Characterization of recombinant rabies virus carrying double glycoprotein genes

A recombinant rabies virus carrying double glycoprotein (G) genes, R(NPMGGL) strain, was generated by a reverse genetics system utilizing cloned cDNA of the RC-HL strain, and the biological properties of the virus were compared to those of the recombinant RC-HL (rRC-HL) strain. The extents of virus growth in cultured cells and virulence for adult mice of the R(NPMGGL) strain were almost same as those of the rRC-HL strain, while G protein content of the purified R(NPMGGL) virion and G protein expression level in R(NPMGGL)-infected cells were 1.5-fold higher than those of the rRC-HL strain. As a result of serial passages of the R(NPMGGL) strain in cultured cells, the expression level of G protein in cultured cells infected with the passaged R(NPMGGL) strain was maintained and virus titers rose with adaptation to the cultured cells. Furthermore, analysis of neutralization titers in mice immunized with UVinactivated virus suggested that the R(NPMGGL) strain had higher immunogenicity than that of the rRC-HL strain. The results suggest that the R(NPMGGL) strain carrying double G genes might be a useful candidate for development of a new inactivated rabies vaccine.     

Inheritance of photo-control of conidial development in the fungusBipolaris oryzae

The inheritance of light dependence for conidial development inBipolaris oryzae was analyzed using single-ascospore isolates. When a photo-induced strain was crossed with another photo-induced strain, only photo-induced progeny were produced. When a photo-induced strain was crossed with a non-photo-induced (I) strain, photo-induced and nonphoto-induced (I) progeny were produced in a ratio of 1∶1. However, when a photo-induced strain was crossed with a non-photo-induced (II) strain, a non-photo-induced (I) progeny were formed in addition to parental types. It was suggested that genes for photo-control of conidiophore induction and genes for photo-control of conidiophore maturation are located on the same chromosome.     

Mechanical properties of trabecular bone. Dependency on strain rate.

The effect of strain rate (epsilon) and apparent density (rho) on stiffness (E), strength (sigma u), and ultimate strain (epsilon u) was studied in 60 human trabecular bone specimens from the proximal tibia. Testing was performed by uniaxial compression to 5% specimen strain. Six different strain rates were used: 0.0001, 0.001, 0.01, 0.1, 1, and 10 s-1. Apparent density ranged between 0.23 and 0.59 g cm-3. Linear and non-linear regression analyses using strength, stiffness and ultimate strain as dependent variables (Y) and strain rate and apparent density as independent variables were performed using the following models: Y = a rho b epsilon c, Y = rho b(a + c epsilon; Y = (a + b rho)epsilon c, Y = a rho 2 epsilon c, E = a rho 3 epsilon c. The variations of strength and stiffness were explained equally well by the linear and the power function relationship to strain rate. The exponent was 0.07 in the power function relationship between strength and strain rate and 0.05 between stiffness and strain rate. The variation of ultimate strain was explained best using a power function relationship to strain rate (exponent = 0.03). The variation of strength and stiffness was explained equally well by the linear, power function and quadratic relationship to apparent density. The cubic relationship between stiffness and apparent density showed a less good fit. Ultimate strain varied independently of apparent density.     

副粘病毒Tianjin株NP蛋白的表达及分析

1999年,本实验室从群体性暴发的急性呼吸道感染素致死的普通棉耳绒猴肺组织中分离到一株副粘病毒,命名为副粘病毒 Tianjin株.经研究发现该动物中心的工作人员都有此病毒抗体,且正常人群(献血员)抗体阳性率高达46%,急性呼吸道感染的患儿抗体阳性率为19.28%,提示此病原体与人类可能有密切的关系.     

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.     

Trypanosoma cruzi: Correlation of Growth Kinetics to Zymodeme Type in Clones Derived from Various Sources*†

SYNOPSIS. Trypanosoma ( Schizotrypanum ) cruzi clones were derived from isolates of an acute human case of Chagas' disease (strain Esmereldo), a human case of T. cruzi infection (strain CAN-III) and from a naturally infected opossum (strain WA-250). The isoenzyme patterns and growth rates of the clones were stable during long-term cultivation, by serial passages, of the parasites in liquid medium. Both clones of strain Esmereldo were zymodeme II; the 2 clones of strain CAN-III, zymodeme III; and the 5 clones of strain WA-250, zymodeme I. The range in doubling times of the parasite populations in liquid medium were 36–49. 7 h (strain WA-250), 117.2–133.7 h (Esmereldo clones) and 169–208 h (CAN-III clones).     

