Identification and analysis of three virulence-associated TonB-dependent outer membrane receptors of Pseudomonas fluorescens.

Pseudomonas fluorescens is a Gram-negative bacterium that can infect a wide range of farmed fish. However, very little is known about the virulence mechanism of P. fluorescens as a fish pathogen. In this study, we identified and analyzed 3 TonB-dependent outer membrane receptors (TDRs) from a pathogenic P. fluorescens strain isolated from fish. In silico analysis revealed that all 3 proteins (named Tdr1 to 3) possess structural domains typical of TDRs. Quantitative real time RT-PCR analysis showed that tdr1, tdr2, and tdr3 expressions were upregulated under iron-depleted conditions. Compared to the wild type, mutants defective in tdr1, tdr2, and tdr3 were retarded in growth to different extents. Infection in a turbot Scophthalmus maximus model showed that all 3 mutants were impaired in their ability to desseminate into and colonize host tissues. In addition, the tdr1 and tdr3 mutants exhibited significantly reduced virulence. When used as subunit vaccines, purified recombinant proteins of Tdr1, Tdr2, and, in particular, Tdr3 elicited significant protection in turbot against lethal P. fluorescens challenge. The vaccinated fish produced specific serum antibodies, which, when incubated with P. fluorescens, blocked infection of P. fluorescens in fish cells. Together these results indicate that Tdr1, Tdr2, and Tdr3 are iron-regulated factors that participate in bacterial virulence and induce protective immunity as subunit vaccines.

[Construction of a new oncolytic virus oHSV2hGM-CSF and its anti-tumor effects].

OBJECTIVE: The aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF. METHODS: oHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded. RESULTS: Both oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P < 0.01). The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree. CONCLUSION: The findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vive to the transplanted B16R tumor models.

[Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP (HSV(GFP))].

OBJECTIVE: To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)). METHODS: Tumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. RESULTS: HSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol. CONCLUSION: Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

[Generation of a herpes simplex virus-permissive mouse melanoma cell line B16RHSV].

OBJECTIVE: To generate an oncolytic herpes simplex virus (oHSV) permissive mouse melanoma cell line B16RHSV, preserving the tumorigenic ability in syngeneic mice. METHODS: The herpes simplex virus entry mediator (HVEM) gene was amplified by PCR from human melanoma cell line A375, and cloned into pGEM-T Easy vector for sequencing. The HVEM gene was then cloned into pcDNA3 vector to generate pcDNA3-HVEM for transfection of mouse melanoma cell line B16-F10 cells. After that, the putative transfected cells were selected in full growth medium containing G418. The HVEM-expressing cells were isolated by immunomagnetic bead separation. The mouse melanoma cell line expressing oHSV receptor-HVEM, designated as B16RHSV, was generated. The permissibility of B16RHSV cells to oHSV infection was examined with green fluorescence protein (GFP)-expressing oHSV (oHSVGFP). To investigate the tumorigenic ability of both cells in vivo, 2×10(5) cells in 100 µl were subcutaneously inoculated into the right flanks of C57/BL mice. RESULTS: In vitro, the B16RHSV mouse melanoma cells were shown by fluorescence microscopy capable of being infected by oHSVGFP. In vivo, the B16RHSV cells, like their wild type counterpart, grew to form melanoma in syngeneic mice. CONCLUSION: A herpes simplex virus-permissive mouse melanoma cell line was established. Its tumorigenicity remained unchanged.

Taxonomic notes on Cyperaceae of Nepal: new records of a genus, six species and other noteworthy species.

This paper reports on the presence of one generic and six specific new records of Cyperaceous species for the flora of Nepal. Amongst the new discoveries are the genus Machaerina, alongside species: Eleocharisochrostachys, Fimbristylisacuminata, F.ferruginea, F.nutans, F.thomsonii and Scleriarugosa. The taxonomy and distribution of Actinoscirpusgrossus, Fimbristylissalbundia and Fuirenaumbellata in Nepal are clarified through notes on nomenclature, description, distribution, specimen examination, identification keys and photographs.

[Zeta chain level of T and NK cells in patients with renal-cell carcinoma and bladder cancer and its clinical significance].

