Antitumor activity of recombinant antimicrobial peptide penaeidin-2 against kidney cancer cells.

Penaeidin-2 (Pen-2) is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides (AMPs) exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2 (rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 µg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers.

[Invasive ductal carcinomas of breast showing partial reversed cell polarity are associated with lymphatic tumor spread].

OBJECTIVE: To investigate the relationship between partial reversed cell polarity (PRCP) and lymphatic tumor spread in invasive ductal carcinoma (IDC), not othervise specified (NOS). METHODS: Immunohistochemistry (EnVision method) was used to examine the expression of epithelial membrane antigen (EMA) and the reversed cell polarity in 199 cases of IDC. RESULTS: Of the 199 cases, including five cases with micropapillary differentiation,30 cases with PRCP and 164 cases of IDC-NOS (without micropapillary differentiation and/or PRCP), lymphovascular invasion was seen in four (4/5), 13(43.3%) and 30 cases (18.3%) respectively; nodal metastasis was seen in four (4/5), 19 (63.3%) and 56 cases (34.1%) respectively. The rates of lymphovascular invasion and nodal metastasis were significantly higher in IDC with PRCP or IMPC than IDC-NOS (P = 0.00); there was however no significant difference between IDC with PRCP and IMPC for lymphovascular invasion and nodal metastasis (P = 0.18, P = 0.64). CONCLUSIONS: IDC with PRCP, similar to IMPC, is more likely to show lymphovascular invasion and nodal metastasis. Complete or partial reversal of cell polarity may play a significant role in lymphatic tumor spread.

TAT-mediated oral subunit vaccine against white spot syndrome virus in crayfish.

White spot syndrome virus is a highly pathogenic virus that infects crayfish and other crustaceans. VP28 is one of its major envelope proteins, and plays a crucial role in viral infection. Cell-penetrating peptides are short peptides that facilitate cellular uptake of various molecular cargoes, and one well known example is TAT peptide from HIV-1 TAT protein. In this study, recombinant plasmids were constructed and transformed into Escherichia coli strain BL21 (DE3) to express TAT-VP28, VP28, TAT-VP28-EGFP and VP28-EGFP fusion proteins. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry methods were used to confirm that TAT fusion proteins can translocate from the intestine to the hemolymph of the crayfish Cambarus clarkii. After immunization, activities of phenoloxidase and superoxide dismutase were analyzed, and it was found that rTAT-VP28 produced the most pronounced increase in both C. clarkii were vaccinated by oral administration of rTAT-VP28 and rVP28 for 7 and 14 days, and rTAT-VP28 resulted in the highest relative percent survival (RPS) (63.3% at 7 days, and 67.8% at 14 days), compared with rVP28 (44.4% at 7 days, and 53.6% at 14 days) following challenge with WSSV after the last day of feeding. This study reports the use of TAT-derived peptide as an oral delivery method of a subunit vaccine against WSSV in C. clarkii.

Improve bioavailability of Harpin protein on plant use PLGA based nanoparticle.

Harpins can induce systemic acquired resistance (SAR) pathway on scores of non-host plant, provide protection against a range of pathogens. In this study, we demonstrated that applied recombinant HarpinZ Pseudomonas syringae pv. tomato (rHrpZ) on tobacco with three kinds of methods: infiltrating from micro-pore into leaf; injecting into petiole, and spraying on leaf, there is great difference in assimilation of protein because of the poor osmosis of tobacco leaves, and with multi-application of rHrpZ, the stimulation effect decreased. We prepared poly d,l-lactide-co-glycolide nanoparticles containing rHrpZ (rHrpZ PLGA NPs). To study the drug effect, we analyzed the change ratio of phenylalanine ammonia lyase (PAL) activity and PR-5dB gene expression after administration of rHrpZ and rHrpZ PLGA NPs on tobacco leaves. The results show that rHrpZ could elicit a rapid and transient increase in both PAL activity and PR-5dB expression, but the effect decreased after multi-application. While sprayed rHrpZ PLGA NPs on leaves, both the change ratio of PAL activity and PR-5dB expression were comparatively smooth and durable. Our study suggested that rHrpZ NPs could help protein enter leaf epidermis and cell wall, release rHrpZ in situ continuously, and enhance the bioavailability of rHrpZ.

[Primary malignant tumor of the appendix: clinicopathological analysis of 22 cases].