Morphological and Molecular Differences in Two Strains of <Emphasis Type="Italic">Ustilago esculenta</Emphasis>

Ustilago esculenta is a fungal endophyte of Zizania latifolia that plays an important agricultural role in this vegetable crop. The purpose of this study was to characterize sporidial (T) and mycelial (M-T) strains of U. esculenta isolated from sporulating and non-sporulating galls on plants growing in Zhejiang province, China. Morphological comparisons of the T strain and M-T strain were made by optical and scanning electron microscope observation. Genetic differences were examined by sequencing the ITS region of the fungus and examining differential protein expression by two-dimensional gel electrophoresis and MALDI-TOF-MS/MS. The sporidial (T) and mycelial (M-T) strains differed in morphological characteristics of their in vitro single colony formations and in cell shape. Alignment of ITS sequences of the T strain and M-T strain revealed a single mutation between the T strain and M-T strain, but the sequences were the same within strains. A total of 146 proteins were only expressed in the M-T strain, and 242 proteins were only expressed in the T strain isolated from infected plants. A total of 222 proteins were up-regulated or down-regulated in the T strain when compared with the M-T strain. Of these, 18 proteins were identified and eight were associated with processes involving energy metabolism and the cytoskeleton. Two morphology-related proteins, MAP kinase kinase and actin, were differentially expressed. The differences noted in the T strain and M-T strain may lead to a better understanding of the life cycle and morphogenesis in U. esculenta.     

Enzyme production-based approach for determining the functions of microorganisms within a community

The functions of specific microorganisms in a microbial community were investigated during the composting process. Cerasibacillus quisquiliarum strain BLx(T) and Bacillus thermoamylovorans strain BTa were isolated and characterized in our previous studies based on their dominance in the composting system. Strain BLx(T) degrades gelatin, while strain BTa degrades starch. We hypothesized that these strains play roles in gelatinase and amylase production, respectively. The relationship between changes in the abundance ratios of each strain and those of each enzyme activity during the composting process was examined to address this hypothesis. The increase in gelatinase activity in the compost followed a dramatic increase in the abundance ratio of strain BLx(T). Zymograph analysis demonstrated that the pattern of active gelatinase bands from strain BLx(T) was similar to that from the compost. Gelatinases from both BLx(T) and compost were partially purified and compared. Homologous N-terminal amino acid sequences were found in one of the gelatinases from strain BLx(T) and that of compost. These results indicate strain BLx(T) produces gelatinases during the composting process. Meanwhile, the increase in the abundance ratio of strain BTa was not concurrent with that of amylase activity in the compost. Moreover, the amylase activity pattern of strain BTa on the zymogram was different from that of the compost sample. These results imply that strain BTa may not produce amylases during the composting process. To our knowledge, this is the first report demonstrating that the function of a specific microorganism is directly linked to a function in the community, as determined by culture-independent and enzyme-level approaches.     

Characterization of a high‐growth‐rate mutant strain of Pyropia yezoensis using physiology measurement and transcriptome analysis

A mutant strain of Pyropia yezoensis, strain E, was isolated from the free‐living conchocelis of a pure strain (NA) treated with ethyl methane sulfonate. The incremental quantities of young strain E blades were higher than those of NA after 14 d of cultivation, indicating that young blades of mutant strain E released more archeospores. The mean length and weight of large E blades were both over three times greater than those of NA after 4 weeks of cultivation. The photosynthetic parameters (Fv/Fm, Y[I], Y[II], and O₂ evolution rate) and pigment contents (including phycoerythrin and phycocyanin) of strain E blades were higher than those of NA (P < 0.05). The cellular respiratory rate of strain E blades was lower than that of NA (P < 0.05). In order to investigate the causes of changes in strain E blades, total RNA in strain E and NA blades were sequenced using the Illumina Hiseq platform. Compared with NA, 1,549 unigenes were selected in strain E including 657 up‐regulated and 892 down‐regulated genes. According to the physiology measurement and differentially expressed genes analysis, cell respiration in strain E might decrease, whereas anabolic‐like photosynthesis and protein biosynthesis might increase compared with NA. This means substance accumulation might be greater than decomposition in strain E. This might explain why strain E blades showed improved growth compared with NA. In addition, several genes related to stress resistance were up‐regulated in strain E indicating that strain E might have a higher stress resistance. The sequencing dataset may be conducive to Pyropia yezoensis molecular breeding research.     