OBJECTIVE: To study the relationship between zeta chain level of peripheral blood T/NK cells and tumor progression in renal-cell carcinoma and bladder cancer and its clinical significance. METHODS: The peripheral blood mononuclcear cells in 58 patients with renal cell carcinoma, 22 patients with bladder cancer and 14 healthy blood donors were examined by flow cytometry with fluorescent anti-CD3 (for T cells), or anti-CD56 (for NK cells) and anti-zeta chain monoclonal antibodies. RESULTS: The zeta chain expression of T cell and NK cell in stage I, II and III renal-cell carcinoma decreased to 59.5%, 37.6%, 21.3% and 62.2%, 27.1%, 18.8% of the healthy control level, respectively. That in stage I, II and III bladder cancer decreased to 37.6%, 29.5%, 18.9% and 35.4%, 20.8%, 5.8% of the control level, respectively. T and NK cell zeta chain levels in 17.5% (14/80) of the patients were within the normal range. T/NK ratios of peripheral blood in stage III patients were remarkably lower than those of the healthy donor. CONCLUSION: Reduced T and NK zeta chain levels and T/NK ratio in the renal-cell carcinoma and bladder cancer are generally consistent with tumor progression. The patients with normal T and NK zeta chain level may be indicated for immunotherapy.

[Antitumor effect of gene gun-mediated DNA vaccine pWRG-neu immunization in C57BL/6 mice].

OBJECTIVE: To investigate the inhibitory effect of DNA vaccine immunization on neu-overexpressed melanoma growth in prophylactic treatment and anti-lung-metastasis experiments in C57BL/6 mice. METHODS: pcDNA-neu transfected into B16F10 with transfection reagent Fugene 6, neu-overexpressed cell clone B16F10-neu was selected with limited dilution method. The growth curve was drawn to analyse its proliferating character in vitro. With Helios gene gun system, DNA vaccine pWRG-neu was immunized to 8-week-old C57BL/6 mice in the shaved abdominal skin for 3 times at two-weekly interval. After immunization, the life span was analyzed. Using MTT assay, the cytolysis activity of the DNA immunized mice spleen cells was compared. RESULTS: One clone of neu-overexpressed B16F10-neu was selected and its proliferating character was the same as B16F10 and B16F10-pcDNA. In prophylactic, treatment and anti-lung-metastasis experiments, gene gun-mediated pWRG-neu immunization could exhibit antitumor effects. The growth and metastasis of neu-overexpressed melanoma was reduced dramatically. The spleen cells of the immuned mice showed cytotoxic T lymphocyte (CTL) activity. CONCLUSION: Gene gun-mediated gene transfer is effective and practicable. DNA vaccine pWRG-neu is potent in preventing subsequent tumor cells challenge, inhibiting the tumor growth and metastasis.

Identification of a lens-specific cis-acting element within the basal promoter of the human lens intrinsic membrane protein MP19 gene (LIM2).

Using overlapping and mutant oligonucleotides as probes, gel mobility assays and competition experiments identified a sequence from -47 to -32 bp upstream of the LIM2 CAP site, which a lens protein complex bound with high affinity which appeared to bind only to the "sense" strand of the double-stranded DNA molecule. This sequence consisted of a string of four guanine residues followed by seven other nucleotides (AACCTAA) and followed by another four guanines, i.e. GGGGAACCTAAGGGG, called the Hsu element. Promoter-CAT constructs containing this sequence or mutations of the sequence indicated that the Hsu element is located within the basal promoter, and is essential for expression of the LIM2 gene. The trans factors binding to the Hsu element are present throughout development, and appear to be lens-specific. Since the LIM2 gene promoter does not contain a classic TATA box, the Hsu element may serve as the site for binding the RNA polymerase complex.

[Construction of eukaryotic expression vector of murine SLC gene and characterization of its chemotactic function].

AIM: To construct the eukaryotic expression vector of murine SLC gene and study the chemotactic function of murine SLC in-vitro and in-vivo. METHODS: Murine SLC gene was cloned by RT-PCR from the thymus tissue of a C57BL/6 mouse. Eukaryotic expression vector of SLC gene-pcDNA3.1 mSLC was constructed and transfected into B16F10 cells by gene gun. Culture supernatant was collected 48 hours after the transfection and chemotactic function of expression product to lymphocytes was detected by a chemotaxis chamber. SLC expression was detected by RT-PCR. Lymphocytic infiltration was observed in SLC gene transfected murine abdominal skin. RESULTS: The gene cloned from the C57BL/6 mouse thymus tissue was SLC gene Scya21b. Transfected cells expressed SLC mRNA, and culture supernatant of those cells had a potent chemotactic function to lymphocytes. Histological examination of transfected skin showed obvious lymphocytic infiltration. CONCLUSION: The SLC gene was cloned from the mouse thymus tissue and could be expressed in B16F10 cells. The expression product in-vitro and in-vivo had a remarkable chemotactic function to lymphocytes.