Soluble expression of active human β-defensin-3 in Escherichia coli and its effects on the growth of host cells

Background Human β-defensin-3 （HBD3） is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coil （E. colt） is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD3 protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways. Methods We first cloned the complementary DNA coding for the mature chain of HBD3, inserted it into the vector PGEX-KG then transformed E. coil BL21 （DE3） with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-β-D-galactopyranoside （IPTG） the transformed E. cofiproduced a recombinant glutathione S-transferase and HBD3 （GST-HBD3） fusion protein. The fusion protein was treated with thrombin to produce pure HBD3 protein then the antimicrobial activity of HBD3 was evaluated in a liquid microdilution assay. Results The fusion protein GST-HBD3 was efficiently cleaved by thrombin and yielded HBD3 that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 μg/ml. The E. coil strain expressing the recombinant protein did not grow slower than the empty vector strain. Conclusion Active HBD3 in E. coil by expressing the recombinant protein GST-HBD3 could be produced, and suicide did not occur in the E. coli strain expressing the recombinant protein.

Prediction of recurrence risk in early breast cancer using human epidermal growth factor 2 and cyclin A2

Background Human epidermal growth factor 2 （HER2） is one of the most important prediction factors, but only 25%-30% of breast cancer patients HER2 are positive. It is unknown whether there are other molecular markers that could be used to predict prognosis and recurrence in HER2 negative patients.This study investigated correlations of cyclin A2 and HER2 levels with clinical outcomes in 281 patients with invasive breast cancer in order to identify whether cyclin A2 can serve as a prognostic factor in HER2 negative patients. Methods Immunohistochemical staining was used to detect cyclin A2 and HER2 expression in 281 patients. Cyclin A2 and HER2 gene amplifications were analyzed using gene analysis and RT-PCR in 12 patients. Risk and survival estimates were analyzed using Log-rank, Kaplan-Meier, and Cox regression analysis; cyclin A2 and HER2 consistency with survival were analyzed using Kappa analysis. Results Patients with higher cyclin A2 and HER2 expressions had significantly shorter disease-free survival periods （P=0.047 and P=-0.05, respectively）. Kappa analysis performed that cyclin A2 and HER2 showed a low Kappa index （kappa=0.37）, allowing us to conclude that cyclin A2 and HER2 detect different pathologies. Gene analysis and RT-PCR showed that cyclin A2 was upregulated in patients with early relapse; the average increase was 3.69-2.74 fold. Conclusions Cyclin A2 and HER2 are associated with proliferation and high recurrence, particularly when combined. Cyclin A2 is easily detected by nuclear staining and might be a useful biomarker for recurrence risk in HER2 negative patients.

Clinicopathological significance of altered metallothionein 2A expression in gastric cancer according to Lauren＇s classification

Background Dysregulated metallothionein 2A （MT2A） has been implicated in carcinogenesis. The purpose of this study was to investigate the expression of MT2A in gastric cancer （GC） and its correlation with prognosis. Methods Reverse transcription-polymerase chain reaction and real-time polymerase chain reaction were used to detect the mRNA expression of MT2A in 12 GC cell lines, normal gastric epithelial GES-1 cells, and 36 GC and adjacent normal tissues. MT2A protein expression was determined in 258 GC tissues and 171 adjacent normal tissues by immunohistochemistry. Results MT2A mRNA expression was lower in GC cells and primary tumors than in GES-1 cells and adjacent normal tissues, respectively, High protein expression of MT2A was present in 130 of 171 normal tissues （76.0%） and in 56 of 258 GC tissues （21,7%; P 〈0.001）. MT2A protein expression was higher in well/moderately differentiated GC （22/54; 40.7%） than in poorly differentiated GC （34/204; 16.7%; P 〈0.001）. Moreover, the protein expression of MT2A was lower in diffuse-type GC （6/82; 7.3%） than in intestinal-type GC （50/176; 28.4%; P=0.0001）. Importantly, MT2A expression was an independent prognostic factor for GC, and decreased MT2A expression was associated with poor clinical outcome （P 〈0.001）. The expression status of MT2A could predict prognosis in intestinal and diffuse-type GCs. Conclusion Expression status of MT2A might be a useful prognostic biomarker for GC, especially when used in combination with Lauren＇s classification.