Characterization of human umbilical cord mesenchymal stem cells following tissue mass culture.

The human umbilical cord represents a promising resource of mesenchymal stem cells (MSCs). In order to improve our understanding of MSCs derived from human umbilical cord (UC-MSCs), we isolated UC-MSCs from human umbilical cord tissues through a direct culture approach. We performed a comprehensive characterization of these cells based on analyses of morphology, growth features, cell surface antigen markers and differentiation capacity. All these analyses validated their stem cell nature. The UC-MSCs presented a spindle-shaped morphology and could be subcultured for up to 15 passages without losing their cellular features. Moreover, these UC-SMCs presented an expression profile of cell surface antigens similar to other MSCs: positive for CD44, CD90, and CD105 expression and negative for CD34, CD31, and CD45 expression. Differentiation assays further validated the multipotency of UC-MSCs by inducing these cells into osteoblasts, adipocytes and functional hepatocytes. Our studies clearly demonstrated that UC-MSCs resemble other types of MSCs in many aspects and have a great potential to be applied in tissue engineering and regenerative medicine.

The preparation and characterization of an antimicrobial polypeptide from the loach, Misgurnus anguillicaudatus.

Using Sephadex G-50 gel filtration, DEAE-52 cellulose ion-exchange chromatography, and an improved polyacrylamide gel electrophoresis together with electroelution, a novel polypeptide with antimicrobial activity in vitro was isolated and characterized from loach, Misgurnus anguillicaudatus. The polypeptide, named MAPP, contains about 94 residues containing l0 different amino acids, of which cysteine was the most abundant. No alkaline residue was found in MAPP. MAPP is a single-chain polypeptide with Mw of about 9800Da and pI of about 4.78; the N-terminus of MAPP was CFGWN. MAPP showed good inhibition of various bacteria including Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. MAPP is thermally stable with more than 70% inhibitory bioactivity remaining after treatment at 60 degrees C for 30min. In addition, MAPP could inhibit the autoxidation of pyrogallol with a high efficiency. Similarity searches by comparing amino acid composition, MS-fingerprint, and the N-terminus of MAPP demonstrated that no protein exactly matched MAPP in databases around the world.

Electrotransformation of Clostridium acetobutylicum ATCC 824 using high-voltage radio frequency modulated square pulses.

Molecular biological improvement of industrial solventogenic clostridia could be enhanced by a higher efficiency of electrotransformation. In this research, we used a new approach to determine the frequency spontaneously generated by Clostridium acetobutylicum ATCC 824 cells during the application of a square high-voltage pulse. Once the frequency of 100 kHz was determined we transformed clostridial cells with pSOS84 plasmid DNA using radio-frequency modulated high-voltage square pulses (electric field strength 12 kVcm-1; pulse duration 22.5 ms; frequency of pulse modulation 100 kHz) to reach an efficiency exceeding 106 transformants microg-1 of plasmid DNA. We propose a possible role for cellular membrane structures in affecting the transformation yield.

Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.

Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein. Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxadiene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a single step with a His-binding metal affinity column. The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.

Transcriptional regulation of c-Fes in myeloid leukemia cells.

The c-Fes proto-oncogene encodes a myeloid-specific protein-tyrosine kinase that is expressed preferentially in differentiated myeloid cells, but not in early myeloblast progenitor cells. To examine the basis for the phenotypic expression of c-Fes, the transcription initiation sites of the human c-Fes gene were mapped in myeloid leukemia cells and regulatory elements in the genomic c-Fes sequence were characterized. Two major transcription initiation sites were found in the myeloid leukemia cell line THP-1 which delineated exon 1 to be 72-83 bp. When the activity of the CAT reporter gene under the control of the c-Fes promoter region, untranslated exon 1 and intron 1 was measured in TF-1, K562 and MCF-7 cells, only TF-1 cells exhibited chloramphenicol acetyltransferase activity. In contrast, all cell lines supported reporter gene activity when intron 1 was deleted. Deletion analyses revealed a negative regulatory region in intron 1, which was localized by Southwestern analysis and DNA footprinting to a 14 bp region. This negative regulatory region suppressed reporter CAT activity in K562 and TF-1 cells when inserted downstream to the SV40 early promoter. These results suggest that the tissue-specific expression of c-Fes may result, in part, from the negative regulation of transcription in myeloid and nonmyeloid cells.

Molecular cloning and heterologous expression of the gene encoding dihydrogeodin oxidase, a multicopper blue enzyme from Aspergillus terreus.

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (+)- geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in lambda gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis.