Comparative characteristics of three human embryonic stem cell lines

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.     

Genomic segments cloning and analysis of Cotesia plutellae polydnavirus using plasmid capture system

Cotesia plutellae polydnaviruses (CpBV) has a segmented genome consisting of multiple circular double stranded DNAs. Recently, we have developed an easy, simple, and convenient system based on Tn7 transposition in order to clone genomic segments of CpBV in Escherichia coli cell and designated plasmid capture system (PCS). The PCS donor-S transferred a pUC19 origin of replication and an ampicillin resistance marker into CpBV genomic DNA by in vitro transposition. Through PCS system, we were able to clone 53 genomic clones ranging from 0.1 to 25.5 kb and further they were classified into 29 segments by their sizes and restriction endonuclease patterns. Among them, a complete nucleotide sequence of CpBV-S28 segment was determined and 10 putative genes were predicted from this segment. Interestingly, 9 of 10 putative ORFs had high level of similarities with catalytic domain of protein tyrosine phosphatase. Also, ORF2807 showed similarity with EP1-like proteins of C. congregata polydnavirus.     

Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.     

Evaluation of 3,4-dihydroquinazoline-2(1H)-thiones as inhibitors of α-MSH-induced melanin production in melanoma B16 cells

Novel 3,4-dihydroquinazoline-2(1H)-thiones (QNTs) 1 were found to be potent inhibitors of α-MSH-induced melanin production. The effect of QNTs to inhibit melanin formation in B16 melanoma cells was screened in the presence of α-MSH. In defining the mechanism of activity, the effects on tyrosinase activity, on tyrosinase synthesis and on the depigmentation of melanin were evaluated. QNTs did not affect the catalytic activity of tyrosinase, but rather acted as an inhibitor of tyrosinase synthesis.     

Complete Genome Sequence of Halalkalicoccus jeotgali B3T,an Extremely Halophilic Archaeon

Halalkalicoccus jeotgali B3^T, isolated from salt-fermented seafood from South Korea, is an extremely halophilic archaeon belonging to the family Halobacteriaceae. Here, we present the complete genome sequence of the type strain H. jeotgali B3^T (3,698,650 bp, with a G+C content of 62.5%), which consists of one chromosome and six plasmids. This is the first complete genome sequence of the Halalkalicoccus species.Extremely halophilic archaea (haloarchaea) are adapted to hypersaline environments and grow optimally in NaCl solutions of 2.6 M or higher (12). These haloarchaea are classified within the family Halobacteriaceae in the order Halobacteriales; currently, this family comprises 28 genera (3), and only 11 complete genome sequences in Halobacteriaceae have been reported. In a study of archaeal diversity in salt-fermented small shrimp or shellfish from South Korea, our laboratory isolated and characterized 5 novel, extremely halophilic archaeal strains of Halobacteriaceae. These strains included Natronococcus jeotgali (9), Halalkalicoccus jeotgali (11), Halorubrum cibi (7), Haloterrigena jeotgali (10) and Haladaptatus cibarius (8). We have now sequenced the genome of Halalkalicoccus jeotgali B3^T; genome sequencing had not been completed or initiated for any strain in this genus when our sequencing project was begun. The genus Halalkalicoccus currently contains only two species, Halalkalicoccus tibetensis (13) and H. jeotgali, and these species exhibit 98.6% gene sequence similarity in their 16S rRNA. The genome of H. jeotgali B3^T is the first of this genus to be sequenced.The complete genome sequence of H. jeotgali B3^T was determined by a whole-genome shotgun strategy using Roche 454 GS (FLX Titanium) pyrosequencing (898,168 reads totaling ∼348 Mb; ∼94-fold coverage of the genome) and a fosmid library (514 reads totaling ∼680 kb) at the Genome Resource Center, KRIBB (Korea Research Institute of Bioscience and Biotechnology). Genome sequences from pyrosequencing were processed by Roche''s software according to the manufacturer''s instructions, and sequences from the fosmid library were processed by PESTAS (6). A total of 898,196 reads were assembled using Newbler Assembler 2.3 (454 Life Science), which generated 54 large contigs (>100 bp in size) with bases having quality scores of 40 and above. The gaps between contigs were closed by primer walking and sequencing of PCR products across the gaps. The annotation was done by merging results obtained from the RAST (Rapid Annotation using Subsystem Technology) pipeline (1), Glimmer 3.02 (2), tRNAscan-SE 1.21 (5), and RNAmmer 1.2 (4).The H. jeotgali B3^T genome is 3,698,650 bases long with a 62.5% G+C content. The chromosome consists of a single circular chromosome (2,809,118 bp, with a G+C content of 65.0%) and six plasmids (406,285 bp, 55.3%; 363,534 bp, 54.2%; 44,576 bp, 58.9%; 44,459 bp, 54.9%; 23,727 bp, 47.6%; 6,951 bp, 60.6%). The genome contains 3,860 predicted coding sequences and 52 RNA genes (determined using RAST). The chromosome is predicted to contain 3,101 coding sequences with a coding intensity of 90.0%, including 47 tRNA genes, 1 5S rRNA gene, 1 16S rRNA gene, and 1 23S rRNA gene. The largest plasmid contains 466 coding sequences with a coding intensity of 81.2% and 2 tRNA genes, while the other five plasmids contain 425, 44, 48, 29, and 5 coding sequences with coding intensities of 80.2%, 84.2%, 83.0%, 69.6%, and 22.8%, respectively (determined using Glimmer3). More detailed analysis of this genome and comparative analysis with other haloarchaea will provide further insight into the genomic differences and metabolism of the extremely halophilic archaea.     

