Production of helper-dependent adenovirus vector relies on helper virus structure and complementing

Background

The helper‐dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD‐Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD‐Ad vector is generally lower than that of an E1‐deleted first‐generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD‐Ad vector.

Methods

To study this question and improve HD‐Ad vector production, we have generated four different helper viruses with a wild‐type or deleted E3 region, and with a relocated loxP. We have also constructed a first‐generation vector with a wild‐type E3 region and without the loxP site. We compared the replication of these viruses in Cre‐positive and ‐negative cells and studied their complementing for HD‐Ad vector production.

Results

Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD‐Ad vector was obtained when a helper virus with wild‐type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability.

Conclusions

Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD‐Ad vector can be further improved by modifications in helper virus structure. Copyright © 2002 John Wiley & Sons, Ltd.

     

Malonate metabolism: biochemistry,molecular biology,physiology, and industrial application

Malonate is a three-carbon dicarboxylic acid. It is well known as a competitive inhibitor of succinate dehydrogenase. It occurs naturally in biological systems, such as legumes and developing rat brains, which indicates that it may play an important role in symbiotic nitrogen metabolism and brain development. Recently, enzymes that are related to malonate metabolism were discovered and characterized. The genes that encode the enzymes were isolated, and the regulation of their expression was also studied. The mutant bacteria, in which the malonate-metabolizing gene was deleted, lost its primary function, symbiosis, between Rhizobium leguminosarium bv trifolii and clover. This suggests that malonate metabolism is essential in symbiotic nitrogen metabolism, at least in clover nodules. In addition to these, the genes matB and matC have been successfully used for generation of the industrial strain of Streptomyces for the production of antibiotics.     

Discrete forms of amylose are synthesized by isoforms of GBSSI in pea

Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.     

Hormone trajectories leading to human birth

The mechanisms regulating human parturition and labor remain unknown. This ignorance is expensive as preterm birth is responsible for 70% of neonatal mortality and 50% of cerebral palsy. Methods for the prediction of preterm birth and treatments for women in preterm labor have poor efficacy reflecting our limited knowledge of the mechanisms involved. Recent research has supported the view that parturition is a cascade of events that commences early in pregnancy and involves the mother, fetus, placenta, membranes, cervix and myometrium. Although a number of the key hormones and proteins involved have been identified, the relationships between these factors in time and tissues remain unclear. Placental production of Corticotropin-releasing hormone (CRH) is proposed as an early event regulating the cascade of events. Central to the onset of parturition will be a mechanism for progesterone withdrawal and estrogen activation in human. Two forms of progesterone receptor with opposing actions exist in the human myometrium. Progesterone receptor A (PR-A) is a dominant negative repressor of progesterone receptor B (PR-B). Preliminary studies strongly support the hypothesis that the onset of human parturition is initiated by rising concentrations of PR-A in the myometrium.     

Importance of ambient saturation deficits in an epizootic of the fungus Neozygites floridana in cassava green mites (Mononychellus tanajoa)

The mite-pathogenic fungus Neozygites floridana Fisher (Entomophthorales: Neozygitaceae) is considered to have potential for the biological control of the cassava green mite, Mononychellus tanajoa (Bondar). However, its activity is sporadic and laboratory data suggest a strong dependence on night-time saturation deficits for transmission. We report on an epizootic of this fungus in a mite population in northeastern Brazil. During the epizootic, host populations appeared to be limited by a combination of the pathogen and a predatory mite Neoseiulus idaeus (Acari: Phytoseiidae). When temperatures increased, the epizootic finished and the host population began to grow. Abiotic conditions could not explain the variation in host mortality following pickup of infective propagules in this epizootic. However, night-time saturation did help to explain the variation in transmission from infective cadavers to newly killed hosts. This supports laboratory observations that horizontal transmission between hosts is determined mainly by saturation deficits, while the process of infection is little affected by abiotic conditions. A further field observation was the near-absence of resting spores in dead mites (ca. 0.1% of cadavers), suggesting that the pathogen population was unsuccessful in producing inoculum to infect future M. tanajoa populations. The implications are that this pathogen will only be effective as a biological control agent in periods of high relative humidity, and establishment in new areas may be limited by resting spore formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03

The Bacillus subtilis strain KS03 was isolated, and identified as a biological control agent that inhibits the anthracnose disease fungus Gloeosporium gloeosporioides. The antifungal compound was purified from its culture broth through butanol extraction, diethylaminoethyl (DEAE) Sepharose CL-6B chromatography, and preparative thin layer chromatography. Tandem mass spectrometric analyses (MS/MS), with matrix-assisted laser desorption ionization (MALDI) time-of-fight/time-of-flight (TOF/TOF) mass spectrometry, showed that the antifungal compound was iturin A, a cyclic lipopeptide antibiotic. The major compound, with a molecular mass of 1042 Da, was identified as iturin A(2).     

Stilbenes from the roots of Pleuropterus ciliinervis and their antioxidant activities

Two stilbene glycosides, pieceid-2"-O-gallate and pieceid-2"-O-coumarate, were isolated from the MeOH extract of the roots of Pleuropterus ciliinervis Nakai (Polygonaceae), together with two known compounds, resveratrol and pieceid. Their structures were determined spectroscopically, particularly by 2D NMR spectroscopic analysis. The antioxidant activities of stilbenes isolated were determined in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, superoxide radicals and by determining their lipid peroxidation inhibitory activities. Among the compounds isolated, pieceid-2"-O-gallate had the most potent inhibitory scavenging effect on DPPH, superoxide radicals and upon lipid peroxidation inhibition with IC50 values of 16.5, 23.9 and 5.1 microM, respectively.