus was only slightly susceptible (LC₅₀ > 10⁶ spores/ml), and A. aegypti was refractory. Susceptibility of Aedes mosquitoes to strain SSII-1 was less variable, with LC₅₀s against A. aegypti, A. canadensis, A. stimulans, and A. triseriatus all being between 10⁴ and 10⁶ vegetative cells + spores/ml. All species of mosquitoes tested were, in general, highly susceptible to B. thuringiensis var. israelensis (LC₅₀s 2.3 × 10³−2.5 × 10⁴ spores/ml). In B. sphaericus toxicity tests, decreased temperatures resulted in up to a 16-fold increase in LC₅₀ and a substantial reduction in probit line slope. First-instar A. aegypti larvae were more susceptible to B. sphaericus strain SSII-1 than the three later instars, which were approximately equally susceptible; however, no significant difference was observed in the susceptibility of the four instars of A. triseriatus.     

Bovine toxoplasmosis: experimental infections.

Three calves dosed per os with 0.75 × 10⁶, 0.75 × 10⁶ and 1.5 × 10⁶Toxoplasma oocysts developed high titres of Toxoplasma antibodies as measured by the indirect fluorescent antibody test and the dye test. Toxoplasma gondii was reisolated from 2 of these animals. Two calves given 1 × 10³ tissue cysts and 2 × 10⁶ tachyzoites intramuscularly did not develop such high fitres, but T. gondii was reisolated from the calf injected with tachyzoites.Of 4 pregnant cows given 7 × 10⁴, 7 × 10⁴ and 1.6 × 10⁵ oocysts and 1.5 × 10² tissue cysts only 2 developed significant levels of Toxoplasma antibodies. There was no evidence of Toxoplasma infection in the calves born by these cows.It was concluded that cattle do not readily acquire persistent T. gondii infections, and this may be due, in part at least, to early elimination of Toxoplasma from their tissues.     

Levels of aflatoxin B1, bacteria and fungi in feed and food in 1971 — 1987

Totally 39% out of 8371 feed and their component samples were contaminated by aflatoxin B₁. Mean contamination was 36μg/kg with maximum yield 10100 μg/kg. Contamination of samples by total count of organisms, mean contamination and maximum yield, respectively was: 1) bacteria 99%, 2.2×10⁶, 2.4×10⁸; 2) proteolytic bacteria 94%, 1.2×10⁵, 3.0×10⁶;3) moulds 98%, 1.3×10⁵, 9.0×10⁶; 4) yeasts 44 %, 3.3×10⁴, 3.6×10⁶. The samples were contaminated in 92 % byAspergillus spp, in 71% byAspergillus flavus, in 83% byPenicillium spp, and in 20% byFusarium spp with mean contamination 8.3×104, 1.1×10³, 4.2×10⁴, 5.0×10³ , and maximum yield 6.8×10⁶, 1.0×10⁵, 5.0×10⁶, 1.5×10⁶, respectively. Totally 8.5% of strains were aflatoxinogenic and 4.4% of the strains were isolated from feed and 21 % of the strains from grain/nut.