Preparation and properties of the specific anti-influenza virus transfer factor.

Specific anti-influenza virus and normal transfer factors prepared in an experimental animal model, the pig, have been tested for their components, characteristics, and activity of known specificity. Two transfer factors are small molecular mixture which consist entirely or partly of polypeptides and polynucleosides. Moreover, the biological activity of transfer factors could be approved by Rosettes test and specific skin test. The study would lay a foundation for the research and development of other specific transfer factor.

Effect of oral Lactococcus lactis containing endostatin on 1, 2-dimethylhydrazine-induced colon tumor in rats.

AIM: To investigate the effects of oral Lactococcus lactis (L lactis) containing endostatin on 1, 2-dimethylhydrazine (DMH)-induced rat colorectal cancer. METHODS: Recombinant endostatin was produced by the expression of L lactis NZ9000. Sixty male Wistar rats were injected with DMH (40 mg/kg body weight) subcutaneously once a week for 10 wk to induce colorectal cancer. The rats were gavaged with 1 mL of endostatin at a dose of 1 x 10(8)/d and fed with the basal diet. The animals were killed after 22 wk for histopathological examination. The total time of experimental observation was 58 wk. RESULTS: Rat endostatin protein was expressed in L lactis. Recombinant endostatin exhibited a significant effect on colorectal cancer (P<0.05). Furthermore, the mean survival time of the rats treated with endostatin was longer than that of the animals treated with DMH. There was no statistically significant difference between the rats treated with endostatin and those treated with DMH. The results showed that endostatin could not result in complete cure.

Lack of inhibitory effects of lactic acid bacteria on 1,2-dimethylhydrazine-induced colon tumors in rats.

AIM: A myriad of healthful effects has been attributed to the probiotic lactic acid bacteria, perhaps the most controversial issue remains that of anticancer activity. This study was aimed at investigating the putative anti-cancer effects of lactic acid bacteria strains on the progression of colon tumor in 1,2-dimethylhydrazine (DMH)-treated animals. METHODS: The strain of lactic acid bacteria used in this study was lactic acid bacteria NZ9000 that conformed to the characteristics of plasmid free. Sixty male Wistar rats were given subcutaneous injections of DMH at a dose of 40 mg/kg body wt or saline once a week for 10 weeks. The rats were divided into 6 experimental groups. After the last DMH injection, animals in groups 1 and 4 were gavaged with 1 ml of lactic acid bacteria at a dose of 5 X 10(9) per day or vehicle until sacrifice at the end of week 22 or week 52. Animals in groups 1-3 were killed at the end of week 22 for histopathological examination. The whole period of experimental observation was 52 weeks. RESULTS: By the end of 22nd week, final average body weights of the rats treated with DMH alone and all animals receiving lactic acid bacteria were significantly decreased compared with the vehicle control (P<0.05). No differences in tumor incidence, multiplicity, dimensions and stage in the colonic mucosa were observed among the groups. At week 52, the survival rate of the rats administered lactic acid bacteria was lower than that of the rats treated with DMH that were fed on control fluids of non-lactococcus lactis. The mean survival time of lactic acid bacteria-treated animals was 39 weeks. CONCLUSION: These results indicate that lactic acid bacteria lacks inhibitory effects on the progression of colon tumor in DMH-treated animals, and does not support the hypothesis that alteration of colonic flora may exert an influence on the progression of colon tumor.

Characterization of a chitin-binding protein GP37 of Spodoptera litura multicapsid nucleopolyhedrovirus.

The GP37 amino acid sequence of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) was compared with other baculovirus GP37, entomopoxvirus fusolin, the enhancing factor of Pseudaletia separata entomopoxvirus, and Alteromonas sp. chitin-binding protein 1. In these proteins, five 'conserved regions' previously reported constitute a chitin-binding domain. SpltMNPV GP37 effectively bound to purified crab shell chitin and the dissociation constant (Kd) for binding was 0.28 microM. Immunofluorescence analysis indicated that SpltMNPV GP37 was located in both cytoplasm and nucleus. Immunoblot analysis revealed that this protein was present in the envelopes of both occlusion body-derived virus and budded virus. Further analysis suggested that GP37 may bind to the chitin component of the peritrophic membrane of S. litura larvae.

[Cloning and expression of a novel ubiquitin fusion gene Uba256].

Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene is the only Ub-gp37 fusion gene in the genome of insect viruses. With the specific primers designed for Uba256 gene that was reported recently, the coding regions of Uba256, N-terminal ubiquitin and C-terminal GP37 that lacking the signal sequence, were amplified from SpltMNPV genomic DNA by PCR. The Uba256 coding region was expressed using the expression vector pBV220, and a band of 38 kD was detected with Western blot analysis, indicating that ubiquitin-GP37 fusion protein did not undergo post-transcriptional processing in E. coli. The ubiquitin and GP37 coding region were highly expressed, respectively, using pQE30 expression vector. Antibody to the purified ubiquitin reacted not only to the recombinant ubiquitin but also to bovine ubiquitin, and an antibody to the purified GP37 also reacted to the recombinant GP37. The results indicated that the purified ubiquitin and GP37 retained their antigenicity. Western blot analysis results of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed in this insect cell line to yield free ubiquitin and GP37 protein.

Characterization of a novel ubiquitin-fusion gene Uba256 from Spodoptera litura nucleopolyhedrovirus.

The complete nucleotide sequence of Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene, encoding ubiquitin fused to GP37 protein of 256 amino acids was determined. The first 76 amino acids of the SpltMNPV ubiquitin showed 78-88, 77 and 81-84% amino acid sequence identity to baculovirus, Melanoplus sanguinipes entomopoxvirus and eukaryotes ubiquitins, respectively. The deduced amino acid sequence of SpltMNPV GP37 protein was similar to other baculovirus GP37 proteins and to entomopoxvirus fusolin proteins. The GP37 protein also showed a distant similarity to Pseudaletia separata entomopoxvirus enhancing factor, bacterial chitinase B and chitin-binding protein 1, but the significance of this is unclear. The mRNA start site of Uba256 fusion gene was mapped within a consensus baculovirus late promoter sequence (ATAAG), commonly found for baculovirus late genes. Uba256 transcripts were present from 48 h p.i. and remained detectable until 72 h p.i. Western blot analysis of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed to free ubiquitin and GP37 protein. Whereas expression Uba256 gene in Escherichia coli did not result in processing of the fusion protein. Tunicamycin treatment of SpltMNPV-infected cells confirmed that SpltMNPV GP37 protein is N-glycosylated. These findings provide additional information on the evolution of ubi genes and insight into genomic variation in baculoviruses.

Cloning and Sequence Analysis of the Chitinase Gene of Spodoptera litura Nuclear Polyhedrosis Virus.

Using AcMNPV chiA-containing fragment as a probe, the chitinase gene of Spodoptera litura nuclear polyhedrosis virus (SpltNPV) was localized in two contiguous fragments, XbaI 5.1 kb and 2.1 kb, and the intact SpltNPV chiA gene was obtained by cloning and sequencing those two fragments. The complete open reading frame of the gene was 1 695 nucleotide long, encoding a putative protein of 564 amino acids with molecular weight of 62.9 kD. Sequence analysis further revealed that the 5' noncoding region had a baculovirus late promoter motif TAAG. A polyadenylation signal, AATAAA, was located dowmnstream of the translation stop codon. A putative signal peptide was present at the N-terminus of the protein. This gene shares 57% homology with AcMNPV chitinase gene.