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A novel and effective method for target cells capture and detection is developed in a label-free and real-time mode. The cultured cell line (TZMbl) expressed as CD4+ was used as the positive CD4+ T lymphocytes for target cells capture and detection experiments. The target cells were captured on various surfaces (such as glass, polydimethylsiloxane, and metal) based on a specific immunity method. The gold surface of the surface plasmon resonance (SPR) sensing chip modified with a nano denaturalised bovine serum albumin layer had the best capture capacity. A total of 90.9% positive CD4+ T lymphocytes were captured with a slow flow rate of 5 μl/min, whereas 84.1% of cells were kept with a fast flow rate of 2 ml/min. Furthermore, the captured positive cells on the SPR sensing chip were detected on-line by using author's home-made SPR biosensor. From the experimental results, 75 cells/μl could produce an obvious change in the SPR response signal, which holds great potential for target cells capture, reorganisation and further analysis of CD4+ T lymphocytes in human peripheral blood. [ABSTRACT FROM AUTHOR]

Toll-like receptors mediate immune responses via recognition of pathogen-associated molecular patterns. Polymorphisms of toll-like receptors may affect their recognition of pathogen-associated molecular patterns, leading to varied host resistance to pathogenic infections. However, little is known about the polymorphisms of chicken toll-like receptors (ChTLR) among breeds. In this study, we cloned ChTLR2 type 1 and type 2 genes from 7 chicken breeds and analyzed their sequences. It was found that there were 10 amino acid polymorphism sites in ChTLR2 type 1 and type 2, among which 6 sites were in type 1 (5 sites in the extracellular domain and 1 site in the cytoplasmic domain) and 4 sites were in type 2 (all 4 sites in the extracellular domain). These results demonstrate that ChTLR2 type 1 and type 2 genes are polymorphic among chicken breeds, suggesting a varied resistance among breeds of chicken. We found that Laiwu Black chicken breeds have distinctive polymorphic sites at I699T in the type 1 and H561R in the type 2 ChTLR2 gene. Beijing Fatty chickens have distinctive sites at Q45R in type 1 and V66L in type 2. Nongda No.3 Chickens have distinctive sites at L115P, H232Y, and T494A in type 1. Hy-Line variety brown chickens have distinctive sites at I311V in type 2. Beijing White 939 chickens have distinctive sites at E284G in type 1 and A22V in type 2. These findings may provide some clues toward understanding the resistance of lighter chickens to salmonellosis. [ABSTRACT FROM AUTHOR]

The tissue-type plasminogen activator (t-PA) has been found to bind reversibly to human fibronectin (Fn). To locate the binding site on Fn for t-PA, the Fn was degraded with N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin, and the resulting fragments were monitored by the enzyme-linked immunosorbent assay method for t-PA binding activities. A 20-kDa fragment with t-PA binding activity was identified, separated, and purified. It was subjected to further degradation with Staphylococcus aureus proteinase V8. An active 10-kDa fragment was finally purified by reverse-phase high pressure liquid chromatography on a C3 column. The dissociation constants of the binding of Fn and the 10-kDa fragment to t-PA were estimated by Scatchard plot to be 1.13 x 10(-8) and 2.08 x 10(-8) M, respectively. The 10-kDa fragment was sequenced and proved to be located at the 8-9th domains of type I homology of Fn. Based on the structural analysis of the 8-9th domains, a heptadecapeptide corresponding to the sequence Thr535-Glyl551 of Fn, which resided at the large disulfide loop of domain (I-9), was designed and synthesized. Both the 10-kDa fragment and the synthetic peptide could competitively inhibit the binding of Fn to t-PA. The synthetic peptide showed about one-tenth of the binding activity of Fn to t-PA with a dissociation constant of 1.35 x 10(-7) M and was proved to be the binding region of Fn for t-PA. In addition, like the intact Fn, both the 10-kDa fragment and the synthetic peptide could remarkably enhance the amidolytic activity of t-PA in a dose-dependent manner, as shown by using S-2288 as a chromogenic substrate.