Network Relationships of Bacteria in a Stable Mixed Culture

We investigated the network relationships of bacteria in a structurally stable mixed culture degrading cellulose. The mixed culture consists of four bacterial strains (a cellulose-degrading anaerobe [strain S], a saccharide-utilizing anaerobe [strain F], a peptide- and acetate-utilizing aerobe [strain 3] and a peptide-, glucose-, and ethanol-utilizing aerobe [strain 5]). Interspecies interactions were examined by analyzing the effects of culture filtrates on the growth of the other strains and by comprehensively analyzing population dynamics in the mixed-culture systems with all possible combinations of the four bacterial strains. The persistence of strain S depends on the effects of strain 5. However, strain 5 is a disadvantaged strain because strain 3 has bacteriocidal activity on strain 5. The extinction of strain 5 is indirectly prevented by strain F that suppresses the growth of strain 3. Although strain F directly has suppressive effects on the growth of strain S, strain F is essential for the persistence of strain S, considering the indirect effects (maintaining strain 5, which is essential for the survival of strain S, by inhibiting strain 3). These indirect relationships form a bacterial network in which all the relationships including suppressive effects were well balanced to maintain the structural stability. In addition to direct metabolite interactions, such kind of indirect relationships could have a great impact on microbial community structure in the natural environment.     

In situ development and application of cDNA-AFLP to isolate genes of Candida oleophila (strain O) potentially involved in antagonistic properties against Botrytis cinerea

The yeast Candida oleophila (strain O) presents a high level of protective activity against Botrytis cinerea (gray mold) on postharvest apples. The cDNA-AFLP technique allows the comparison of mRNA populations extracted from cells grown in different conditions. In order to isolate yeast genes potentially involved in biological control properties, that technique was applied on strain O cells growing on apple wounds. The biological control properties of 8 C. oleophila strains and strain O were assessed in order to compare the gene expression of a non antagonistic strain against gene expression of strain O. In the absence of a non-antagonistic strain, an other comparison model was designed. It was based on the growth of strain O in different in situ conditions: strain O applied on apple wounds (O), strain O applied on apple wounds in presence of B. cinerea (B) and B. cinerea alone on apple wounds (F). A recovering technique, based on the washing of cells in the wound and a RNA extraction method followed by a DNase treatment were optimised before cDNA-AFLP application. Thirteen primer pairs were used. Their application resulted in an average of 54 and 55 bands for O and B respectively whereas no bands were observed for F. Among these bands, 8 were expressed more intensely in presence of the pathogen (1.1% of the fragments).     

大熊猫犬瘟热病毒附着或血凝蛋白基因的序列分析

首次对犬瘟热病毒（ＣＤＶ）大熊猫（ＧＰ）毒株附着或血凝蛋白（Ｈ）基因进行了序列测定并与疫苗株Ｏｎｄｅｒｓｔｅｐｏｏｒｔ进行了比较。我们设计合成了４对引物,对ＧＰ株进行了ＲＴ－ＰＣＲ扩增与测序。Ｈ蛋白基因全长为１９４６ｂｐ,开放阅读框架（ＯＲＦ）始于２１－２３位的ＡＴＧ,终止于１８４２－１８４４位的ＴＧＡ,编码６０７个氨基酸,该基因序列已被ＧｅｎＢａｎｋ。将ＧＰ毒株与ＧｅｎＢａｎｋ中疫苗弱毒株Ｏｎｄ     

Isolation and characterization of recombinants between attenuated and virulent aphthovirus strains.

