Simulated microgravity reduces proliferation and reorganizes the cytoskeleton of human umbilical cord mesenchymal stem cells.

The cytoskeleton plays a key role in cellular proliferation, cell-shape maintenance and internal cellular organization. Cells are highly sensitive to changes in microgravity, which can induce alterations in the distribution of the cytoskeletal and cell proliferation. This study aimed to assess the effects of simulated microgravity (SMG) on the proliferation and expression of major cell cycle-related regulators and cytoskeletal proteins in human umbilical cord mesenchymal stem cells (hucMSCs). A WST-1 assay showed that the proliferation of SMG-exposed hucMSCs was lower than a control group. Furthermore, flow cytometry analysis demonstrated that the percentage of SMG-exposed hucMSCs in the G0/G1 phase was higher than the control group. A western blot analysis revealed there was a downregulation of cyclin A1 and A2 expression in SMG-exposed hucMSCs as well. The expression of cyclin-dependent kinase 4 (cdk4) and 6 (cdk6) were also observed to be reduced in the SMG-exposed hucMSCs. The total nuclear intensity of SMG-exposed hucMSCs was also lower than the control group. However, there were no differences in the nuclear area or nuclear-shape value of hucMSCs from the SMG and control groups. A western blot and quantitative RT-PCR analysis showed that SMG-exposed hucMSCs experienced a downregulation of bata-actin and alpha-tubulin compared to the control group. SMG generated the reorganization of microtubules and microfilaments in hucMSCs. Our study supports the idea that the downregulation of major cell cycle-related proteins and cytoskeletal proteins results in the remodeling of the cytoskeleton and the proliferation of hucMSCs.

Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants.

To study the role of the hinge region in nisin and to obtain mutants that exhibit altered or new biological activities and functional properties, we changed certain amino acids in the hinge region by performing site-directed mutagenesis with the nisinZ structural gene ( nisZ). The results showed that the nisinZ mutants had decreased antimicrobial activities against Micrococcus flavus NCIB8166 and Streptococcus thermophilus. Interestingly, compared with wild nisinZ, mutant N20K nisinZ and M21K nisinZ displayed antimicrobial activity against gram-negative Shigella, Pseudomonas and Salmonella; and they had a higher solubility than wild-type nisinZ. At pH 8, the solubilities of N20K nisinZ and M21K nisinZ were, respectively, three-fold higher and five-fold higher than that of nisinZ. Mutant N20Q nisinZ and M21G nisinZ were considerably more stable than nisinZ at higher temperatures and neutral or alkaline pH. These mutants provided information that the central hinge region in nisinZ plays an important role in providing the conformational flexibility required for the antimicrobial activity on the membrane. Our finding documented that it may well be worth considering the construction of the new nisin mutants with changed inhibitory activity against a wide range of gram-negative bacteria and the improvement of functional properties by site-directed mutagenesis.

[cDNA cloning and sequence analysis of human lactoferrin].

Human lactoferrin (hLF) cDNA was amplified by RT-PCR from normal human mammary tissue obtained from Daxing County of Beijing City of China, and then subcloned into pGEM-T vector. hLF cDNA sequence was determined, which consists of 2136 bp. Comparison with five other hLF cDNA sequences registered in GenBank shows 99% homology in DNA sequence. However, there are two base substitutions (nucleotide 1740 G-->C, nucleotide 1756 T-->C), one of which subsequently leads to an amino acid change (residue 580 Glu-->Asp).

[Fusion expression of a peptide antibiotic-apidaecin gene in Lactococcus lactis].

The ubiquitin fusion of apidaecin was expressed in Lactococcus lactis, using a novel nisin-inducible expression system. After induction, a specific band could be detected in the extracts of the host strain by Tricine-SDS-PAGE and Western blotting. Production of the fusion was up to 7.2% of the total soluble protein of the host strain. While the fusion was cut by ubiquitin specific protease-UBP1, the product had distinct antibacterial activity.

[Cloning and expression of nisZ gene in Lactococcus lactis].

The gene encoding the precursor of nisin was amplified by PCR using the lambda HJ-3 DNA as the template, which contained the entire nisin biosynthesis gene cluster from Lactococcus lactis AL2 with high yield of nisin, and was cloned into pMG36e. The recombinant plasmid pHJ201 was introduced into Lactococcus lactis NZ9800 by electroporation. pHJ201 is very stable in L. lactis NZ9800. Antimicrobial activity test and Tricine-SDS-PAGE analysis revealed that L. lactis NZ9800 harbouring pHJ201 restored ability of nisin production, but the production level was markedly lower than L. lactis AL2. The result of DNA sequence analysis indicated that Nisin Z is produced by L. lactis AL2.

[Cloning and expression of Streptomyces lividans promoters].

BamHI restriction fragments from Streptomyces lividans TK24 chromosome DNA have been cloned into BamHI site of promoter probe plasmid pIJ486. Transformants were selected on the medium containing 5 micrograms/ml of neomycin. Four recombinant plasmids pMG1(10.6 kb), pMG40(7.6 kb), pMG50(10.8 kb) and pMG88(7.92 kb), were found and designated respectively. The inserted fragments in pMG40 and pMG50 were reduced to 0.78kb and 2.2 kb by BglII digestion and rejoining. The different levels of neomycin and kanamycin resistance of these recombinant plasmids were determined. The results revealed that pMG50-25 showed a high level of neomycin resistance (90 micrograms/ml) and kanamycin resistance (500 micrograms/ml).