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680

Regiospecific 3′‐hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K_m and k_cat values of CYP105D7 for daidzein were 21.83 ± 6.3 µM and 15.01 ± 0.6 min^?1 in the presence of 1 µM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO‐difference spectra at 450 nm using the whole cell extract. When the whole‐cell reaction for the 3′‐hydroxylation reaction of daidzein was carried out with 100 µM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6‐fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3′,4′‐trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h. Biotechnol. Bioeng. 2010. 105: 697–704. © 2009 Wiley Periodicals.     

Pivotal role of RanBP9 in integrin-dependent focal adhesion signaling and assembly

Accumulation of the amyloid β (Aβ) peptide derived from the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease (AD). We previously reported that the scaffolding protein RanBP9 is markedly increased in AD brains and promotes Aβ generation by scaffolding APP/BACE1/LRP complexes together and accelerating APP endocytosis. Because APP, LRP, and RanBP9 all physically interact with β-integrins, we investigated whether RanBP9 alters integrin-dependent cell adhesion and focal adhesion signaling. Here, we show that RanBP9 overexpression dramatically disrupts integrin-dependent cell attachment and spreading in NIH3T3 and hippocampus-derived HT22 cells, concomitant with strongly decreased Pyk2/paxillin signaling and talin/vinculin localization in focal adhesion complexes. Conversely, RanBP9 knockdown robustly promotes cell attachment, spreading, and focal adhesion signaling and assembly. Cell surface biotinylation and endocytosis assays reveal that RanBP9 overexpression and RanBP9 siRNA potently reduces and increases surface β1-integrin and LRP by accelerating and inhibiting their endocytosis, respectively. Primary hippocampal neurons derived from RanBP9-transgenic mice also demonstrate severely reduced levels of surface β1-integrin, LRP, and APP, as well as neurite arborization. Therefore, these data indicate that RanBP9 simultaneously inhibits cell-adhesive processes and enhances Aβ generation by accelerating APP, LRP, and β1-integrin endocytosis.     

Human nuclear clusterin mediates apoptosis by interacting with Bcl-XL through C-terminal coiled coil domain

Clusterin (CLU), a glycoprotein, is involved in apoptosis, producing two alternatively spliced isoforms in various cell types. The pro-apoptotic CLU appears to be a nuclear isoform (nuclear clusterin; nCLU), and the secretory CLU (sCLU) is thought to be anti-apoptotic. The detailed molecular mechanism of nCLU as a pro-apoptotic molecule has not yet been clear. In the current study, overexpressed nCLU induced apoptosis in human kidney cells. Biochemical studies revealed that nCLU sequestered Bcl-XL via a putative BH3 motif in the C-terminal coiled coil (CC2) domain, releasing Bax, and promoted apoptosis accompanied by activation of caspase-3 and cytochrome c release. These results suggest a novel mechanism of apoptosis mediated by nCLU as a pro-apoptotic molecule.