A guanidine-resistant mutant of the attenuated strain of aphthovirus type 01 strain Campos and the original wild-type strain were crossed to generate recombinant viruses. Two independently derived recombinant viruses were isolated. One isolate (RI) contained the P1 (structural proteins) gene region of attenuated strain and P3 (polymerase precursor) gene region of the wild-type strain. The other isolate (RII) had a genomic structure complementary to that of RI, this is, P1 of the wild-type strain and P3 of the attenuated virus. Recombinant RII inherited some in vitro phenotypic markers that were characteristic of the attenuated strain, whereas the RI recombinant had in vitro behavior that was similar to that of the wild-type strain. The data obtained suggest that the polymerase precursor (P3) of the attenuated strain (01 Campos) could be involved in the determination of the attenuated phenotype for fetal bovine kidney cells and, eventually, for cattle.     

Effect of light intensity on the oviposition rhythm of the altitudinal strains of Drosophila ananassae

The sensitivity of the circadian photoreceptors mediating entrainment of the eclosion rhythm and phase shifts of oviposition rhythm of the high altitude (HA) strain of Drosophila ananassae originating from Badrinath (5123 m above sea level) in the Himalayas was compared with the low altitude (LA) strain from Firozpur (179 m above sea level). Reduced photic sensitivity of the HA strain is regarded as the result of natural selection, which led to the weakening of the coupling mechanism between the circadian pacemaker and light at the high altitude of origin. The present study was designed to determine whether or not the photic entrainment of the oviposition rhythm of the HA strain of D. ananassae is also altered by the high altitude of its origin, and the results are compared with those of the LA strain. The effects of light intensity on the phase angle difference (Ψ), degree of rhythmicity (R), the percent oviposition in photophase, the threshold light intensity (i.e., the intensity at which stable entrainment occurred), and the saturation light intensity (i.e., the intensity beyond which the values of Ψ or amplitude of rhythm remained unaltered) were determined. Entrainment was studied in light-dark cycles in which the light intensity of 12 h of photophase varied from 1 to 1000 lux, and complete darkness prevailed in all scotophases. The oviposition rhythm of the HA strain was arrhythmic from 1 to 90 lux, weakly rhythmic at 95 lux, but rhythmic at or above 100 lux, while that of the LA strain was weakly rhythmic at 1 lux but rhythmic at or above 2 lux. Oviposition of the HA strain occurred mostly in the photophase, while that of the LA strain occurred in the scotophase; as a result, the oviposition medians of the HA strain were around the subjective forenoons while those of the LA strain were around the subjective evenings. The percent of oviposition in photophase increased from 68 to 98 in the HA strain and from 5 to 33 in the LA strain as light intensity increased from 1 to 1000 lux. In the HA strain, the Ψ values were significantly less and values of R and percent oviposition in photophase were significantly more than those of the LA strain at each level of light intensity. Threshold and saturation intensities for Ψ were 100 and 700 lux, respectively, for the HA strain, but just 2 and 45 lux, respectively, for the LA strain. The saturation intensity for R was 650 and 700 lux for the HA and LA strains, respectively. These results extend the confirmation that the reduced photic sensitivity of the HA strain might have been acquired through natural selection in response to environmental conditions at the high altitude of its origin.     

Studies on the Bacteriophage 2 Receptors of Pseudomonas aeruginosa

The lysogenization of Pseudomonas aeruginosa strain BI with phage 2 resulted in the loss of the capacity to adsorb the same phage. The absence of phage 2 receptors on the surface of the lysogenized strain BI(2)(8) was confirmed by the failure of purified slime polysaccharide (SPB) or lipopolysaccharide (LPS) to inactivate phage 2. SPB and LPS from a phage 2-resistant strain also failed to inactivate phage 2 in contrast to the phage inactivation exhibited by the SPB and LPS obtained from the wild-type strain BI. Chemically, quantitative differences were apparent when the SPB and LPS of strains BI(2)(8) and BI/2S(2) were compared with those of the wild-type strain BI. The most striking difference noted was the absence of amino sugars in the SPB of strain BI/2S(2). The SPB of strain BI(2)(8) also contained a lower percentage of amino sugars compared with the SPB of the wild-type strain BI.     

