Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line

The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. The evaluation of gene copy numbers by a qPCR method, is one of the most common approaches used to assess the consistency of transgenes in a constructed cell line. The cell line AV529-19 is a Vero-based cell line specifically engineered to express the HSV-1 UL5 and UL29 open reading frames. AV529-19 is used to support the replication of a defective HSV-2 viral candidate vaccine called HSV529. To assess the genetic stability of the UL5 and UL29 transgenes in AV529-19 cells, a digital PCR-based approach was developed. During characterization of the test method, the specificity, accuracy, and intermediate precision of the assay was investigated based on regulatory guidelines. The developed assay was used to monitor the stability of the transgenes in the manufactured AV529-19 cell lines by comparison of transgene copy numbers in the master cell bank (MCB) with their copy numbers in the extended cell bank (ECB). Results showed that the UL29 and UL5 transgenes are stable in that there are one and three copies of the UL29 and UL5 genes, respectively, per cell in both the AV529-19 MCB and ECB.     

Effect of streptozotocin diabetes on some urea cycle enzymes

Streptozotocin induced diabetes in rats increased the activities of the three mitochondrial enzymes, carbamylphosphate synthetase, ornithine transcarbamylase and N-acetylglutamate synthetase, but not of the cytosolic N-acetylglutamate deacylase. Levels of both N-acetylglutamate and arginine, which are activators of carbamylphosphate synthetase and N-acetylglutamate synthetase respectively, increased in diabetes. These results serve to explain the increase both of mitochondrial citrulline and urea formation in hepatocytes and the increased urea excretion in diabetes.     

Chemical composition and rumen degradability of three corn hybrids treated with insecticides against the European corn borer (Ostrinia nubilalis)

This experiment determined the chemical composition, rumen degradability (aNDF in stalks and starch in kernels) and in vitro gas production of kernels from three corn hybrids treated (TT) or not treated (control, CTR) with insecticides against the European corn borer (ECB, Ostrinia nubilalis). Two whole-plant silage hybrids belonging to the FAO rating 600 and 700 maturity class (S600 and S700, respectively) and one selected for grain production (G600, FAO rating 600, Dekalb-Monsanto Agricoltura S.p.A., Lodi, Italy) were sown in two main plots (TT and CTR) of an experimental field. Two subsequent treatments of pyrethroids (25 and 1.2 g/ha of cyfluthrin and deltamethrin, respectively) were applied to the TT plots. The insecticide treatment reduced the number of damaged plants (4.5 broken plants/plot versus 0.3 broken plants/plot, P<0.01) and increased the total grain yield by 11% (13.8 t/ha versus 12.4 t/ha), while hybrids did not differ. ECB larvae which bored into the stalk tunnels modified the chemical composition of stalks and kernels. In stalks, total sugars content (i.e. glucose, fructose, sucrose) was about twice that in TT versus CTR plants (123 g/kg versus 60 g/kg DM, P<0.01), while aNDF content was higher in CTR stalks (765 versus 702 g/kg DM, P<0.01). DM degradability after 48 h of incubation of stalks was higher in TT than in CTR, both in vitro (0.360 versus 0.298, P<0.01) and in situ (0.370 versus 0.298, P<0.05), while there were no differences in aNDF degradability. Kernels from TT plots contained less DM (615 g/kg versus 651 g/kg, P<0.01) and more CP (84 g/kg and 78 g/kg DM, P<0.05) than those from CTR plots, while in situ rumen starch disappearance and in vitro gas production were similar. Corn hybrid selected for yield of grain (G600) differed from S600 and S700 due to a higher (P<0.01) content of aNDF, ADF and lignin(sa) in the stalks, and a higher starch content (696 g/kg versus 674 and 671 g/kg DM, P<0.01) and CP (87 g/kg versus 77 and 76 g/kg DM, P<0.05) in grain. The G600 hybrid produced stalks with a lower (P<0.01) aNDF rumen degradability than the S600 and S700.     

Far from Inert: Membrane Lipids Possess Intrinsic Reactivity That Has Consequences for Cell Biology

In this article, it is hypothesized that a fundamental chemical reactivity exists between some non-lipid constituents of cellular membranes and ester-based lipids, the significance of which is not generally recognized. Many peptides and smaller organic molecules have now been shown to undergo lipidation reactions in model membranes in circumstances where direct reaction with the lipid is the only viable route for acyl transfer. Crucially, drugs like propranolol are lipidated in vivo with product profiles that are comparable to those produced in vitro. Some compounds have also been found to promote lipid hydrolysis. Drugs with high lytic activity in vivo tend to have higher toxicity in vitro. Deacylases and lipases are proposed as key enzymes that protect cells against the effects of intrinsic lipidation. The toxic effects of intrinsic lipidation are hypothesized to include a route by which nucleation can occur during the formation of amyloid fibrils.     

