Monoclonal antibody-based serological assays for detection of Potato virus S in potato plants.

Potato virus S (PVS) often causes significant losses in potato production in potato-growing countries. In this study, the ordinary strain of PVS (PVSO) was purified from PVS-infected potato plants and used as the immunogen to produce hybridomas secreting monoclonal antibodies (MAbs). Five highly specific and sensitive murine MAbs (1A3, 16C10, 18A9, 20B12, and 22H4) against PVS were prepared using conventional hybridoma technology. Using these MAbs, tissue print-enzyme-linked immunosorbent assay (ELISA), dot-ELISA, and double-antibody sandwich (DAS)-ELISA were developed for sensitive and specific detection of PVS infection in potato plants. The results of sensitivity assays revealed that PVS could be reliably detected in PVS-infected leaf crude extracts diluted at 1:10 240 and 1:163 840 (w/v, g/ml) in phosphate buffer saline (PBS) by dot-ELISA and DAS-ELISA, respectively. Twenty-two samples collected from potato fields in Yunnan Province, China were tested for PVS infection using the serological assays we had developed, and 14 of them were found to be positive. This indicates that PVS is now prevalent in potato fields in Yunnan Province.

Low-protein diet supplemented with ketoacids reduces the severity of renal disease in 5/6 nephrectomized rats: a role for KLF15.

Dietary protein restriction is an important treatment for chronic kidney disease. Herein, we tested the effect of low-protein or low-protein plus ketoacids (KA) diet in a remnant kidney model. Rats with a remnant kidney were randomized to receive normal protein diet (22%), low-protein (6%) diet (LPD), or low-protein (5%) plus KA (1%) diet for 6 months. Protein restriction prevented proteinuria, decreased blood urea nitrogen levels, and renal lesions; however, the LPD retarded growth and decreased serum albumin levels. Supplementation with KA corrected these abnormalities and provided superior renal protection compared with protein restriction alone. The levels of Kruppel-like factor-15 (KLF15), a transcription factor shown to reduce cardiac fibrosis, were decreased in remnant kidneys. Protein restriction, which increased KLF15 levels in the normal kidney, partially recovered the levels of KLF15 in remnant kidney. The expression of KLF15 in mesangial cells was repressed by oxidative stress, transforming growth factor-ß, and tumor necrosis factor (TNF)-α. The suppressive effect of TNF-α on KLF15 expression was mediated by TNF receptor-1 and nuclear factor-κB. Overexpression of KLF15 in mesangial and HEK293 cells significantly decreased fibronectin and type IV collagen mRNA levels. Furthermore, KLF15 knockout mice developed glomerulosclerosis following uninephrectomy. Thus, KLF15 may be an antifibrotic factor in the kidney, and its decreased expression may contribute to the progression of kidney disease.

Synergistic effects of tea polyphenols and ascorbic acid on human lung adenocarcinoma SPC-A-1 cells.

Tea polyphenols have been shown to have anticancer activity in many studies. In the present study, we investigated effects of theaflavin-3-3'-digallate (TF(3)), one of the major theaflavin monomers in black tea, in combination with ascorbic acid (AA), a reducing agent, and (-)-epigallocatechin-3-gallate (EGCG), the main polyphenol presented in green tea, in combination with AA on cellular viability and cell cycles of the human lung adenocarcinoma SPC-A-1 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay showed that the 50% inhibition concentrations (IC(50)) of TF(3), EGCG, and AA on SPC-A-1 cells were 4.78, 4.90, and 30.62 micromol/L, respectively. The inhibitory rates of TF(3) combined with AA (TF(3)+AA) and EGCG combined with AA (EGCG+AA) at a molar ratio of 1:6 on SPC-A-1 cells were 54.4% and 45.5%, respectively. Flow cytometry analysis showed that TF(3)+AA and EGCG+AA obviously increased the cell population in the G(0)/G(1) phase of the SPC-A-1 cell cycle from 53.9% to 62.8% and 60.0%, respectively. TF(3)-treated cells exhibited 65.3% of the G(0)/G(1) phase at the concentration of its IC(50). Therefore, TF(3)+AA and EGCG+AA had synergistic inhibition effects on the proliferation of SPC-A-1 cells, and significantly held SPC-A-1 cells in G(0)/G(1) phase. The results suggest that the combination of TF(3) with AA or EGCG with AA enhances their anticancer activity.

A low-protein diet supplemented with ketoacids plays a more protective role against oxidative stress of rat kidney tissue with 5/6 nephrectomy than a low-protein diet alone.

