Simple and Novel Assay of the Host-Guest Complexation of Homocysteine with Cucurbit[7]uril.

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH2CH2SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

A novel 1-acyl-sn-glycerol-3-phosphate O-acyltransferase homolog for the synthesis of membrane phospholipids with a branched-chain fatty acyl group in Shewanella livingstonensis Ac10.

1-Acyl-sn-glycerol-3-phosphate O-acyltransferase (PlsC) plays an essential role in the formation of phosphatidic acid, a precursor of various membrane phospholipids (PLs), in bacteria by catalyzing the introduction of an acyl group into the sn-2 position of lysophosphatidic acid. Various bacteria produce more than one PlsC. However, the physiological significance of the occurrence of multiple PlsCs is poorly understood. A psychrotrophic bacterium, Shewanella livingstonensis Ac10, which produces eicosapentaenoic acid at low temperatures, has five putative PlsCs (PlsC1-5). We previously showed that PlsC1 is responsible for the production of PLs containing an eicosapentaenoyl group. Here, we characterized another putative PlsC of this bacterium named PlsC4. We generated a plsC4-disrupted mutant and found that PLs containing 13:0 found in the parental strain were almost completely absent in the mutant. The loss of these PLs was suppressed by introduction of a plsC4-expression plasmid. PLs containing 15:0 were also drastically decreased by plsC4 disruption. Gas chromatography-mass spectrometry analysis of fatty acyl methyl esters derived from PLs of the parental strain showed that the 13:0 and 15:0 groups were an 11-methyllauroyl group and a 13-methylmyristoyl group, respectively. Phospholipase A2 treatment revealed that these fatty acyl groups were linked to the sn-2 position of PLs. Thus, PlsC4 is a new type of PlsC homolog that is responsible for the synthesis of PLs containing a branched-chain fatty acyl group at the sn-2 position and plays a clearly different role from that of PlsC1 in vivo.

Development of collagenase-resistant collagen and its interaction with adult human dermal fibroblasts.

Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenecity, made collagen the primary source in biomedical application. Collagen has been widely used in the crosslinked form to extend the durability of collagen. The chemical treatment influences the structural integrity of collagen molecule resulting in the loss of triple helical characteristic. The structural characteristic of collagen is importantly related to its biological function for the interaction with cell. In this study, structural stability of collagen was enhanced thought EGCG treatment, resulting in high resistance against degradation by bacterial collagenase and MMP-1, which is confirmed by collagen zymography. The triple helical structure of EGCG-treated collagen could be maintained at 37 degrees C in comparison with collagen, which confirmed by CD spectra analysis, and EGCG-treated collagen showed high free-radical scavenging activity. Also, with fibroblasts culture on EGCG-treated collagen, the structural stability of EGCG-treated collagen provided a favorable support for cell function in cell adhesion and actin filament expression. These observations underscore the need for native, triple helical collagen conformation as a prerequisite for integrin-mediated cell adhesion and functions. According to this experiment, EGCG-treated collagen assumes to provide a practical benefit to resist the degradation by collagenase retaining its structural characteristic, and can be a suitable biomaterial for biomedical application.