Isolation and characterization of two forms of Met-tRNAf deacylase from rabbit reticulocyte ribosomes

We have separated and purified two forms of Met-tRNA_f deacylase (or two separate enzymes), an activity that mediates in part the suppression of polypeptide chain initiation that occurs in heme deficiency or with double-stranded RNA, 1000-fold from the 0.5 M KCl wash of rabbit reticulocyte ribosomes. Deacylase I is a minor activity with an S_20,w of 5.9, D_20,w of 4.9 and M_r of 110 000, while deacylase II is the major activity with an S_20,w of 3.3, D_20,w of 7.1 and M_r of 43 000. Both convert crude reticulocyte or pure yeast, wheat germ, and E. coli [³⁵S]Met-tRNA_f to [³⁵S]methionine and tRNA^Met_f and have no effect on reticulocyte [³⁵S]fMet-tRNA_f, [³H]Ala-tRNA or [³H]Lys-tRNA. However, while deacylase I has similar activity throughout the pH range of 6.1–8.1, deacylase II has a sharp pH optimum at 7.9 and is almost completely inactive at 6.1. In addition, deacylase II shows a much greater affinity for pure Met-tRNA_f than deacylase I (K_m of 1.5–3 nM vs. 100 nM), and, while deacylase II is selectively inhibited by tRNA^Met_f, deacylase I is inhibited similarly by any added tRNA.     

A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate

Tyrosyl-tRNA synthetase catalyzes the attachment of tyrosine to the 3′ end of tRNA^Tyr, releasing AMP, pyrophosphate, and l-tyrosyl-tRNA as products. Because this enzyme plays a central role in protein synthesis, it has garnered attention as a potential target for the development of novel antimicrobial agents. Although high-throughput assays that monitor tyrosyl-tRNA synthetase activity have been described, these assays generally use stoichiometric amounts of tRNA, limiting their sensitivity and increasing their cost. Here, we describe an alternate approach in which the Tyr-tRNA product is cleaved, regenerating the free tRNA substrate. We show that cyclodityrosine synthase from Mycobacterium tuberculosis can be used to cleave the l-Tyr-tRNA product, regenerating the tRNA^Tyr substrate. Because tyrosyl-tRNA synthetase can use both l- and d-tyrosine as substrates, we replaced the cyclodityrosine synthase in the assay with d-tyrosyl-tRNA deacylase, which cleaves d-Tyr-tRNA. This substitution allowed us to use the tyrosyl-tRNA synthetase assay to monitor the aminoacylation of tRNA^Tyr by d-tyrosine. Furthermore, by making Tyr-tRNA cleavage the rate-limiting step, we are able to use the assay to monitor the activities of cyclodityrosine synthetase and d-tyrosyl-tRNA deacylase. Specific methods to extend the tyrosyl-tRNA synthetase assay to monitor both the aminoacylation and post-transfer editing activities in other aminoacyl-tRNA synthetases are discussed.     

Evolving enzyme technology for pharmaceutical applications: case studies

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent. Received 07 March 1997/ Accepted in revised form 15 June 1997     

DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex

The highly purified DNA Pol- from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488–1492]. Binding of ConA and RCA to human recombinant DNA polymerase- showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231–6237].The catalytic polypeptide, DNA polymerase- of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase- from embryonic chicken brain (ECB) contains an -galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase- reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567–573] by the treatment with methyl--galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (M _r = 186 kDa) with an -galactose binding, 56 kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA.Pol- as determined by immunostaining with the polymerase--specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA- and a complete separation of polymerase complex and primase.     

酰胺水解酶基因工程菌的构建及其对棘白菌素B的转化

棘白菌素B0(ECB)去侧链母核为重要抗真菌药物阿尼芬净的半合成前体.本实验室从保存的犹他游动放线菌(Actinoplanes utahensis)SIPI-A.2001 基因组中克隆到ECB酰胺水解酶及其上下游基因,并将其构建入表达质粒pTGV2,通过接合转移的方法将此表达质粒导入到变铅青链霉菌(Streptomyc...     

In vivo effect of mixtures of allelochemicals on the life cycle of the European corn borer, Ostrinia nubilalis

The phenylpropanoid, dillapiol, a compound capable of inhibiting the activity of cytochrome P-450 epoxidase in microsomal preparations of midguts of the European corn borer (ECB), Ostrinia nubilalis Hubner (Lepidoptera), did not enhance the toxicity of two insecticidal allelochemicals from the Asteraceae: the sesquiterpene lactone, tenulin or the phenylacetylene, phenylheptatriyne, when ingested simultaneously. Dillapiol alone was toxic to ECB larvae, and inhibited their growth. Possible reasons for the lack of synergism or potentiation by dillapiol of the insecticidal activity of the other allelochemicals are considered.

---

Résumé Le dillapiole, un phénylpropanoide monomère d'un lignan et contenant un groupement méthylènedioxyhphényl (MDP), a la capacité d'inhiber in vitro les enzymes d'oxydations multiples à cytochrome P-450 (PSMO) utilisées par la pyrale du maïs (ECB), Ostrinia nubilalis, pour detoxifier les substances allélochimiques. Les composés contenant un groupe MDP comptent parmi les plus nombreux et puissants synergistes d'insecticides. Cependant, aucun synergisme n'est observé lorsque le dillapiole est ingéré par la pyrale en association avec l'une ou l'autre ou les deux substances secondaires insecticides des Astéraceae, la lactone sesquiterpène ténuline ou/et le polyacétylène phénylheptatriyne (PHT). Plutôt, le dillapiole est une toxine qui diminue la prise de poids des larves et retarde leur développement. Les effets toxiques sont plus importants avec le dillapiole seul que lorsque celui-ci est associé à la ténuline ou/et au PHT. Les expications possibles de ces résultats sont discutées.