Selection of phage-display peptides that bind specifically to the outer coat protein of Rice black streaked dwarf virus.

Several peptides that could bind specifically to the outer coat protein encoded by the S10 gene of Rice black streaked virus (RBSDV) were isolated from a phage-display random 12-mer peptide library. The sequence analysis showed that the amino acid motif (K)K**(*)P, the asterisk denoting any amino acid, might be the core sequence by which the peptides bind to the target protein. The peptide 1 that had a high affinity to RBSDV outer coat protein was synthesized by a chemical method and its fusion protein with glutathione-S-transferase (GST) was produced in an Escherichia coli expression system. The dot and Western blot analyses indicated that RBSDV could be detected with a high sensitivity in crude extracts of diseased plant leaves using a purified GST fusion protein. The circular dichroism (CD) spectroscopy revealed that the synthesized binding peptide but not a nonbinding peptide could bring about a marked change in the conformation of outer coat RBSDV protein. Since the protein functions only when it has correct conformation, the peptides binding specifically to it could possibly disturb the function of the virus outer coat protein and might be used to block the transmission pathway of the virus. Summing up, as these peptides showed a high specificity and sensitivity and diagnostic potential for RBSDV, they may represent the basis of a novel strategy for development of resistance to RBSDV.

Identification of rice black streaked dwarf virus in different cereal crops with dwarfing symptoms in China.

This report describes isolation of virus particles from plants of rice, maize, wheat and sorghum with symptoms of dwarfing collected from two provinces of China, purification of double-stranded RNA (dsRNA) from the virus particles, and synthesis of full-length cDNAs of genome segments 9 (S9) and 10 (S10) by reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis showed that the S9 sequences of the Chinese isolates and a Japanese rice black-streaked disease virus (RBSDV) isolate were very similar (89.1-89.6% homology at nucleotide level and 92.3-92.9% and 95.8-98.6% homology at amino acid level for ORF1 and ORF2, respectively). Analogical similarity was found also for the S10 sequences of the isolates under comparison: 93.0-95.4% homology at nucleotide level and 96.2-97.0% homology at amino acid level. However, there was a relatively lower similarity for S9 and S10 segments ofthe Chinese isolates and an Italian maize rough dwarf virus (MRDV) isolate. The phylogenetic analysis indicated that the Chinese isolates that infect rice, maize, wheat and sorghum and cause similar symptoms could represent the same virus species, RBSDV.

Selection and expression of peptides which can change the conformation of p20 protein of rice stripe virus.

Phages with high affinity to the P20 protein of rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of phage display screening. Nine different peptides from the enriched library were selected by enzyme-linked immunosorbent assay (ELISA). The P20 protein from raw extracts of rice leaves infected with RSV could be detected by those 9 peptides displayed on the phage, which suggested that a peptide could be an effective tool for diagnosis of RSV in rice and planthopper. Circular dichroism (CD) spectra of P20 fusion proteins with the binding phages and non-binding phages showed that the conformation of P20 protein was changed after binding to each of the 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of the P20 protein. Thereafter, those peptides might be used to develop plant resistance and disrupt virus transmission. Three of the 12-mer peptide genes were fused with the glutathione-S-transferase (GST) gene in the vector pGEX 3X. The fusion proteins were obtained from an Escherichia coli expression system and purified. The fusion proteins might have a potential to develop a plant peptide-based resistance to its pathogens and virus diagnosis. It also provided a tool (i) to confirm the inhibition of the function of P20 protein by the fusion peptides in vivo, and (ii) to detect the function of P20 protein and the interaction between the virus and its vector.

[Evaluating cervical ripening in late pregnancy by transvaginal B-ultrasonic scan].

The ripening of the uterine cervix was studied by using trans vaginal B-ultrasonic scanning against Bishop's scoring in 60 pregnancies in the 3rd trimester, yielding a conformity rate of 70%. All cases with a score > or = 6 by transvaginal B-ultrasound delivered naturally or shortly after induction. There were 22 cases shown immature by Bishop's scoring, of which, 18 found to be mature by B-ultrasonic scan. 5 of the 18 ended naturally and 13 following induction, while the 4 immature according to B-ultrasonic scan failed to come into labor. This proved that vaginal B-ultrasonic scanning is more accurate than Bishop's scoring. B-ultrasonic scanning on the perineum could display the internal cervical os but not the external, unless the body position was changed in different ways. Transabdominal B-ultrasound scanning did not clearly display the cervix and only one case revealed in the cervical canal, and the amniotic membrane. Thus, vaginal B-ultrasonic scanning has a higher rate of visualization and clearer pictures, being able to reflect correctly and objectively the cervical ripening, thereby providing a more reliable method for cervical ripening evaluation.

Microsatellite analysis in post-transplantation lymphoproliferative disorder to determine donor/recipient origin.

Post-transplantation lymphoproliferative disorders (PTLD) are a group of heterogeneous diseases that occur after organ transplantation. Determination of the origin of the tumor cells not only provides clues to its possible pathogenetic mechanism, but also gives prognostic guidance in the clinical management of patients. We reviewed the clinicopathological features of four cases of PTLD that developed after solid organ transplantation. Using microsatellite analysis performed on paraffin-embedded tissue and using multiple, highly polymorphic markers, we have successfully determined the recipient/donor origin of the tumor cells in all of them. The time of onset of the PTLD ranged from 5 to 11 mo. All cases were diffuse large cell lymphomas of B-cell lineage, and the two cases that have been tested for EBV by in situ hybridization were positive. Three of the 4 PTLD were of donor origin and these three patients died of diseases unrelated to PTLD. The single patient with PTLD of recipient origin died of disseminated PTLD. The mean survival length of the three patients with donor origin was 26.3 mo, whereas that of the patient with recipient origin was 12 mo. Our results indicate a relatively high incidence of PTLD of donor origin among our patients with solid organ transplantation, as compared to other reported series. Moreover, the finding of the relatively indolent nature of PTLD of donor origin supports that determination of the donor/recipient origin of PTLD is of prognostic significance.