Dietary protein restriction is one major therapy in chronic kidney disease (CKD), and ketoacids have been evaluated in CKD patients during restricted-protein diets. The objective of the present study was to compare the efficacy of a low-protein diet supplemented with ketoacids (LPD+KA) and a low-protein diet alone (LPD) in halting the development of renal lesions in CKD. 5/6 Nephrectomy Sprague-Dawley rats were randomly divided into three groups, and fed with either 22 % protein (normal-protein diet; NPD), 6 % protein (LPD) or 5 % protein plus 1 % ketoacids (LPD+KA) for 24 weeks. Sham-operated rats were used as controls. Each 5/6 nephrectomy group included fifteen rats and the control group included twelve rats. Proteinuria, decreased renal function, glomerular sclerosis and tubulointerstitial fibrosis were found in the remnant kidneys of the NPD group. Protein restriction ameliorated these changes, and the effect was more obvious in the LPD+KA group after 5/6 nephrectomy. Lower body weight and serum albumin levels were found in the LPD group, indicating protein malnutrition. Lipid and protein oxidative products were significantly increased in the LPD group compared with the LPD+KA group. These findings indicate that a LPD supplemented with ketoacids is more effective than a LPD alone in protecting the function of remnant kidneys from progressive injury, which may be mediated by ketoacids ameliorating protein malnutrition and oxidative stress injury in remnant kidney tissue.

[Effects of shexiang baoxin pill on function and nitric oxide secretion of endothelial progenitor cells].

OBJECTIVE: To investigate the effects of Shexiang Baoxin Pill (SBP) on function of endothelial progenitor cells (EPCs) and its nitric oxide (NO) secretion. METHODS: Total mononuclear cells were isolated from human peripheral blood by ficoll density gradient centrifugation and inoculated on the human fibro-ligandin encrusting plate. After 7 days of in vitro culture, adherent cells were collected and incubated with SBP for 24 h. The proliferation, migration, adhesive activity, vasculogenesis capacity and NO secretion of EPCs were assayed using MTT, Transwell chamber, adhesion determination, in vitro vasculogenesis kit and nitrate reductase method, respectively. RESULTS: EPCs incubated with SBP showed the capacities higher than those of control in proliferation, migration, adhesion, in vitro vasculogenesis, and with a higher NO concentration in the culture supernatant. CONCLUSION: SBP can improve the function of EPCs, which might be a novel mechanism of its effects in improving vascular endothelial function and promoting angiogenesis.

Identification of a movement protein of the tenuivirus rice stripe virus.

Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.

Generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus.

To seek a new delivery system of vaccine for infectious bronchitis virus (IBV), transgenic potato expressing full-length spike (S) protein of IBV was produced and its immunogenicity in chickens was investigated. One to three copies of S gene of IBV were randomly and stably inserted into potato (Solanum tuberrosum cv. Dongnong 303) genome by Agrobacterium tumefaciens-mediated transformation. Transcription and translation of S gene for IBV were confirmed by Northern blot and Western blot analyses in transgenic plantlets. Chickens immunized orally and intramuscularly with transgenic potato tubers expressing S protein generated the detectable levels of serum neutralizing antibodies and were protected against the challenge with the virulent IBV. In vitro secretion of interleukin 2 and T lymphocyte proliferation of spleen cells from the immunized chickens varied with the dose and the manner of vaccination with S protein derived from transgenic plants. The results indicated that S protein expressed in transgenic plants might be a new source for the production of Coronaviridae IBV vaccine.

[Transgenic potato containing immunogenic gene of avian coronavirus and its immunogenicity in mice].

To check the feasibility of expression of the immunogenic gene of avian coronavirus infectious bronchitis virus (IBV) in plants, the transformation of S1 gene of IBV into potato and the immunogenicity of its expression product was studied. The S1 gene of IBV-ZJ971 strain was inserted into plasmid pBI121 under the control of 35 S promoter. Agrobacterium fumefaciens EHA105 with the recombinant vector pBI121 was obtained by tri-parental mating method. So, an efficient potato transformation system mediated by Agrobacterium fumefaciens was established. The rates of calli and shoots differentiation were 100%, and more than 95% respectively, for transgenic potato with S1 gene of IBV. PCR and Southern blot analyses showed that IBV S1 gene was integrated into genomic DNA of the potato plant and most transgenic plants had two copies of S1 gene of IBV. In our experiments, 47 transgenic plantlets have been obtained. Northern blot and ELISA analyses indicated that most transgenic plants could normally transcribe and translate S1 gene of IBV, though the levels of transcription and translation were different in various transgenic plants. Immunity assay with BALB/C mice showed that expression products of transgenic potato with S1 gene of IBV were immunogenic, and ELISA antibody titer reached 1:20 to 1:40 and 1:80 to 1:160 with doses of 0.5 g and 1 g, respectively. Virus neutralization (VN) antibodies were detected by tracheal organ cultures, and the results showed that VN titers reached respectively 1:160 to 1:320 and 1:320 to 1:2048 with doses of 0.5 g and 1 g.

Expression of immunogenic S1 glycoprotein of infectious bronchitis virus in transgenic potatoes.

The expression of infectious bronchitis virus (IBV) S1 glycoprotein in potatoes and its immunogenicity in mice and chickens were investigated. Potato plants were genetically transformed with a cDNA construct encoding the IBV S1 glycoprotein with the Agrobacterium system. Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the foreign cDNA into the potato genome, as well as its transcription. Mice and chickens vaccinated with the expressed IBV S1 glycoprotein produced antibodies that neutralized IBV infectivity. After three immunizations, vaccinated chickens were completely protected from virulent IBV infection. These results demonstrate that transgenic potatoes expressing IBV S1 glycoprotein can be used as a source of recombinant antigen for vaccine production.