Cell surface characteristics of two halotolerant strains of Sinorhizobium meliloti

The halotolerant Sinorhizobium meliloti strain Rmd201 and its variant Rmd201 a were examined for their cell surface properties. The variant strain formed rough colonies and was found to be more hydrophobic. Growth of the variant strain was not affected appreciably when NaCl concentration of the medium was increased from 2 mM to 700 mM. Exopolysaccharide (EPS) and the lipopolysaccharide (LPS) content of the variant strain was found to be 7 and 14 times less, respectively, than the parent strain. However, enhanced synthesis of high molecular weight LPS bands were observed in SDS-PAGE analysis in the variant strain when the NaCl concentration was raised from 2 mM to 700 mM. Ribose and glucosamine were present in the variant LPS only. Mannose appeared as a major LPS constituent of the variant when grown in high salt containing medium. All these cell surface characteristics indicated that there were significant differences between the halotolerant strains of S. meliloti. The changes in the cell surface of the variant strain indicated the possible mutation in the gene(s) of cell surface polysaccharide biosynthesis.     

Relative contribution of detoxifying enzymes to pyrethroid resistance in a resistant strain of Helicoverpa armigera

Abstract:  The relative contribution of oxidases and esterases to pyrethroid resistance was studied in a YS-FP strain of Helicoverpa armigera from China. The YS-FP strain was derived from a field-collected strain (YS) by 16 generations of selection with a mixture of fenvalerate and phoxim. Compared with the YS strain, the YS-FP strain showed 1850- to >7140-fold resistance to four ester-bonded phenoxybenzyl alcohol pyrethroids (fenvalerate, deltamethrin, cypermethrin and cyhalothrin), >205-fold resistance to a non-ester phenoxybenzyl alcohol pyrethroid (etofenprox) and only 19-fold resistance to an ester-bonded methylated biphenyl alcohol pyrethroid (bifenthrin). The oxidase inhibitor piperonyl butoxide eliminated most the of resistance to fenvalerate, deltamethrin, cypermethrin, cyhalothrin and etofenprox, whereas the esterase inhibitor S,S,S -tributylphosphorothioate had a small synergistic effect for fenvalerate and cyhalothrin only. This suggests that the resistance to these pyrethroids in the YS-FP strain was mainly because of enhanced oxidative detoxification. The monooxygenase activities of the midguts of sixth-instar larvae of the YS-FP strain to substrates p -nitroanisole, ethoxycoumarin and methoxycoumarin were 3.7-, 4.7- and 10-fold, respectively, compared with that of the YS strain. Glutathione S -transferase activity and esterase activity were not significantly altered in the YS-FP strain. This confirms that enhanced oxidative detoxification was a major mechanism contributing to pyrethroid resistance in the YS-FP strain.     

Differences in the Composition of the Cell Walls of Two Morphologically Different Lines of Suspension-Cultured Catharanthus roseus Cells

Differences in the composition of cell walls of two morphologicallydifferent lines (A and B) of suspension-cultured Catharanthusroseus cells, which have the same origin, were investigated.The cells of strain A are nearly spherical, while those of strainB are cylindrical. In strain A, the amount of cell wall pergram fresh weight of cells increased during the logarithmicphase. In strain B, the amount of cell wall per cell decreasedduring the logarithmic phase. The level of matrix polysaccharides increased markedly duringthe logarithmic phase in strain A. The amount of cellulose incell wall was relatively larger in strain B than in strain A.The following differences in sugar composition between the twostrains were observed: (a) there was an increase in the relativelevels of 4-linked galactose in the EDTA-soluble fraction andof 3-linked glucose in the 5% KOH-soluble fraction during thelogarithmic phase in strain A; (b) there were significantlyhigher levels of arabinose, probably derived from 2,5- and/or3,5-linked arabinan, in the EDTA-soluble fraction and in theextracellular polysaccharides in strain B; (c) there were decreasesin the relative amounts of some kinds of sugar, probably thosederived from xyloglucan, during the stationary phase in strainB. (Received March 31, 1989; Accepted October 12, 1989)