OBJECTIVE: To investigate the clinicopathological and immunohistochemical features of primary malignant tumor of the appendix. METHODS: The clinical data were reviewed; and histopathological and immunohistochemical features were analyzed in 22 cases with primary malignant tumor of the appendix. RESULT: In 22 cases of primary malignant tumor of the appendix, 19 cases were carcinoid and 3 were adenocarcinoma. Immunohistochemistry showed that the carcinoid was positively reacted to the neuroendocrine markers, and the adenocarcinoma was negatively reacted to the neuroendocrine markers. CONCLUSION: Immunohistochemistry is useful in diagnosis of primary malignant tumor of the appendix, a rare type of cancer.

Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium.

Protective immune responses in shrimp induced by DNA vaccines against white spot syndrome virus (WSSV) with intramuscular injection have been reported in recent reports. In this study, we investigated the utilities of attenuated Salmonella enterica serovar Typhimurium (Salmonella typhimurium) as a bactofection vehicle for the oral delivery of a DNA vaccine plasmid to crayfish (Cambarus clarkii). The DNA vaccine plasmid pcDNA3.1-VP28, encoding viral envelope protein VP28, was transformed to an attenuated S. typhimurium strain SV4089 and the resulting recombinant bacteria named SV/pcDNA3.1-VP28 were used to orally immunize crayfish with coated feed. Successful delivery of the DNA vaccine plasmid was shown by the isolation of recombinant bacteria SV/pcDNA3.1-VP28 from the vaccinated crayfish. The distribution analysis of plasmid pcDNA3.1-VP28 in different tissues revealed the effective release of DNA vaccine plasmid into crayfish. RT-PCR and immunoflurescence results confirmed the expression of protein VP28 in the vaccinated crayfish. Challenge experiments with WSSV at 7, 15, 25 days post-vaccination demonstrated significant protection in immunized crayfish with relative survival rate 83.3%, 66.7% and 56.7%, respectively. Studies on stability and safety of SV/pcDNA3.1-VP28 showed the recombinant bacteria could exist in crayfish at least 7 days but not more than 10 days and without any observable harm to the host. Our study here demonstrates, for the first time, the ability of attenuated Salmonella as a live vector to orally deliver a DNA vaccine against WSSV into the arthropod crayfish and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.

[Significance of galectin-3 and CD44v6 expression in differential diagnosis of thyroid nodules].

OBJECTIVE: To investigate the difference of galectin-3 and CD44v6 expression between benign and malignant thyroid nodules, and to evaluate their clinical value in distinguishing thyroid cancer from benign thyroid nodules. METHODS: The expression of galectin-3 and CD44v6 was immunohistochemically detected by the ABC method in 143 benign and malignant thyroid nodule samples. RESULTS: Expression of these two markers in benign thyroid nodules: galectin-3 was negative in 10 cases of para-cancer normal tissue and 14 cases of benign nodules found in the other benign thyroid disease. It was weakly positive in 4 of 52 nodular goiter (7.7%). Also it was weakly positive in 2 of 22 follicular adenomas (9.1%). But all three eosinophilic follicular adenomas were diffusely or focally positive for galectin-3. CD44v6 was negative in 10 cases of para-cancer normal tissue, but positive in 4 of 14 nodular lesions found in benign thyroid diseases (28.6%). It was also positive in 16 of 52 nodular goiters (30.8%), and weakly positive in 7 of 22 follicular adenomas (31.8%). The two markers in malignant lesions: galectin-3 was positive in 50 of 52 thyroid adenocarcinoma (96.2%), CD44v6 was positive in 42 of 52 thyroid adenocarcinoma (80.8%). The positive rate of galectin-3 and CD44v6 expression in thyroid cancer was significantly higher than that in benign thyroid nodule and normal tissue (P < 0.001). The sensitivity, specificity and accuracy of galectin-3 combined with CD44v6 in differentiating benign from malignant thyroid nodule were 80.8%, 93.4%, 88.8%; they were 96.2%, 90.1%, 92.3% for Galectin-3 alone. CONCLUSION: The immunohistochemical expression of galectin-3 and CD44v6 by the ABC method is significantly higher in thyroid cancers than in benign thyroid nodules, especially galectin-3 in thyrocyte being helpful in differentiating benign thyroid nodule from thyroid cancer.