Characterisation of a novel plasmid containing a florfenicol resistance gene in Haemophilus parasuis.

Thirty clinical isolates of H. parasuis from pig farms in eastern China were screened for antimicrobial susceptibility. A novel plasmid, designated pHPSGC, was extracted from one isolate with evidence of resistance (elevated minimum inhibitory concentration) to florfenicol. DNA sequencing demonstrated that pHPSGC (5297 base pairs) contains three open reading frames (ORFs), corresponding to the genes rep, floR and lysR. The rep gene of pHPSGC shared 99% sequence identity with the rep gene of pHPS1019. In addition, the region containing floR and lysR in pHPSGC shared 99% similarity with the corresponding region of pCCK381. pHPSGC may be derived from a recombination event between pHPS1019 and pCCK381. A florfenicol resistance gene in H. parasuis may have been transferred via recombination between different plasmids.

Bone Regeneration of Blood-derived Stem Cells within Dental Implants.

Peripheral blood (PB) is known as a source of mesenchymal stem cells (MSCs), as is bone marrow (BM), and is acquired easily. However, it is difficult to have enough MSCs, and their osteogenic capacity with dental implantations is scarce. Therefore, we characterized peripheral blood mesenchymal stem cells (PBMSCs) cultured on a bone marrow-derived mesenchymal stem cell (BMMSC) natural extracellular matrix (ECM) and demonstrated the osteogenic capability in an experimental chamber implant surgery model in rabbits. We isolated PBMSCs from rabbits by culturing on a natural ECM-coated plate during primary culture. We characterized the PBMSCs using a fluorescence-activated cell scanner, cell proliferation assay, and multiple differentiation assay and compared them with BMMSCs. We also analyzed the osteogenic potential of PBMSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) by transplanting them into immunocompromised mice. Then, the mixture was applied to the canals. After 3 and 6 wk, we analyzed new bone (NB) formation inside the chambers using histological and histomorphometric analyses. The PBMSCs had a similar rate of BrdU-positive cells to BMMSCs, positively expressing CD90 but negative for CD14. The PBMSCs also showed osteogenic, adipogenic, and chondrogenic ability in vitro and osteogenic ability in vivo. Histological and histomorphometric results illustrated that the PBMSC and BMMSC groups showed higher NB than the HA/TCP and defect groups in the upper and lower chambers at 6 wk and in the upper canal at 3 wk; however, there was no difference in NB among all groups in the lower canal at 3 wk. The PBMSCs have characteristics and bone regeneration ability similar to BMMSCs both in vitro and in vivo. ECM was effective for obtaining PBMSCs. Therefore, PBMSCs are a promising source for bone regeneration for clinical use.

Bone regeneration at dental implant sites with suspended stem cells.

During the maintenance of bone marrow-derived mesenchymal stem cells (BMMSCs), suspended cells are discarded normally. We noted the osteogenic potential of these cells to be like that of anchorage-dependent BMMSCs. Therefore, we characterized suspended BMMSCs from rabbit bone marrow by bioengineering and applied the suspended BMMSCs to double-canaled dental implants inserted into rabbits. After primary isolation of BMMSCs, we collected the suspended cells during primary culture on the third day. The cells were transferred and maintained on an extracellular-matrix-coated culture plate. The cells were characterized and compared with BMMSCs by colony-forming-unit fibroblast (CFU-f) and cell proliferation assay, fluorescence-activated cell sorter (FACS), in vitro multipotency, and reverse transcription polymerase chain reaction (RT-PCR). We also analyzed the osteogenic potential of cells mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and transplanted into immunocompromised mice. We compared the viability and proliferation of the suspended BMMSCs and BMMSCs on the titanium implant surface and observed cell morphology. Then, the cells mixed with HA/TCP were applied to the double-canaled implants during installation into rabbit tibia. Four weeks later, we analyzed bone formation inside the canal by histomorphometry. The suspended cells showed higher CFU-f on the extracellular matrix (ECM)-coated culture plate and similar results of proliferation capacity compared with BMMSCs. The cells also showed osteogenic, adipogenic, and chondrogenic ability. The suspended cells showed levels of attachment survival and proliferation on the surfaces of titanium implant discs to be higher than or similar to those of BMMSCs. The suspended cells as well as BMMSCs showed stronger bone formation ability in both upper and lower canals of the implants compared with controls on double-canaled implants inserted into rabbit tibia. In this study, we showed that suspended cells after primary BMMSC isolation have bone regeneration capacity like that of BMMSCs, not only in vitro but also in vivo. ECM was valuable for propagation of MSCs for cell-based bone regeneration. Therefore, the suspended cells could also be useful tools for bone regeneration after implant surgery.

R-enantioselective hydrolysis of 2,2-dimethylcyclopropanecarboxamide by amidase from a newly isolated strain Brevibacterium epidermidis ZJB-07021.

AIMS: To isolate new micro-organisms with R-stereospecific amidase activity and to examine their potential as biocatalysts in enantioselective hydrolysis of 2,2-dimethylcyclopropanecarboxamide (1). METHODS AND RESULTS: A novel R-stereospecific amidase-producing strain ZJB-07021 was isolated through a sophisticated colorimetric screening method. Based on morphology, physiological tests, Biolog system (GP2) and 16S rRNA sequence, the new isolate was identified as Brevibacterium epidermidis. After 70 min of bioconversion at 35 degrees C, kinetic resolution of (R,S)-1 by the amidase afforded (S)-1 in 41.1% yield (>99% ee) and (R)-2 in 49.9% yield (69.7% ee) with an average E-value of 23. The enantioselectivity was found to be temperature dependent and enhanced from 12.6 at 45 degrees C to 65.9 at 14 degrees C. CONCLUSIONS: A novel bacterial strain of B. epidermidis ZJB-07021 producing R-stereospecific amidase was isolated and characterized. The isolate exhibited high E values for kinetic resolution of racemic-1 to (S)-1. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this was the first report on the species B. epidermidis that harboured R-stereospecific amidase. Strain ZJB-07021 could be further improved as a suitable biocatalyst for the stereoselective bioconversion of racemic-1 after optimization of culture and biotransformation process.

Stability study on the nitrile hydratase of Nocardia sp. 108: from resting cell to crude enzyme preparation.

In recent years, nitrile hydratases (NHases) have drawn increasing attentions due to their critical roles in organic synthesis. In present paper, extensive investigation on the stability and activity of the NHase from Nocardia sp. 108, which is succeed in the industrial application in China, were conducted by the bioconversion of acrylonitrile to acrylamide in a batch manner. Cultivation study demonstrated that biosynthesis of NHase changed significantly with culture time, and the optimal NHase biosynthesis phase was 45 h after inoculation with NHase activity of 1209.8 U/g of biomass. Stability study indicated that crude enzyme preparation both exhibit a good stability when exposed to the pH 7.2 tris-HCl buffer at 4 degrees C for 4 h.